cd45 congenic recipient mice 1 d  (Worthington Biochemical)


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    Name:
    Reverse Transcriptase Recombinant HIV
    Description:
    Chromatographically purified dimeric form with M W of 66 kDa and 51 kDa A solution in 10 mM potassium phosphate pH 7 4 1 mM DTT and 20 glycerol
    Catalog Number:
    ls05000
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    Source:
    E. coli plasmid pRC-RT
    Cas Number:
    9068.38.6
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    Structured Review

    Worthington Biochemical cd45 congenic recipient mice 1 d
    Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into <t>CD45</t> <t>congenic</t> recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P
    Chromatographically purified dimeric form with M W of 66 kDa and 51 kDa A solution in 10 mM potassium phosphate pH 7 4 1 mM DTT and 20 glycerol
    https://www.bioz.com/result/cd45 congenic recipient mice 1 d/product/Worthington Biochemical
    Average 86 stars, based on 65 article reviews
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    cd45 congenic recipient mice 1 d - by Bioz Stars, 2020-08
    86/100 stars

    Images

    1) Product Images from "A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation"

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20132267

    Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P
    Figure Legend Snippet: Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P

    Techniques Used: Mouse Assay, Transduction, Expressing, Injection, Staining

    Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P
    Figure Legend Snippet: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Genome Wide, Generated, Magnetic Cell Separation, FACS, Irradiation, Purification

    Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P
    Figure Legend Snippet: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P

    Techniques Used: Injection, Mouse Assay, FACS, Staining, Fluorescence, In Vivo

    Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.
    Figure Legend Snippet: Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.

    Techniques Used: Injection, Expressing, Staining, Mouse Assay

    2) Product Images from "Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *"

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M510504200

    Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.
    Figure Legend Snippet: Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.

    Techniques Used: Sequencing

    Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.
    Figure Legend Snippet: Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.

    Techniques Used: Sequencing

    Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.
    Figure Legend Snippet: Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.

    Techniques Used: Generated, Labeling

    3) Product Images from "Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity"

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.01848-17

    ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).
    Figure Legend Snippet: ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).

    Techniques Used: Cell Attachment Assay, Labeling, Staining, Binding Assay

    The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis

    The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Incubation, SDS Page, Western Blot

    The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Incubation, Infection, Plaque Assay, SDS Page, Staining

    The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Quantitative RT-PCR

    ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Generated

    The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).
    Figure Legend Snippet: The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).

    Techniques Used: Mutagenesis, Incubation, Fluorescence

    The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Activity Assay, Incubation, Infection, Plaque Assay, Western Blot, SDS Page, Staining

    The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).
    Figure Legend Snippet: The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).

    Techniques Used: Mutagenesis, SDS Page, Western Blot, Infection, Immunofluorescence

    4) Product Images from "Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs"

    Article Title: Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1901165116

    AUF1 expression is essential for myoblast differentiation and myotube formation. ( A ) Expression of AUBP mRNAs during differentiation at 96 h determined by qRT-PCR ( n = 3), normalized to invariant GAPDH mRNA. ( B ) Expression of AUBP proteins during differentiation corresponding to A , n = 4. Numbers below immunoblot: Twist1 fold increase normalized to WT cells. ( C ) Immunoblot of CTCF, AUF1, MyoD, and β-tubulin control during C2C12 myoblast differentiation. CTCF was silenced in C2C12 cells by lentiviral-mediated shRNA. Numbers under immunoblot correspond to AUF1 fold change relative to β-tubulin levels, normalized to 0-h Nsi time point. n = 4. ( D ) Immunoblot of CTCF and AUF1 with increasing transfection of a CTCF cDNA expression plasmid in HEK 293 cells. Overexpression of CTCF was performed using pMy-CTCF (Addgene), n = 3. CTCF Chromatin-IP (ChIP) analysis using Auf1 promoter in C2C12 cells at 48 h after induction of differentiation. DNA enrichment in fragmented ChIP assay with anti-CTCF antibody relative to anti-rabbit IgG IP control, normalized to intron signal measured by qRT-PCR. n = 3. * P
    Figure Legend Snippet: AUF1 expression is essential for myoblast differentiation and myotube formation. ( A ) Expression of AUBP mRNAs during differentiation at 96 h determined by qRT-PCR ( n = 3), normalized to invariant GAPDH mRNA. ( B ) Expression of AUBP proteins during differentiation corresponding to A , n = 4. Numbers below immunoblot: Twist1 fold increase normalized to WT cells. ( C ) Immunoblot of CTCF, AUF1, MyoD, and β-tubulin control during C2C12 myoblast differentiation. CTCF was silenced in C2C12 cells by lentiviral-mediated shRNA. Numbers under immunoblot correspond to AUF1 fold change relative to β-tubulin levels, normalized to 0-h Nsi time point. n = 4. ( D ) Immunoblot of CTCF and AUF1 with increasing transfection of a CTCF cDNA expression plasmid in HEK 293 cells. Overexpression of CTCF was performed using pMy-CTCF (Addgene), n = 3. CTCF Chromatin-IP (ChIP) analysis using Auf1 promoter in C2C12 cells at 48 h after induction of differentiation. DNA enrichment in fragmented ChIP assay with anti-CTCF antibody relative to anti-rabbit IgG IP control, normalized to intron signal measured by qRT-PCR. n = 3. * P

    Techniques Used: Expressing, Quantitative RT-PCR, shRNA, Transfection, Plasmid Preparation, Over Expression, Chromatin Immunoprecipitation

    Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)
    Figure Legend Snippet: Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)

    Techniques Used: Cell Culture, Mouse Assay, Staining, Isolation, Transduction, Expressing

    AUF1 targeted decay of Twist1 mRNA partially restores myogenesis. Relative expression of TWIST1 mRNA ( A ) and protein levels in WT C2C12 myoblasts and AUF1 KO C2C12 myoblasts ( B ), n = 3. Numbers under blot refer to fold increase in AUF1 normalized to GAPDH protein. ** P
    Figure Legend Snippet: AUF1 targeted decay of Twist1 mRNA partially restores myogenesis. Relative expression of TWIST1 mRNA ( A ) and protein levels in WT C2C12 myoblasts and AUF1 KO C2C12 myoblasts ( B ), n = 3. Numbers under blot refer to fold increase in AUF1 normalized to GAPDH protein. ** P

    Techniques Used: Expressing

    Targeted decay of RGS5 and Twist1 mRNAs restores myotube differentiation and maturation in the absence of AUF1. ( A ) Immunoblot of AUF1 and RGS5 and RGS5 quantification relative to GAPDH in WT and ( Left ) AUF1 KO ( Right ) C2C12 myoblasts 96 h after differentiation, n = 3. ( B ) RGS5 mRNA decay rate. WT C2C12 cells, dotted line; AUF1 KO C2C12 cells, solid line. n = 3, ±SEM, ** P
    Figure Legend Snippet: Targeted decay of RGS5 and Twist1 mRNAs restores myotube differentiation and maturation in the absence of AUF1. ( A ) Immunoblot of AUF1 and RGS5 and RGS5 quantification relative to GAPDH in WT and ( Left ) AUF1 KO ( Right ) C2C12 myoblasts 96 h after differentiation, n = 3. ( B ) RGS5 mRNA decay rate. WT C2C12 cells, dotted line; AUF1 KO C2C12 cells, solid line. n = 3, ±SEM, ** P

    Techniques Used:

    5) Product Images from "SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES"

    Article Title: SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2009.11.072

    Relatively passive mechanism for strand displacement synthesis of HIV-1 RT
    Figure Legend Snippet: Relatively passive mechanism for strand displacement synthesis of HIV-1 RT

    Techniques Used:

    Sequence dependent strand displacement synthesis of HIV-1 RT
    Figure Legend Snippet: Sequence dependent strand displacement synthesis of HIV-1 RT

    Techniques Used: Sequencing

    Active and passive mechanisms for strand displacement DNA synthesis by HIV-1 RT near hairpin locations
    Figure Legend Snippet: Active and passive mechanisms for strand displacement DNA synthesis by HIV-1 RT near hairpin locations

    Techniques Used: DNA Synthesis

    DNA replication on flow-stretched ssDNA by HIV-1 RT
    Figure Legend Snippet: DNA replication on flow-stretched ssDNA by HIV-1 RT

    Techniques Used: Flow Cytometry

    6) Product Images from "Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template"

    Article Title: Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm767

    Comparison of the RNaseH activity of HIV-1 RT with (lanes 1–4) and without (lanes 5–8) dNTPs at different enzyme concentrations: 0.5 U (lanes 1, 3, 5, 7) and 2.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U), Mod = abasic RNA template (X = rAS).
    Figure Legend Snippet: Comparison of the RNaseH activity of HIV-1 RT with (lanes 1–4) and without (lanes 5–8) dNTPs at different enzyme concentrations: 0.5 U (lanes 1, 3, 5, 7) and 2.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U), Mod = abasic RNA template (X = rAS).

    Techniques Used: Activity Assay

    Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h P ss : primer ss, P rs : primer rs, T m : abasic RNA template (X = rAS), T n : non-damaged RNA template (X = U).
    Figure Legend Snippet: Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h P ss : primer ss, P rs : primer rs, T m : abasic RNA template (X = rAS), T n : non-damaged RNA template (X = U).

    Techniques Used:

    Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.
    Figure Legend Snippet: Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

    Techniques Used:

    7) Product Images from "Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein"

    Article Title: Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1045

    Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) ( 70 ). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer ( 9 ). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.
    Figure Legend Snippet: Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) ( 70 ). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer ( 9 ). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.

    Techniques Used: Sequencing

    Effect of HIV-1 NC on the kinetics of 591R primer extension catalyzed by WT RT and RNase H-minus RT. The data were plotted as % FL DNA versus Time (min). Symbols. WT RT: minus NC, squares; plus NC, triangles. RNase H-minus RT: minus NC, circles; plus NC, diamonds.
    Figure Legend Snippet: Effect of HIV-1 NC on the kinetics of 591R primer extension catalyzed by WT RT and RNase H-minus RT. The data were plotted as % FL DNA versus Time (min). Symbols. WT RT: minus NC, squares; plus NC, triangles. RNase H-minus RT: minus NC, circles; plus NC, diamonds.

    Techniques Used:

    Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. ( A ) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. ( B ) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32 P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.
    Figure Legend Snippet: Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. ( A ) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. ( B ) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32 P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.

    Techniques Used: Synthesized, Incubation, Radioactivity, Labeling, Concentration Assay

    8) Product Images from "Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA"

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp782

    RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).
    Figure Legend Snippet: RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, Northern Blot

    9) Product Images from "Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties"

    Article Title: Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt945

    Effect of A3A on HIV-1 RT-catalyzed extension of a (−) SSDNA oligonucleotide. The bar graph shows the percent of DNA extension product without (−) or with (+) RT and/or A3A. The positive control reaction with RT only is shown as a white bar. Reactions with RT contain either 5 µM (black bar) or 10 µM (gray bar) A3A (WT and E72Q mutant).
    Figure Legend Snippet: Effect of A3A on HIV-1 RT-catalyzed extension of a (−) SSDNA oligonucleotide. The bar graph shows the percent of DNA extension product without (−) or with (+) RT and/or A3A. The positive control reaction with RT only is shown as a white bar. Reactions with RT contain either 5 µM (black bar) or 10 µM (gray bar) A3A (WT and E72Q mutant).

    Techniques Used: Positive Control, Mutagenesis

    Deamination of dC in ss regions of a 40-bp DNA duplex and the effect of SSB proteins. ( A ) Schematic representation of a series of TBs in a ds nucleic acid. Unpaired bases are located in the center of a 40-bp DNA duplex that contains the TT C A sequence in the ss region of one strand. ( B ) Deaminase assay performed using duplexes (40 bp) containing TBs with different lengths of unpaired bases (1–9 nt). These duplexes were generated by heat annealing the ssDNA substrate (JL913) to oligonucleotides containing 1 nt (JL1088; TB-1), 3 (JL1089; TB-3), 5 (JL1090; TB-5) and 9 (JL1091; TB-9) that are not complementary to the corresponding residues in the other DNA strand. ( C ) Deaminase assay using the ssDNA substrate (JL913; 180 nM) after preincubation with SSB proteins (HIV-1 NC, T4 Gene 32 or E. coli SSB; each protein at 500 nM) for 15 min at 37°C before addition of A3A and incubation for 1 h. Bars: 1, 5 and 9, no proteins (negative control); 2, 6 and 10, A3A only (positive control); 3, 7, 11 and 4, 8, 12, A3A and ssDNA preincubated with 2.5 or 5 µM SSB protein, respectively.
    Figure Legend Snippet: Deamination of dC in ss regions of a 40-bp DNA duplex and the effect of SSB proteins. ( A ) Schematic representation of a series of TBs in a ds nucleic acid. Unpaired bases are located in the center of a 40-bp DNA duplex that contains the TT C A sequence in the ss region of one strand. ( B ) Deaminase assay performed using duplexes (40 bp) containing TBs with different lengths of unpaired bases (1–9 nt). These duplexes were generated by heat annealing the ssDNA substrate (JL913) to oligonucleotides containing 1 nt (JL1088; TB-1), 3 (JL1089; TB-3), 5 (JL1090; TB-5) and 9 (JL1091; TB-9) that are not complementary to the corresponding residues in the other DNA strand. ( C ) Deaminase assay using the ssDNA substrate (JL913; 180 nM) after preincubation with SSB proteins (HIV-1 NC, T4 Gene 32 or E. coli SSB; each protein at 500 nM) for 15 min at 37°C before addition of A3A and incubation for 1 h. Bars: 1, 5 and 9, no proteins (negative control); 2, 6 and 10, A3A only (positive control); 3, 7, 11 and 4, 8, 12, A3A and ssDNA preincubated with 2.5 or 5 µM SSB protein, respectively.

    Techniques Used: Sequencing, Generated, Incubation, Negative Control, Positive Control

    10) Product Images from "Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids"

    Article Title: Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0802607105

    Biosynthesis of 28:0 and 30:0 from 24:0 in cardiomyocytes and ARPE-19 cells expressing Elovl4 transgene. Cardiomyocytes or ARPE-19 cells were transduced with or without recombinant Elovl4 or GFP viruses for 24 h and then cultured in 24:0-supplemented
    Figure Legend Snippet: Biosynthesis of 28:0 and 30:0 from 24:0 in cardiomyocytes and ARPE-19 cells expressing Elovl4 transgene. Cardiomyocytes or ARPE-19 cells were transduced with or without recombinant Elovl4 or GFP viruses for 24 h and then cultured in 24:0-supplemented

    Techniques Used: Expressing, Transduction, Recombinant, Cell Culture

    Transgenic expression of mouse ELOVL4 in rat cardiomyocytes and ARPE-19 cells. ( A ) Comparison of quantitative expression of Elovl4 gene in different rat tissues and in ARPE-19 cells by qRT-PCR, and presented relative to the expression of the housekeeping
    Figure Legend Snippet: Transgenic expression of mouse ELOVL4 in rat cardiomyocytes and ARPE-19 cells. ( A ) Comparison of quantitative expression of Elovl4 gene in different rat tissues and in ARPE-19 cells by qRT-PCR, and presented relative to the expression of the housekeeping

    Techniques Used: Transgenic Assay, Expressing, Quantitative RT-PCR

    Biosynthesis of VLC-PUFA in cardiomyocytes expressing Elovl4 transgene. GC-MS allowed identification of the VLC-PUFA derived from sample equivalent to 2.0 mg of protein from cardiomyocytes treated with 20:5n3 or 22:5n3 for 72 h after transduction with
    Figure Legend Snippet: Biosynthesis of VLC-PUFA in cardiomyocytes expressing Elovl4 transgene. GC-MS allowed identification of the VLC-PUFA derived from sample equivalent to 2.0 mg of protein from cardiomyocytes treated with 20:5n3 or 22:5n3 for 72 h after transduction with

    Techniques Used: Expressing, Gas Chromatography-Mass Spectrometry, Derivative Assay, Transduction

    11) Product Images from "?-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication via PKA-Dependent Control of Viral RTA"

    Article Title: ?-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication via PKA-Dependent Control of Viral RTA

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.21.13538-13547.2005

    Role of the β-adrenergic receptor and cAMP/PKA signaling pathway in norepinephrine-induced reactivation of KSHV. (A) To define the role of β-adrenergic receptors in norepinephrine-mediated reactivation of lytic gene expression, BCBL-1 cells were treated with indicated concentrations of the β-antagonist propranalol for 1 h before exposure to 10 μM norepinephrine, and then assayed for KSHV lytic protein expression by Western blot 48 h later (primary antibody: Kaposi's sarcoma patient serum). Results show induction of the major KSHV lytic protein species at MW 49,800 which was the only band diagnostic of reactivation by the PMA positive control. (B) Specificity of adrenergic receptor involvement was tested by treating KS-1 cells with the α-antagonist phentolamine or the β-antagonist propranalol for 1 h prior to norepinephrine (NE) exposure; 24 h later, concentrations of mRNA for the immediate-early ORF50/ RTA and late ORF29 were assessed by real-time RT-PCR (values normalized to GAPDH and expressed as a ratio relative to vehicle-treated controls). (C) Expression of mRNA for β 1 , β 2 , and β 3 adrenergic receptors was assessed in untreated KS-1 and BCBL-1 cells by real-time RT-PCR. Products were resolved on a 3.5% agarose gel and compared to positive control PCR products (parallel amplification of genomic DNA for β 1 and β 3 adrenergic receptors or a PBMC cDNA library for β 2 adrenergic receptors and GAPDH). To verify that RT-PCR results were free of contaminating DNA, BCBL-1 and KS-1 RNA samples were amplified in parallel in the absence of reverse transcriptase (No RT). Data are representative of four independent experiments in which β 1 adrenergic receptors were consistently detected at high levels, and β 2 and β 3 receptors were not significantly expressed. (D) To evaluate the role of PKA and PKC in norepinephrine activation of KSHV lytic gene expression, KS-1 cells were pretreated with the PKA antagonist KT5720 (1 μM) or the PKC antagonists chelerythrine chloride (Chel, 1 μM) or bisindolylmaleimide HCl (300 nM) for 1 h prior to norepinephrine exposure. Expression of mRNA for the late lytic gene ORF29 was assayed by real-time RT-PCR 24 h later. PKA blockade inhibited norepinephrine-induced lytic gene expression by > 90%, but PKC inhibitors failed to block norepinephrine effects. Chelerythrine and bisindolylmaleimide (data not shown) efficiently blocked PMA induction of ORF29, verifying that inhibitors were capable of blocking known PKC activators. KT5720 failed to block PMA-mediated ORF29 expression, indicating that norepinephrine/PKA and PMA/PKC signaling pathways are functionally independent in PEL cells. (E) To determine whether cAMP activity was sufficient to induce lytic gene expression, KS-1 cells were treated with graded doses of pharmacological db-cAMP or with indicated concentrations of physiological cAMP inducers prostaglandin E 2 (PGE 2 ) and histamine (H 2 ) expressing the catalytic subunit of PKA (PKA c ). At 48 h following transduction with an empty vector, or vector bearing bicistronic EGFP and PKA c , EGFP-positive cells were quantified by flow cytometry to assess transduction efficiency. Cells transduced with EGFP alone showed transduction efficiencies comparable to comparable to EGFP plus PKA c (data not shown). (G) Also at 48 h posttransduction, concentrations of mRNA for immediate-early ORF50/ RTA and late lytic ORF29 were quantified by real-time RT-PCR (data normalized to GAPDH and expressed as a ratio relative to empty vector controls). Data represent the mean (± standard error) of three independent experiments, with statistical significance evaluated by paired t test. Similar effects were observed with BCBL-1 cells (data not shown).
    Figure Legend Snippet: Role of the β-adrenergic receptor and cAMP/PKA signaling pathway in norepinephrine-induced reactivation of KSHV. (A) To define the role of β-adrenergic receptors in norepinephrine-mediated reactivation of lytic gene expression, BCBL-1 cells were treated with indicated concentrations of the β-antagonist propranalol for 1 h before exposure to 10 μM norepinephrine, and then assayed for KSHV lytic protein expression by Western blot 48 h later (primary antibody: Kaposi's sarcoma patient serum). Results show induction of the major KSHV lytic protein species at MW 49,800 which was the only band diagnostic of reactivation by the PMA positive control. (B) Specificity of adrenergic receptor involvement was tested by treating KS-1 cells with the α-antagonist phentolamine or the β-antagonist propranalol for 1 h prior to norepinephrine (NE) exposure; 24 h later, concentrations of mRNA for the immediate-early ORF50/ RTA and late ORF29 were assessed by real-time RT-PCR (values normalized to GAPDH and expressed as a ratio relative to vehicle-treated controls). (C) Expression of mRNA for β 1 , β 2 , and β 3 adrenergic receptors was assessed in untreated KS-1 and BCBL-1 cells by real-time RT-PCR. Products were resolved on a 3.5% agarose gel and compared to positive control PCR products (parallel amplification of genomic DNA for β 1 and β 3 adrenergic receptors or a PBMC cDNA library for β 2 adrenergic receptors and GAPDH). To verify that RT-PCR results were free of contaminating DNA, BCBL-1 and KS-1 RNA samples were amplified in parallel in the absence of reverse transcriptase (No RT). Data are representative of four independent experiments in which β 1 adrenergic receptors were consistently detected at high levels, and β 2 and β 3 receptors were not significantly expressed. (D) To evaluate the role of PKA and PKC in norepinephrine activation of KSHV lytic gene expression, KS-1 cells were pretreated with the PKA antagonist KT5720 (1 μM) or the PKC antagonists chelerythrine chloride (Chel, 1 μM) or bisindolylmaleimide HCl (300 nM) for 1 h prior to norepinephrine exposure. Expression of mRNA for the late lytic gene ORF29 was assayed by real-time RT-PCR 24 h later. PKA blockade inhibited norepinephrine-induced lytic gene expression by > 90%, but PKC inhibitors failed to block norepinephrine effects. Chelerythrine and bisindolylmaleimide (data not shown) efficiently blocked PMA induction of ORF29, verifying that inhibitors were capable of blocking known PKC activators. KT5720 failed to block PMA-mediated ORF29 expression, indicating that norepinephrine/PKA and PMA/PKC signaling pathways are functionally independent in PEL cells. (E) To determine whether cAMP activity was sufficient to induce lytic gene expression, KS-1 cells were treated with graded doses of pharmacological db-cAMP or with indicated concentrations of physiological cAMP inducers prostaglandin E 2 (PGE 2 ) and histamine (H 2 ) expressing the catalytic subunit of PKA (PKA c ). At 48 h following transduction with an empty vector, or vector bearing bicistronic EGFP and PKA c , EGFP-positive cells were quantified by flow cytometry to assess transduction efficiency. Cells transduced with EGFP alone showed transduction efficiencies comparable to comparable to EGFP plus PKA c (data not shown). (G) Also at 48 h posttransduction, concentrations of mRNA for immediate-early ORF50/ RTA and late lytic ORF29 were quantified by real-time RT-PCR (data normalized to GAPDH and expressed as a ratio relative to empty vector controls). Data represent the mean (± standard error) of three independent experiments, with statistical significance evaluated by paired t test. Similar effects were observed with BCBL-1 cells (data not shown).

    Techniques Used: Expressing, Western Blot, Diagnostic Assay, Positive Control, Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Blocking Assay, Activity Assay, Transduction, Plasmid Preparation, Flow Cytometry, Cytometry

    Regulation of RTA promoter activity by PKA. (A) Activity of the RTA promoter was assessed by luciferase reporter assays in which pRpluc (firefly luciferase coding sequence controlled by ∼3 kb of KSHV genomic DNA upstream of ORF50) was electroporated into the Ramos or DG75 B cell lines, which are known to be free of KSHV and Epstein-Barr virus, or into BC3 PEL cells containing latent KSHV. Following electroporation, cells were incubated for 18 h in medium supplemented as indicated with the PKA activator db-cAMP (300 μM) or the PKC activator PMA (20 ng/ml). Firefly luciferase activity was normalized to Renilla luciferase activity generated by pRLCMV ( Renilla luciferase under control of the CMV promoter), and the statistical significance of triplicate determinations was evaluated by t test. Results showed significant PKA-mediated induction of RTA promoter activity in both cell types, but db-cAMP up-regulated RTA promoter activity more strongly KSHV-containing BC3 cells (as indicated by a cell type × db-cAMP interaction term from a factorial analysis of variance, P
    Figure Legend Snippet: Regulation of RTA promoter activity by PKA. (A) Activity of the RTA promoter was assessed by luciferase reporter assays in which pRpluc (firefly luciferase coding sequence controlled by ∼3 kb of KSHV genomic DNA upstream of ORF50) was electroporated into the Ramos or DG75 B cell lines, which are known to be free of KSHV and Epstein-Barr virus, or into BC3 PEL cells containing latent KSHV. Following electroporation, cells were incubated for 18 h in medium supplemented as indicated with the PKA activator db-cAMP (300 μM) or the PKC activator PMA (20 ng/ml). Firefly luciferase activity was normalized to Renilla luciferase activity generated by pRLCMV ( Renilla luciferase under control of the CMV promoter), and the statistical significance of triplicate determinations was evaluated by t test. Results showed significant PKA-mediated induction of RTA promoter activity in both cell types, but db-cAMP up-regulated RTA promoter activity more strongly KSHV-containing BC3 cells (as indicated by a cell type × db-cAMP interaction term from a factorial analysis of variance, P

    Techniques Used: Activity Assay, Luciferase, Sequencing, Electroporation, Incubation, Generated

    12) Product Images from "Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition"

    Article Title: Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition

    Journal: Retrovirology

    doi: 10.1186/s12977-016-0322-5

    a Force-extension curves for dsDNA stretch ( solid lines ) and return ( dashed lines ) with no protein and in the presence of 30 nM SIV NC or HIV-1 NC. b , c Dependence of the measured transition slope ( b ) and hysteresis area ratio ( c ) on protein concentration (see Additional file 4 ) for HIV-1 NC and SIV NC. The lines in ( b ) are fits to a simple binding isotherm (Additional file 4 : Eq. 6), revealing equilibrium dissociation constants K d = 5.5 ± 0.4 nM for SIV NC and K d = 4.2 ± 0.4 nM for HIV-1 NC. Error bars are standard errors for three or more measurements
    Figure Legend Snippet: a Force-extension curves for dsDNA stretch ( solid lines ) and return ( dashed lines ) with no protein and in the presence of 30 nM SIV NC or HIV-1 NC. b , c Dependence of the measured transition slope ( b ) and hysteresis area ratio ( c ) on protein concentration (see Additional file 4 ) for HIV-1 NC and SIV NC. The lines in ( b ) are fits to a simple binding isotherm (Additional file 4 : Eq. 6), revealing equilibrium dissociation constants K d = 5.5 ± 0.4 nM for SIV NC and K d = 4.2 ± 0.4 nM for HIV-1 NC. Error bars are standard errors for three or more measurements

    Techniques Used: Protein Concentration, Binding Assay

    Plot of the parameters determined from measuring the interaction between HIV-1 NC, SIV NC, and HIV-1 Gag and the indicated HIV-1 and SIV RNAs as a function of salt concentration. The dark gray circles indicate the fitted a K d(1M) (M = molarity) and b Z eff parameters from each individual salt-titration experiment, while each light gray bar is the average of at least three independent trials
    Figure Legend Snippet: Plot of the parameters determined from measuring the interaction between HIV-1 NC, SIV NC, and HIV-1 Gag and the indicated HIV-1 and SIV RNAs as a function of salt concentration. The dark gray circles indicate the fitted a K d(1M) (M = molarity) and b Z eff parameters from each individual salt-titration experiment, while each light gray bar is the average of at least three independent trials

    Techniques Used: Concentration Assay, Titration

    a Method for calculating the compaction force (F c ) induced by protein-DNA interactions. Inset shows stretch ( solid lines ) and return ( dashed lines ) curves for dsDNA in the absence of protein and in the presence of near saturated (30 nM) HIV-1 NC protein. F c is calculated in the low force-extension regime denoted within the gray box in the inset and magnified in the main figure. The DNA only extension curve is fit to the WLC model (Additional file 4 : Eq. 1). The force difference (F c ) between the return curve in the presence of high protein concentration and the DNA only stretching curve is averaged over measured extensions
    Figure Legend Snippet: a Method for calculating the compaction force (F c ) induced by protein-DNA interactions. Inset shows stretch ( solid lines ) and return ( dashed lines ) curves for dsDNA in the absence of protein and in the presence of near saturated (30 nM) HIV-1 NC protein. F c is calculated in the low force-extension regime denoted within the gray box in the inset and magnified in the main figure. The DNA only extension curve is fit to the WLC model (Additional file 4 : Eq. 1). The force difference (F c ) between the return curve in the presence of high protein concentration and the DNA only stretching curve is averaged over measured extensions

    Techniques Used: Protein Concentration

    DLS measurements for HIV-1 and SIV NC proteins in the presence of SIV Psi RNA. The size distributions of NA aggregates formed in the presence of the indicated NC or a no NC control are shown. Each curve represents the average of at least three independent experiments
    Figure Legend Snippet: DLS measurements for HIV-1 and SIV NC proteins in the presence of SIV Psi RNA. The size distributions of NA aggregates formed in the presence of the indicated NC or a no NC control are shown. Each curve represents the average of at least three independent experiments

    Techniques Used:

    Sequence and structural features of HIV-1 and SIV NC proteins. a Schematic diagrams of NC proteins: HIV-1 NL4-3 NC and SIVmne NC. Basic residues are colored blue , acidic residues are colored red , the CCHC residues that coordinate the Zn 2+ ions in the ZFs are colored gray , and the aromatic residue in each finger is underlined. The numbering is based on the sequence of the mature NC protein in each case. b Sequence alignment of HIV-1 and SIV NC proteins. Coloring and underlining are the same as in ( a ). The boxes indicate the sequence comprising each ZF
    Figure Legend Snippet: Sequence and structural features of HIV-1 and SIV NC proteins. a Schematic diagrams of NC proteins: HIV-1 NL4-3 NC and SIVmne NC. Basic residues are colored blue , acidic residues are colored red , the CCHC residues that coordinate the Zn 2+ ions in the ZFs are colored gray , and the aromatic residue in each finger is underlined. The numbering is based on the sequence of the mature NC protein in each case. b Sequence alignment of HIV-1 and SIV NC proteins. Coloring and underlining are the same as in ( a ). The boxes indicate the sequence comprising each ZF

    Techniques Used: Sequencing

    Sequence and mfold-predicted secondary structure of TAR and Psi RNA constructs used in this study. a HIV-1 TARpolyA. b HIV-1 Psi. c SIVmac TAR. d SIVmac Psi. In all cases, numbering refers to the nt position in gRNA. The box in ( a ) indicates HIV-1 TAR RNA. The boxes in ( b , d ) indicate the ∆DIS mutation, where DIS loop residues are replaced with a GNRA-type tetraloop (GAGA) to eliminate dimerization
    Figure Legend Snippet: Sequence and mfold-predicted secondary structure of TAR and Psi RNA constructs used in this study. a HIV-1 TARpolyA. b HIV-1 Psi. c SIVmac TAR. d SIVmac Psi. In all cases, numbering refers to the nt position in gRNA. The box in ( a ) indicates HIV-1 TAR RNA. The boxes in ( b , d ) indicate the ∆DIS mutation, where DIS loop residues are replaced with a GNRA-type tetraloop (GAGA) to eliminate dimerization

    Techniques Used: Sequencing, Construct, Mutagenesis

    Kinetics of SIV minus-strand transfer in the presence of SIV and HIV-1 NC proteins. Reactions were incubated with the indicated concentrations of SIV or HIV-1 NC for 60 min at 37 °C and were analyzed as described in “ Methods ” section. a Representative gels showing DNA species present in reactions with 1.25 µM SIV or HIV-1 NC. The transfer product (T) and (−) SSDNA are indicated to the left of the gel image and these were the only two bands that appeared on the gel. Note that self-priming products [ 18 , 19 , 100 , 114 ] were not formed under the conditions used for these assays. Lane c shows the migration of (−) SSDNA in the absence of other reactants. b , c The % strand transfer product formed was plotted against time of incubation for reactions with SIV NC ( b ) or HIV-1 NC ( c ). Error bars represent the SD from three or more independent experiments
    Figure Legend Snippet: Kinetics of SIV minus-strand transfer in the presence of SIV and HIV-1 NC proteins. Reactions were incubated with the indicated concentrations of SIV or HIV-1 NC for 60 min at 37 °C and were analyzed as described in “ Methods ” section. a Representative gels showing DNA species present in reactions with 1.25 µM SIV or HIV-1 NC. The transfer product (T) and (−) SSDNA are indicated to the left of the gel image and these were the only two bands that appeared on the gel. Note that self-priming products [ 18 , 19 , 100 , 114 ] were not formed under the conditions used for these assays. Lane c shows the migration of (−) SSDNA in the absence of other reactants. b , c The % strand transfer product formed was plotted against time of incubation for reactions with SIV NC ( b ) or HIV-1 NC ( c ). Error bars represent the SD from three or more independent experiments

    Techniques Used: Incubation, Migration

    Kinetics of minus-strand annealing with SIV and HIV-1 substrates in the presence of SIV and HIV-1 NC proteins. a Reconstituted system used to assay minus-strand annealing and transfer. The diagram shows the acceptor RNA with a portion of U3 and the R sequence at the 3′ end of the viral genome annealed to (−) SSDNA with the complementary r sequence and a portion of u5, complementary to the U5 sequence. For the SIV substrates, the nt lengths of u5, R/r, and U3 sequences are as follows: u5, 20 nt; R/r, 176 nt; and U3, 52 nt. For the HIV-1 substrates, the lengths are: u5, 34 nt; R/r, 94 nt; and U3, 54 nt. The asterisk indicates that the (−) SSDNA is labeled at its 5′ end with 32 P. Annealing of the complementary R regions is indicated by vertical lines . The U3 sequence serves as the template for RT-catalyzed extension of annealed (−) SSDNA. The final DNA transfer product is 248 nt (SIV) or 182 nt (HIV-1). The diagram is not drawn to scale. b-1 , b-2 , c-1 , c-2 Reactions were incubated with SIV ( b-1 , b-2 ) or HIV-1 substrates ( c-1 , c-2 ) and different concentrations of SIV NC or HIV-1 NC for 30 min at 37 °C and analyzed as described in “ Methods ” section. Representative gels can be found in Additional file 1 : Fig. S1. The percent (%) annealed product was plotted against time of incubation. Error bars represent the standard deviation (SD) from three or more independent experiments
    Figure Legend Snippet: Kinetics of minus-strand annealing with SIV and HIV-1 substrates in the presence of SIV and HIV-1 NC proteins. a Reconstituted system used to assay minus-strand annealing and transfer. The diagram shows the acceptor RNA with a portion of U3 and the R sequence at the 3′ end of the viral genome annealed to (−) SSDNA with the complementary r sequence and a portion of u5, complementary to the U5 sequence. For the SIV substrates, the nt lengths of u5, R/r, and U3 sequences are as follows: u5, 20 nt; R/r, 176 nt; and U3, 52 nt. For the HIV-1 substrates, the lengths are: u5, 34 nt; R/r, 94 nt; and U3, 54 nt. The asterisk indicates that the (−) SSDNA is labeled at its 5′ end with 32 P. Annealing of the complementary R regions is indicated by vertical lines . The U3 sequence serves as the template for RT-catalyzed extension of annealed (−) SSDNA. The final DNA transfer product is 248 nt (SIV) or 182 nt (HIV-1). The diagram is not drawn to scale. b-1 , b-2 , c-1 , c-2 Reactions were incubated with SIV ( b-1 , b-2 ) or HIV-1 substrates ( c-1 , c-2 ) and different concentrations of SIV NC or HIV-1 NC for 30 min at 37 °C and analyzed as described in “ Methods ” section. Representative gels can be found in Additional file 1 : Fig. S1. The percent (%) annealed product was plotted against time of incubation. Error bars represent the standard deviation (SD) from three or more independent experiments

    Techniques Used: Sequencing, Labeling, Incubation, Standard Deviation

    13) Product Images from "Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications"

    Article Title: Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

    Journal: Virology

    doi: 10.1016/j.virol.2010.06.042

    ). (B and C) Bar graphs showing the percentage (%) of minus-strand transfer product (B) or SP products (C) synthesized as a function of NC, Gag, or Gag WM protein concentrations. Symbols: no protein, open bars; HIV-1 NC, closed bars; Gag, gray bars; Gag WM, hatched bars; C, cross-hatched bar.
    Figure Legend Snippet: ). (B and C) Bar graphs showing the percentage (%) of minus-strand transfer product (B) or SP products (C) synthesized as a function of NC, Gag, or Gag WM protein concentrations. Symbols: no protein, open bars; HIV-1 NC, closed bars; Gag, gray bars; Gag WM, hatched bars; C, cross-hatched bar.

    Techniques Used: Synthesized

    Minus-strand transfer activity of HIV-1 MA, CA, and MACA proteins in the absence or presence of HIV-1 NC added in trans. 33 P-labeled DNA 128 was incubated with RNA 148 for 60 min in the absence or presence of increasing concentrations of MA (2 to 5), CA (7 to 10), and MACA proteins (12 to 15). (A) and (B) Bar graphs show the % of minus-strand transfer product synthesized as a function of the protein concentrations of MA, CA, and MACA in the absence (A) or presence of (B) 0.92 μM NC added in trans. Symbols: open bars, no protein; gray bars, NC only; and closed bars, increasing concentrations of MA, CA, and MACA (concentrations indicated at the bottom of the figure).
    Figure Legend Snippet: Minus-strand transfer activity of HIV-1 MA, CA, and MACA proteins in the absence or presence of HIV-1 NC added in trans. 33 P-labeled DNA 128 was incubated with RNA 148 for 60 min in the absence or presence of increasing concentrations of MA (2 to 5), CA (7 to 10), and MACA proteins (12 to 15). (A) and (B) Bar graphs show the % of minus-strand transfer product synthesized as a function of the protein concentrations of MA, CA, and MACA in the absence (A) or presence of (B) 0.92 μM NC added in trans. Symbols: open bars, no protein; gray bars, NC only; and closed bars, increasing concentrations of MA, CA, and MACA (concentrations indicated at the bottom of the figure).

    Techniques Used: Activity Assay, Labeling, Incubation, Synthesized

    14) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.
    Figure Legend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Techniques Used: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.
    Figure Legend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Techniques Used: Functional Assay, Preserving, Sequencing, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.
    Figure Legend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Techniques Used: Refining, Sequencing

    15) Product Images from "Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration"

    Article Title: Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration

    Journal: Analytical chemistry

    doi: 10.1021/ac201307d

    a,b) Electropherogram demonstrating detection of 6.5 pM HIV-1 RT in buffer, c) Dose response curve of anti-HIV-1RT aptamer with HIV-RT spiked in buffer, error bars represent standard error from duplicate experiments, d) Linear relationship in the log-log
    Figure Legend Snippet: a,b) Electropherogram demonstrating detection of 6.5 pM HIV-1 RT in buffer, c) Dose response curve of anti-HIV-1RT aptamer with HIV-RT spiked in buffer, error bars represent standard error from duplicate experiments, d) Linear relationship in the log-log

    Techniques Used:

    16) Product Images from "Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription"

    Article Title: Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription

    Journal: Virus research

    doi: 10.1016/j.virusres.2014.06.004

    Effect of WT NCp15 and mutants with changes in the C-terminal p6 domain on the kinetics of minus-strand annealing and strand transfer. (A) Primary sequence of HIV-1 p6 and schematic representation of C-terminal NCp15 mutants. The acidic and basic residues
    Figure Legend Snippet: Effect of WT NCp15 and mutants with changes in the C-terminal p6 domain on the kinetics of minus-strand annealing and strand transfer. (A) Primary sequence of HIV-1 p6 and schematic representation of C-terminal NCp15 mutants. The acidic and basic residues

    Techniques Used: Sequencing

    Schematic representation of NC proteins and RNA templates used in this study. (A) Proteins produced by C-terminal cleavage of Gag. HIV-1 Gag is shown with each domain indicated by rectangles depicted as follows: MA, open; CA, dark gray; spacer peptide
    Figure Legend Snippet: Schematic representation of NC proteins and RNA templates used in this study. (A) Proteins produced by C-terminal cleavage of Gag. HIV-1 Gag is shown with each domain indicated by rectangles depicted as follows: MA, open; CA, dark gray; spacer peptide

    Techniques Used: Produced

    Effect of HIV-1 NC proteins on (-) SSDNA synthesis. (A) Reconstituted system used for assay of (-) SSDNA synthesis. The diagram shows annealing (vertical lines) of the 18 nt at the 3’ end of tRNA Lys3 to the complementary 18-nt PBS in RNA 200 (gray
    Figure Legend Snippet: Effect of HIV-1 NC proteins on (-) SSDNA synthesis. (A) Reconstituted system used for assay of (-) SSDNA synthesis. The diagram shows annealing (vertical lines) of the 18 nt at the 3’ end of tRNA Lys3 to the complementary 18-nt PBS in RNA 200 (gray

    Techniques Used:

    Effect of HIV-1 NC proteins on annealing of tRNA Lys3 to RNA 200. Annealing was performed with tRNA Lys3 , uniformly labeled with 33 P. Bar graphs show the % tRNA annealed as a function of NC protein concentration. Symbols: no protein, open bars; NCp7, closed
    Figure Legend Snippet: Effect of HIV-1 NC proteins on annealing of tRNA Lys3 to RNA 200. Annealing was performed with tRNA Lys3 , uniformly labeled with 33 P. Bar graphs show the % tRNA annealed as a function of NC protein concentration. Symbols: no protein, open bars; NCp7, closed

    Techniques Used: Labeling, Protein Concentration

    Minus-strand annealing and transfer activity of NCp7 in the absence or presence of HIV-1 SP2 added in trans . (A) Primary sequence of NCp9. The zinc coordinating residues (Cys and His) are shown in gray. The arrow indicates the PR cleavage site. The final
    Figure Legend Snippet: Minus-strand annealing and transfer activity of NCp7 in the absence or presence of HIV-1 SP2 added in trans . (A) Primary sequence of NCp9. The zinc coordinating residues (Cys and His) are shown in gray. The arrow indicates the PR cleavage site. The final

    Techniques Used: Activity Assay, Sequencing

    17) Product Images from "Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein"

    Article Title: Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm375

    Effect of HIV-1 NC on minus-strand transfer with RNA 50 and RNA 50 mutants. ( A ) RNA 50 and mutants. The predicted Δ G values shown for RNA 50 and mutants represent the values for overall thermodynamic stability. The relevant sequences in each RNA are boxed. ( B ) Gel analysis. DNA 50 was incubated with acceptor RNA 50 and mutants for 60 min in the absence (No) (lanes 1, 6, 11, 16) or presence of increasing concentrations of HIV-1 NC, as follows: lanes 2, 7, 12, 17, 7 nt/NC (0.14 μM); lanes 3, 8, 13, 18, 3.5 nt/NC (0.3 μM); lanes 4, 9, 14, 19, 1.75 nt/NC (0.6 μM); lanes 5, 10, 15, 20, 0.88 nt/NC (1.2 μM). ( C ) Bar graphs showing the percentage (%) of minus-strand transfer product synthesized as a function of NC concentration. Note that the numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Symbols: RNA 50, open bars; RNA 50G46U, hatched bars; RNA 50C49U, cross-hatched bars and RNA 50G48U, gray bars.
    Figure Legend Snippet: Effect of HIV-1 NC on minus-strand transfer with RNA 50 and RNA 50 mutants. ( A ) RNA 50 and mutants. The predicted Δ G values shown for RNA 50 and mutants represent the values for overall thermodynamic stability. The relevant sequences in each RNA are boxed. ( B ) Gel analysis. DNA 50 was incubated with acceptor RNA 50 and mutants for 60 min in the absence (No) (lanes 1, 6, 11, 16) or presence of increasing concentrations of HIV-1 NC, as follows: lanes 2, 7, 12, 17, 7 nt/NC (0.14 μM); lanes 3, 8, 13, 18, 3.5 nt/NC (0.3 μM); lanes 4, 9, 14, 19, 1.75 nt/NC (0.6 μM); lanes 5, 10, 15, 20, 0.88 nt/NC (1.2 μM). ( C ) Bar graphs showing the percentage (%) of minus-strand transfer product synthesized as a function of NC concentration. Note that the numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Symbols: RNA 50, open bars; RNA 50G46U, hatched bars; RNA 50C49U, cross-hatched bars and RNA 50G48U, gray bars.

    Techniques Used: Incubation, Synthesized, Concentration Assay

    Influence of acceptor RNA secondary structure on minus-strand transfer. ( A ) Secondary structures of RNA 70 and RNA 50 acceptor RNAs, based on m Fold analysis and extensive RNase mapping studies ( 28 ). The 20-nt U3 sequences are indicated. (Note that m Fold predicts that two bases from U3, C19 and U20, are part of the stem-loop structure at the 5′ end of RNA 70). The predicted Δ G values are shown beneath the structures. The potential nucleation site at the 5′ end of each RNA is boxed. The arrows point to residues in RNA 70 and RNA 50 (gray shading) that were mutated. ( B ) Secondary structure of DNA 50, based on m Fold analysis and enzymatic mapping studies ( 28 ). The predicted Δ G value is shown on the right. The arrows indicate residues C17 and C18 (gray shading) that were mutated. Note the 11-nt single-stranded sequence at the 3′ end of the DNA. ( C ) NC-mediated minus-strand transfer. DNA 50 and RNA 70 or RNA 50 were present in reactions at a 1:1 ratio of (−) SSDNA to acceptor RNA, each with a final concentration of 10 nM. These nucleic acid concentrations were used in all of the experiments described below. Incubation was for 60 min in the absence or presence of HIV-1 NC (0.88 nt/NC), as described under the Materials and Methods section, and was followed by PAGE and PhosphorImager analysis. The bar graph shows the percentage (%) of minus-strand transfer product synthesized in each reaction. Plus NC, closed bars; minus NC, open bars.
    Figure Legend Snippet: Influence of acceptor RNA secondary structure on minus-strand transfer. ( A ) Secondary structures of RNA 70 and RNA 50 acceptor RNAs, based on m Fold analysis and extensive RNase mapping studies ( 28 ). The 20-nt U3 sequences are indicated. (Note that m Fold predicts that two bases from U3, C19 and U20, are part of the stem-loop structure at the 5′ end of RNA 70). The predicted Δ G values are shown beneath the structures. The potential nucleation site at the 5′ end of each RNA is boxed. The arrows point to residues in RNA 70 and RNA 50 (gray shading) that were mutated. ( B ) Secondary structure of DNA 50, based on m Fold analysis and enzymatic mapping studies ( 28 ). The predicted Δ G value is shown on the right. The arrows indicate residues C17 and C18 (gray shading) that were mutated. Note the 11-nt single-stranded sequence at the 3′ end of the DNA. ( C ) NC-mediated minus-strand transfer. DNA 50 and RNA 70 or RNA 50 were present in reactions at a 1:1 ratio of (−) SSDNA to acceptor RNA, each with a final concentration of 10 nM. These nucleic acid concentrations were used in all of the experiments described below. Incubation was for 60 min in the absence or presence of HIV-1 NC (0.88 nt/NC), as described under the Materials and Methods section, and was followed by PAGE and PhosphorImager analysis. The bar graph shows the percentage (%) of minus-strand transfer product synthesized in each reaction. Plus NC, closed bars; minus NC, open bars.

    Techniques Used: Sequencing, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis, Synthesized

    18) Product Images from "Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G"

    Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G

    Journal: Nature microbiology

    doi: 10.1038/s41564-017-0063-9

    Mapping of A3G-RT interaction sites on A3G protein a) Anti FLAG immunoprecipitation of p51_FLAG and p66_FLAG co-expressed with GST or GST_A3G fusion proteins, recovered proteins were detected with anti-GST (for A3G) or anti-FLAG antibodies as indicated. A3G truncations are indicated and numbers refer to amino acid positions in A3G. b) Co-immunoprecipitation analysis of wild type or mutant A3G with HIV-1 p51_FLAG and p66_FLAG, recovered proteins were detected with anti-HA (for A3G) or anti-FLAG antibodies. One representative out of three experiments is shown. c) FRET-FLIM analysis of wild type or mutant A3G with the p66 subunit of HIV-1 RT. Representative images show green fluorescence (GFP, left panel) and red fluorescence (mCherry, right panel) and GFP lifetime as pseudo-colored images according to the indicated scale (as in Fig 5). White scale bars represent 10 μm. d) Dot plots showing individual FRET efficiencies with their mean and one standard deviation from n=12 cells each. *** indicates p-value of
    Figure Legend Snippet: Mapping of A3G-RT interaction sites on A3G protein a) Anti FLAG immunoprecipitation of p51_FLAG and p66_FLAG co-expressed with GST or GST_A3G fusion proteins, recovered proteins were detected with anti-GST (for A3G) or anti-FLAG antibodies as indicated. A3G truncations are indicated and numbers refer to amino acid positions in A3G. b) Co-immunoprecipitation analysis of wild type or mutant A3G with HIV-1 p51_FLAG and p66_FLAG, recovered proteins were detected with anti-HA (for A3G) or anti-FLAG antibodies. One representative out of three experiments is shown. c) FRET-FLIM analysis of wild type or mutant A3G with the p66 subunit of HIV-1 RT. Representative images show green fluorescence (GFP, left panel) and red fluorescence (mCherry, right panel) and GFP lifetime as pseudo-colored images according to the indicated scale (as in Fig 5). White scale bars represent 10 μm. d) Dot plots showing individual FRET efficiencies with their mean and one standard deviation from n=12 cells each. *** indicates p-value of

    Techniques Used: Immunoprecipitation, Mutagenesis, Fluorescence, Standard Deviation

    Consequences of UDG inhibition on A3G antiviral phenotype and cDNA profiles a) Immunoblot analysis of HIV-1 virion lysates showing increasing amounts of packaged A3G_HA at constant CA levels for virions produced in the presence or absence of a codon optimized (humanized) uracil-DNA glycosylase inhibitor (hUGI). ‘Low’ or ‘High’ A3G refers to a producer cell transfection ratios of 1:10 or 1:1, respectively (A3G expression plasmid to NL4.3/ΔVif). One of three independent sets of virus preparations used for b) and c) is shown. b) Virion infectivity was evaluated by challenging TZM-bl cells and measurement of β-galactosidase activity. c) The abundance of (-)sss containing cDNA in CEM-SS cells at 4 h post-infection was measured by quantitative PCR. For b) and c) each viral preparation was used to infect TZM-bl or CEM-SS target cells with or without hUGI, black dots and grey squares respectively. The individual data points with their mean and standard deviation for three independent viral preparations and infections are shown. d) Sequencing reads from a MiSeq™ library run were analyzed and presented as in Figure 1g . The labeling to the right indicates whether the HEK293T producer cells (Prod) and/or the CEM-SS target (Target) cells expressed hUGI. No A3G indicates the absence of A3G in producer cells and high A3G refers to relative A3G content in the producer cells. Sequencing data are derived from one representative experiment out of two independent repeats.
    Figure Legend Snippet: Consequences of UDG inhibition on A3G antiviral phenotype and cDNA profiles a) Immunoblot analysis of HIV-1 virion lysates showing increasing amounts of packaged A3G_HA at constant CA levels for virions produced in the presence or absence of a codon optimized (humanized) uracil-DNA glycosylase inhibitor (hUGI). ‘Low’ or ‘High’ A3G refers to a producer cell transfection ratios of 1:10 or 1:1, respectively (A3G expression plasmid to NL4.3/ΔVif). One of three independent sets of virus preparations used for b) and c) is shown. b) Virion infectivity was evaluated by challenging TZM-bl cells and measurement of β-galactosidase activity. c) The abundance of (-)sss containing cDNA in CEM-SS cells at 4 h post-infection was measured by quantitative PCR. For b) and c) each viral preparation was used to infect TZM-bl or CEM-SS target cells with or without hUGI, black dots and grey squares respectively. The individual data points with their mean and standard deviation for three independent viral preparations and infections are shown. d) Sequencing reads from a MiSeq™ library run were analyzed and presented as in Figure 1g . The labeling to the right indicates whether the HEK293T producer cells (Prod) and/or the CEM-SS target (Target) cells expressed hUGI. No A3G indicates the absence of A3G in producer cells and high A3G refers to relative A3G content in the producer cells. Sequencing data are derived from one representative experiment out of two independent repeats.

    Techniques Used: Inhibition, Produced, Transfection, Expressing, Plasmid Preparation, Infection, Activity Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Labeling, Derivative Assay

    Interaction of A3G with HIV-1 reverse transcriptase. Co-immunoprecipiation analysis of A3G_HA binding to FLAG tagged HIV-1 RT. Transfected HEK293T cell lysates were subjected to anti-FLAG immunoprecipiation, recovered proteins were detected with anti-HA (for A3G), anti-RT or anti-FLAG antibodies. CD8_FLAG served as an irrelevant protein control. One representative experiment of three repeats is shown. *HC: immunoglobulin heavy chain b) RNase resistance of the A3G-RT complex. Shown are anti-FLAG immunoprecipitations after the bead bound proteins had been subjected to RNase A or RNase Mix treatment, at the indicated concentrations, followed by washing and immunoblotting. One representative experiment of three repeats is shown. Samples without RT_FLAG carry CD8_FLAG as an irrelevant tagged protein control. c) Surface plasmon resonance analysis of purified A3G and p51 on a Biacore T-200 instrument. Association and dissociation curves of p51_FLAG to immobilized A3G_6xHis at the indicated concentrations are shown. The sensorgram indicates specific binding between the two components, and the responses gave good fits to a single interaction binding model with a K d of ~1.6 μM. d)-f) Measurements of FRET efficiency using FLIM in HeLa cells expressing GFP and mCherry fusion proteins. Representative images with GFP fluorescence from multiphoton laser scanning microscopy (left panel) and corresponding wide field CCD camera images of mCherry fluorescence (right panels (e only)) are shown. The center panels represent pseudo-colored images of GFP lifetime (τ) (blue/green, normal/longer GFP lifetime; yellow/red, shorter GFP lifetime indicating FRET). d) Control images demonstrating normal GFP lifetime in the absence of mCherry acceptor. White scale bars represent 10 μm. e) Co-expression of indicated GFP and mCherry fusion proteins and the fluorescence lifetime according to the scale in d) indicating the presence or absence of FRET. f) Dot plot of FRET efficiencies with their mean and standard deviation from n=7 cells each.
    Figure Legend Snippet: Interaction of A3G with HIV-1 reverse transcriptase. Co-immunoprecipiation analysis of A3G_HA binding to FLAG tagged HIV-1 RT. Transfected HEK293T cell lysates were subjected to anti-FLAG immunoprecipiation, recovered proteins were detected with anti-HA (for A3G), anti-RT or anti-FLAG antibodies. CD8_FLAG served as an irrelevant protein control. One representative experiment of three repeats is shown. *HC: immunoglobulin heavy chain b) RNase resistance of the A3G-RT complex. Shown are anti-FLAG immunoprecipitations after the bead bound proteins had been subjected to RNase A or RNase Mix treatment, at the indicated concentrations, followed by washing and immunoblotting. One representative experiment of three repeats is shown. Samples without RT_FLAG carry CD8_FLAG as an irrelevant tagged protein control. c) Surface plasmon resonance analysis of purified A3G and p51 on a Biacore T-200 instrument. Association and dissociation curves of p51_FLAG to immobilized A3G_6xHis at the indicated concentrations are shown. The sensorgram indicates specific binding between the two components, and the responses gave good fits to a single interaction binding model with a K d of ~1.6 μM. d)-f) Measurements of FRET efficiency using FLIM in HeLa cells expressing GFP and mCherry fusion proteins. Representative images with GFP fluorescence from multiphoton laser scanning microscopy (left panel) and corresponding wide field CCD camera images of mCherry fluorescence (right panels (e only)) are shown. The center panels represent pseudo-colored images of GFP lifetime (τ) (blue/green, normal/longer GFP lifetime; yellow/red, shorter GFP lifetime indicating FRET). d) Control images demonstrating normal GFP lifetime in the absence of mCherry acceptor. White scale bars represent 10 μm. e) Co-expression of indicated GFP and mCherry fusion proteins and the fluorescence lifetime according to the scale in d) indicating the presence or absence of FRET. f) Dot plot of FRET efficiencies with their mean and standard deviation from n=7 cells each.

    Techniques Used: Binding Assay, Transfection, SPR Assay, Purification, Expressing, Fluorescence, Laser-Scanning Microscopy, Standard Deviation

    A3G interaction with HIV-1 RT in virions Suspensions of HIV-1 virions with packaged A3G_GFP, GFP_Vpr, GFP_CYPA or A3G_GFP and A3G_mCherry were immobilized on coverslips, fixed and stained with Cy3 labeled anti-RT or anti-CA Fab fragments. a) and b) Representative images show clusters of HIV-1 virions immobilized on fibronectin streaks with green fluorescence (left panel), red fluorescence (Cy3 or mCherry as indicated, right panel) and GFP lifetime as pseudo-colored images according to the indicated scale (as in Figure 3 ). White scale bars represent 10 μm. a) A3G_GFP demonstrates normal lifetime when packaged into HIV-1 virions. b) FRET is detected for the positive control of A3G_GFP and A3G_mCherry (upper left panel) and between A3G_GFP and Cy3 stained RT (lower right panel), but not between Vpr and RT, CYPA and RT, or A3G and CA (upper right panels). The absence of a signal for red fluorescence with HIV-1ΔRT virions confirmed the specificity of the anti-RT Fab fragments (lower left panel). c) Quantification of FRET efficiencies for n=5 areas. Individual measurements with their mean and standard deviation are shown.
    Figure Legend Snippet: A3G interaction with HIV-1 RT in virions Suspensions of HIV-1 virions with packaged A3G_GFP, GFP_Vpr, GFP_CYPA or A3G_GFP and A3G_mCherry were immobilized on coverslips, fixed and stained with Cy3 labeled anti-RT or anti-CA Fab fragments. a) and b) Representative images show clusters of HIV-1 virions immobilized on fibronectin streaks with green fluorescence (left panel), red fluorescence (Cy3 or mCherry as indicated, right panel) and GFP lifetime as pseudo-colored images according to the indicated scale (as in Figure 3 ). White scale bars represent 10 μm. a) A3G_GFP demonstrates normal lifetime when packaged into HIV-1 virions. b) FRET is detected for the positive control of A3G_GFP and A3G_mCherry (upper left panel) and between A3G_GFP and Cy3 stained RT (lower right panel), but not between Vpr and RT, CYPA and RT, or A3G and CA (upper right panels). The absence of a signal for red fluorescence with HIV-1ΔRT virions confirmed the specificity of the anti-RT Fab fragments (lower left panel). c) Quantification of FRET efficiencies for n=5 areas. Individual measurements with their mean and standard deviation are shown.

    Techniques Used: Staining, Labeling, Fluorescence, Positive Control, Standard Deviation

    Phenotypes of packaged L35A and R24A A3G mutant proteins on viral infectivity and cDNA profiles a) Immunoblot analysis of HIV-1 virions showing relative amounts of packaged wild type or mutant A3G_HA at constant CA levels. Ratios refer to the amounts of transfected A3G expression plasmid to proviral plasmid during virus production. b) A3G-L35A, but not A3G-R24A, displays diminished HIV-1 inhibitory activity. A3G packaging was quantified by immunoblot density measurements and the different wild type A3G packaging levels were plotted over measured infectivity. The extent of infection inhibition exerted by the wild type protein at the empirically determined level of packaged mutant protein was then extrapolated (see Supplementary Fig 10 ). Inhibition levels, in % relative to the no A3G control, of wild type A3G (triangles) and L35A or R24A (circles) in eight (L35A) or seven (R24A) independent experiments are shown. A paired, two tailed student t test was performed in GraphPad Prism ® and * indicates p
    Figure Legend Snippet: Phenotypes of packaged L35A and R24A A3G mutant proteins on viral infectivity and cDNA profiles a) Immunoblot analysis of HIV-1 virions showing relative amounts of packaged wild type or mutant A3G_HA at constant CA levels. Ratios refer to the amounts of transfected A3G expression plasmid to proviral plasmid during virus production. b) A3G-L35A, but not A3G-R24A, displays diminished HIV-1 inhibitory activity. A3G packaging was quantified by immunoblot density measurements and the different wild type A3G packaging levels were plotted over measured infectivity. The extent of infection inhibition exerted by the wild type protein at the empirically determined level of packaged mutant protein was then extrapolated (see Supplementary Fig 10 ). Inhibition levels, in % relative to the no A3G control, of wild type A3G (triangles) and L35A or R24A (circles) in eight (L35A) or seven (R24A) independent experiments are shown. A paired, two tailed student t test was performed in GraphPad Prism ® and * indicates p

    Techniques Used: Mutagenesis, Infection, Transfection, Expressing, Plasmid Preparation, Activity Assay, Inhibition, Two Tailed Test

    Effects of A3G on profiles of nascent HIV-1 cDNA products in infected T cells. a) Early steps of the HIV-1 life cycle illustrating three proposed anti-retroviral mechanisms for A3G that are deaminase-dependent (pathways 1 and 2) or -independent (pathway 3). b) Diagram of HIV-1 reverse transcription. The first full intermediate, (-)sss cDNA, is completed in step 3. PBS: primer binding site, PPT: polypurine tract. c) Basic steps of sequencing library preparation. During infection, HIV-1 produces nascent viral cDNAs of increasing length (see step 2 in b). Sequencing reads reveal precise 3’-termini at the points of adaptor-viral DNA ligation (red box). d) Immunoblot analysis of HIV-1 virion lysates from one of six independent virus preparations. ‘Low’ or ‘High’ A3G refers to producer cell transfection ratios of 1:10 or 1:4, respectively (A3G expression plasmid to NL4.3/ΔVif). e) Single-cycle virion infectivity measured by β-galactosidase activity in challenged TZM-bl reporter cells. f) Quantitative PCR measuring cDNA abundance in CEM-SS cells at 4 h post-infection. For e) and f) the individual data points with their mean and standard deviation of eight independent infections from six virus preparations are shown. *** indicates p-value of
    Figure Legend Snippet: Effects of A3G on profiles of nascent HIV-1 cDNA products in infected T cells. a) Early steps of the HIV-1 life cycle illustrating three proposed anti-retroviral mechanisms for A3G that are deaminase-dependent (pathways 1 and 2) or -independent (pathway 3). b) Diagram of HIV-1 reverse transcription. The first full intermediate, (-)sss cDNA, is completed in step 3. PBS: primer binding site, PPT: polypurine tract. c) Basic steps of sequencing library preparation. During infection, HIV-1 produces nascent viral cDNAs of increasing length (see step 2 in b). Sequencing reads reveal precise 3’-termini at the points of adaptor-viral DNA ligation (red box). d) Immunoblot analysis of HIV-1 virion lysates from one of six independent virus preparations. ‘Low’ or ‘High’ A3G refers to producer cell transfection ratios of 1:10 or 1:4, respectively (A3G expression plasmid to NL4.3/ΔVif). e) Single-cycle virion infectivity measured by β-galactosidase activity in challenged TZM-bl reporter cells. f) Quantitative PCR measuring cDNA abundance in CEM-SS cells at 4 h post-infection. For e) and f) the individual data points with their mean and standard deviation of eight independent infections from six virus preparations are shown. *** indicates p-value of

    Techniques Used: Infection, Binding Assay, Sequencing, DNA Ligation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    19) Product Images from "Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity"

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.01848-17

    ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).
    Figure Legend Snippet: ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).

    Techniques Used: Cell Attachment Assay, Labeling, Staining, Binding Assay

    The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis

    The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Incubation, SDS Page, Western Blot

    The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Incubation, Infection, Plaque Assay, SDS Page, Staining

    The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Quantitative RT-PCR

    ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Generated

    The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).
    Figure Legend Snippet: The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).

    Techniques Used: Mutagenesis, Incubation, Fluorescence

    The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).
    Figure Legend Snippet: The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Techniques Used: Mutagenesis, Activity Assay, Incubation, Infection, Plaque Assay, Western Blot, SDS Page, Staining

    The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).
    Figure Legend Snippet: The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).

    Techniques Used: Mutagenesis, SDS Page, Western Blot, Infection, Immunofluorescence

    20) Product Images from "Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice"

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008161

    Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.
    Figure Legend Snippet: Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Techniques Used: Immunofluorescence, Microscopy, Infection, Expressing, Confocal Microscopy, Staining, Immunostaining, Isolation, Mouse Assay

    Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.
    Figure Legend Snippet: Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.

    Techniques Used: Functional Assay, Western Blot, Expressing, Transfection, Derivative Assay, Plasmid Preparation, Infection, Protease Inhibitor, Blocking Assay, Luciferase

    Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.
    Figure Legend Snippet: Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.

    Techniques Used: Imaging, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry, Activity Assay, Real-time Polymerase Chain Reaction

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.
    Figure Legend Snippet: In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.

    Techniques Used: In Vitro, Sequencing, Expressing, FACS, Flow Cytometry, Cytometry, Derivative Assay, Negative Control

    21) Product Images from "Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA"

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp782

    RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).
    Figure Legend Snippet: RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, Northern Blot

    22) Product Images from "Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template"

    Article Title: Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm767

    Comparison of the RNaseH activity of HIV-1 RT with (lanes 1–4) and without (lanes 5–8) dNTPs at different enzyme concentrations: 0.5 U (lanes 1, 3, 5, 7) and 2.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U), Mod = abasic RNA template (X = rAS).
    Figure Legend Snippet: Comparison of the RNaseH activity of HIV-1 RT with (lanes 1–4) and without (lanes 5–8) dNTPs at different enzyme concentrations: 0.5 U (lanes 1, 3, 5, 7) and 2.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U), Mod = abasic RNA template (X = rAS).

    Techniques Used: Activity Assay

    Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h P ss : primer ss, P rs : primer rs, T m : abasic RNA template (X = rAS), T n : non-damaged RNA template (X = U).
    Figure Legend Snippet: Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h P ss : primer ss, P rs : primer rs, T m : abasic RNA template (X = rAS), T n : non-damaged RNA template (X = U).

    Techniques Used:

    Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.
    Figure Legend Snippet: Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

    Techniques Used:

    23) Product Images from "Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G"

    Article Title: Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm750

    Schematic diagram of the events in reverse transcription. Step 1. Reverse transcription is initiated by a cellular tRNA primer ( , in the case of HIV-1), following annealing of the 3′ 18 nt of the tRNA to the 18-nt PBS near the 5′ end of the genome. RT catalyzes synthesis of ( − ) SSDNA, which contains copies of the R sequence and the unique 5′ genomic sequence (U5). Step 2. As the primer is extended, the RNase H activity of RT degrades the genomic RNA sequences that have been reverse transcribed. Step 3. ( − ) SSDNA is transferred to the 3′ end of vRNA (minus-strand transfer). Step 4. Elongation of minus-strand DNA and RNase H degradation continue. Plus-strand synthesis is initiated by the 15-nt PPT immediately upstream of the unique 3′ genomic sequence (U3). Step 5. RT copies the u3, u5 and r regions in minus-strand DNA, as well as the 3′ 18 nt of the tRNA primer, thereby reconstituting the PBS. The product formed is termed (+) SSDNA. Step 6. RNase H removal of the tRNA and PPT primers from minus- and plus-strand DNAs, respectively. Step 7. Plus-strand transfer, facilitated by annealing of the complementary PBS sequences at the 3′ ends of (+) SSDNA and minus-strand DNA, is followed by circularization of the two DNA strands and displacement synthesis. Step 8. Minus- and plus-strand DNAs are elongated, resulting in a linear dsDNA with a long terminal repeat (LTR) at each end. vRNA is shown by an open rectangle and minus-and plus-strand DNAs are shown by black and gray rectangles, respectively. The tRNA primer is represented by a short open rectangle (3′ 18 nt of the tRNA) attached to a ‘clover-leaf’ (remaining tRNA bases). Minus- and plus-strand sequences are depicted in lower and upper case, respectively. The very short white rectangles represent fragments produced by RNase H cleavage of genomic RNA. Adapted from reference ( 43 ) with permission from Elsevier.
    Figure Legend Snippet: Schematic diagram of the events in reverse transcription. Step 1. Reverse transcription is initiated by a cellular tRNA primer ( , in the case of HIV-1), following annealing of the 3′ 18 nt of the tRNA to the 18-nt PBS near the 5′ end of the genome. RT catalyzes synthesis of ( − ) SSDNA, which contains copies of the R sequence and the unique 5′ genomic sequence (U5). Step 2. As the primer is extended, the RNase H activity of RT degrades the genomic RNA sequences that have been reverse transcribed. Step 3. ( − ) SSDNA is transferred to the 3′ end of vRNA (minus-strand transfer). Step 4. Elongation of minus-strand DNA and RNase H degradation continue. Plus-strand synthesis is initiated by the 15-nt PPT immediately upstream of the unique 3′ genomic sequence (U3). Step 5. RT copies the u3, u5 and r regions in minus-strand DNA, as well as the 3′ 18 nt of the tRNA primer, thereby reconstituting the PBS. The product formed is termed (+) SSDNA. Step 6. RNase H removal of the tRNA and PPT primers from minus- and plus-strand DNAs, respectively. Step 7. Plus-strand transfer, facilitated by annealing of the complementary PBS sequences at the 3′ ends of (+) SSDNA and minus-strand DNA, is followed by circularization of the two DNA strands and displacement synthesis. Step 8. Minus- and plus-strand DNAs are elongated, resulting in a linear dsDNA with a long terminal repeat (LTR) at each end. vRNA is shown by an open rectangle and minus-and plus-strand DNAs are shown by black and gray rectangles, respectively. The tRNA primer is represented by a short open rectangle (3′ 18 nt of the tRNA) attached to a ‘clover-leaf’ (remaining tRNA bases). Minus- and plus-strand sequences are depicted in lower and upper case, respectively. The very short white rectangles represent fragments produced by RNase H cleavage of genomic RNA. Adapted from reference ( 43 ) with permission from Elsevier.

    Techniques Used: Sequencing, Activity Assay, Produced

    Effect of A3G on minus-strand transfer reactions. ( A ) Effect of A3G on the time course of RNase H cleavage in the absence or presence of NC. 32 P-labeled TAR RNA (0.1 pmol) and TAR DNA (0.2 pmol) were heat annealed and the hybrid was incubated at 37°C in reaction buffer (see above) with 0.4 pmol HIV-1 RT with or without NC (7 nt/NC, 0.1 µM), with or without A3G (80 nM). Samples were loaded on a 15% denaturing gel. Positions of the major cleavage products are indicated on the right. ( B ) Time course of annealing of 32 P-labeled DNA 128 to RNA 148 incubated in the absence or presence of A3G (80 nM) with or without NC (3.5 nt/NC, 0.4 µM). Symbols: no NC/+A3G (filled circles); +NC/no A3G (open squares); and +NC/+A3G (open circles). ( C ) Schematic diagram illustrating the minus-strand transfer assay system. The R homology is 94 nt; U5 and U3 are 34 and 54 nt, respectively. ( D ) Graph of percent transfer product plotted versus incubation time. To quantify the percentage of strand transfer, the amount of transfer product was divided by the total amount of DNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); no NC/+A3G (open squares); +NC/no A3G (open circles); and +NC/+A3G (open triangles).
    Figure Legend Snippet: Effect of A3G on minus-strand transfer reactions. ( A ) Effect of A3G on the time course of RNase H cleavage in the absence or presence of NC. 32 P-labeled TAR RNA (0.1 pmol) and TAR DNA (0.2 pmol) were heat annealed and the hybrid was incubated at 37°C in reaction buffer (see above) with 0.4 pmol HIV-1 RT with or without NC (7 nt/NC, 0.1 µM), with or without A3G (80 nM). Samples were loaded on a 15% denaturing gel. Positions of the major cleavage products are indicated on the right. ( B ) Time course of annealing of 32 P-labeled DNA 128 to RNA 148 incubated in the absence or presence of A3G (80 nM) with or without NC (3.5 nt/NC, 0.4 µM). Symbols: no NC/+A3G (filled circles); +NC/no A3G (open squares); and +NC/+A3G (open circles). ( C ) Schematic diagram illustrating the minus-strand transfer assay system. The R homology is 94 nt; U5 and U3 are 34 and 54 nt, respectively. ( D ) Graph of percent transfer product plotted versus incubation time. To quantify the percentage of strand transfer, the amount of transfer product was divided by the total amount of DNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); no NC/+A3G (open squares); +NC/no A3G (open circles); and +NC/+A3G (open triangles).

    Techniques Used: Labeling, Incubation

    Effect of A3G on -primed ( − ) SSDNA synthesis. ( A ) Time course of annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. ( B ) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). ( C ) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of ( − ) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.
    Figure Legend Snippet: Effect of A3G on -primed ( − ) SSDNA synthesis. ( A ) Time course of annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. ( B ) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). ( C ) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of ( − ) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.

    Techniques Used:

    Effect of A3G on PPT initiation and plus-strand transfer. ( A ) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α- 32 P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. ( B ) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5 D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).
    Figure Legend Snippet: Effect of A3G on PPT initiation and plus-strand transfer. ( A ) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α- 32 P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. ( B ) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5 D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).

    Techniques Used: DNA Synthesis, Labeling

    24) Product Images from "In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques"

    Article Title: In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.24.13553-13561.2004

    Sequence alignment of amino acids 101 to 240 in the RTs of HIV-1 HXB2 , RT-SHIV mne , SIV mne , and SIV-RT-YY. Dots indicate residues conserved between the different isolates. Boxed residues indicate residues 181 and 188, which are the residues that were changed to tyrosines in SIV-RT-YY.
    Figure Legend Snippet: Sequence alignment of amino acids 101 to 240 in the RTs of HIV-1 HXB2 , RT-SHIV mne , SIV mne , and SIV-RT-YY. Dots indicate residues conserved between the different isolates. Boxed residues indicate residues 181 and 188, which are the residues that were changed to tyrosines in SIV-RT-YY.

    Techniques Used: Sequencing

    In vitro RT inhibition with NNRTIs. Representative data are shown in which JC53 BL13+ cells were infected with HIV-1 NFNSX , SIV mne , RT-SHIV mne , or SIV-RT-YY in the presence or absence of multiple concentrations of EFV (A), NVP (B), or UC781 (C). Infections were performed in duplicate, and luciferase measurements were performed in triplicate. Results are expressed as the percent cells infected for each virus with each dilution of drug compared to infection without drug.
    Figure Legend Snippet: In vitro RT inhibition with NNRTIs. Representative data are shown in which JC53 BL13+ cells were infected with HIV-1 NFNSX , SIV mne , RT-SHIV mne , or SIV-RT-YY in the presence or absence of multiple concentrations of EFV (A), NVP (B), or UC781 (C). Infections were performed in duplicate, and luciferase measurements were performed in triplicate. Results are expressed as the percent cells infected for each virus with each dilution of drug compared to infection without drug.

    Techniques Used: In Vitro, Inhibition, Infection, Luciferase

    25) Product Images from "Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription"

    Article Title: Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.13.7623-7634.2003

    Time course of RNase H-catalyzed RNA PPT primer removal. Aliquots were removed from reaction mixtures at the indicated times, and samples were run in 8% sequencing gels. The amount of 32 P-labeled 20-nt product formed after PPT primer removal was quantified by PhosphorImager analysis. The percent cleavage was calculated by comparing the volume of the 20-nt band at each time point with the value for the volume of the 35-nt product synthesized with T4 DNA polymerase, set at 100%. (A) HIV-1 substrate. Circles, HIV-1 RT; squares, HIV-2 440 RT. (B) HIV-2 substrate. Circles, HIV-1 RT; squares, HIV-2 440 RT; triangles, HIV-2 470 RT; inverted triangles, HIV-2 484 RT. (C) Schematic diagram of the PPT primer removal assay. Step 1, extension by T4 DNA polymerase. The 15-nt RNA PPT and the plus-strand 20-nt DNA extension are represented by an open rectangle and a thick dashed line, respectively. The asterisk indicates that the 20-nt product is internally labeled with 32 P. Step 2, PPT primer removal by RT. The DNA moiety attached to the PPT is shown as a solid rectangle. The vertical arrow indicates the site of cleavage, i.e., at the junction of the 3′ end of the RNA primer and the 5′ end of nascent plus-strand DNA. The 20-nt labeled DNA product and the unlabeled 15-nt primer, which are released, are also shown.
    Figure Legend Snippet: Time course of RNase H-catalyzed RNA PPT primer removal. Aliquots were removed from reaction mixtures at the indicated times, and samples were run in 8% sequencing gels. The amount of 32 P-labeled 20-nt product formed after PPT primer removal was quantified by PhosphorImager analysis. The percent cleavage was calculated by comparing the volume of the 20-nt band at each time point with the value for the volume of the 35-nt product synthesized with T4 DNA polymerase, set at 100%. (A) HIV-1 substrate. Circles, HIV-1 RT; squares, HIV-2 440 RT. (B) HIV-2 substrate. Circles, HIV-1 RT; squares, HIV-2 440 RT; triangles, HIV-2 470 RT; inverted triangles, HIV-2 484 RT. (C) Schematic diagram of the PPT primer removal assay. Step 1, extension by T4 DNA polymerase. The 15-nt RNA PPT and the plus-strand 20-nt DNA extension are represented by an open rectangle and a thick dashed line, respectively. The asterisk indicates that the 20-nt product is internally labeled with 32 P. Step 2, PPT primer removal by RT. The DNA moiety attached to the PPT is shown as a solid rectangle. The vertical arrow indicates the site of cleavage, i.e., at the junction of the 3′ end of the RNA primer and the 5′ end of nascent plus-strand DNA. The 20-nt labeled DNA product and the unlabeled 15-nt primer, which are released, are also shown.

    Techniques Used: Sequencing, Labeling, Synthesized

    ). (A) Gel data. The time points are indicated above the lanes. The positions of (−) SSDNA, SP products (SP), and the primer are indicated to the left of the gel. (B) PhosphorImager analysis of gel data. Total DNA synthesized at each time point was determined by PhosphorImager analysis and was plotted as a function of time. Circles, HIV-1 RT; squares, HIV-2 RT.
    Figure Legend Snippet: ). (A) Gel data. The time points are indicated above the lanes. The positions of (−) SSDNA, SP products (SP), and the primer are indicated to the left of the gel. (B) PhosphorImager analysis of gel data. Total DNA synthesized at each time point was determined by PhosphorImager analysis and was plotted as a function of time. Circles, HIV-1 RT; squares, HIV-2 RT.

    Techniques Used: Synthesized

    DNA-dependent DNA synthesis catalyzed by HIV-1 and HIV-2 RTs. A 15-nt HIV-1 or HIV-2 DNA PPT primer was extended by 20 nt in the presence of [α- 32 P]dATP using the appropriate minus-strand 35-nt DNA template, as described in Materials and Methods. HIV-1 primer-template (P-T), lanes 1 to 6; HIV-2 P-T, lanes 7 to 12. The amounts of enzyme added were as follows: 1 pmol, lanes 1, 2, 7, and 8; 0.1 pmol, lanes 3, 4, 9, and 10; and 0.01 pmol, lanes 5, 6, 11, and 12. The odd-numbered lanes represent reactions with HIV-1 RT, and the even-numbered lanes represent reactions with HIV-2 440 RT. The percentage of total products represented by 35-nt DNA and the percentage of the total amount of DNA synthesized relative to the control are given below each lane; 100% represents the amount of DNA synthesized by 1 pmol of HIV-1 RT with the HIV-1 substrate.
    Figure Legend Snippet: DNA-dependent DNA synthesis catalyzed by HIV-1 and HIV-2 RTs. A 15-nt HIV-1 or HIV-2 DNA PPT primer was extended by 20 nt in the presence of [α- 32 P]dATP using the appropriate minus-strand 35-nt DNA template, as described in Materials and Methods. HIV-1 primer-template (P-T), lanes 1 to 6; HIV-2 P-T, lanes 7 to 12. The amounts of enzyme added were as follows: 1 pmol, lanes 1, 2, 7, and 8; 0.1 pmol, lanes 3, 4, 9, and 10; and 0.01 pmol, lanes 5, 6, 11, and 12. The odd-numbered lanes represent reactions with HIV-1 RT, and the even-numbered lanes represent reactions with HIV-2 440 RT. The percentage of total products represented by 35-nt DNA and the percentage of the total amount of DNA synthesized relative to the control are given below each lane; 100% represents the amount of DNA synthesized by 1 pmol of HIV-1 RT with the HIV-1 substrate.

    Techniques Used: DNA Synthesis, Synthesized

    Time course of 5′-directed RNase H cleavage activity. The 5′-directed RNase H cleavage activities of HIV-l and HIV-2 440 RTs were assayed as described in Materials and Methods. (A) Gel data. The time points are indicated above the lanes. Lane C is the zero-time-point (minus RT) control. The sizes of the RNA bands, shown on the left, were estimated by comparison with an RNA ladder generated by alkaline hydrolysis of the 20-nt RNA, run on the same gel as the samples. The 19-nt band in all samples is a contaminant and represents ∼3% of the total 20-nt RNA preparation. (B) PhosphorImager analysis of the gel data. Total cleavage was calculated by dividing the “volume” of all of the cleavage products in each lane by the total volume and then multiplying by 100. The values for total cleavage were plotted as a function of the time of incubation. Circles, HIV-1 RT; squares, HIV-2 RT. (C) Schematic diagram illustrating the 32 P-labeled substrate. The 20-nt RNA primer is shown as an open rectangle; the 32 P label at the 5′ end of the RNA is indicated by an asterisk. The 55-nt minus-strand DNA is shown as a hatched rectangle.
    Figure Legend Snippet: Time course of 5′-directed RNase H cleavage activity. The 5′-directed RNase H cleavage activities of HIV-l and HIV-2 440 RTs were assayed as described in Materials and Methods. (A) Gel data. The time points are indicated above the lanes. Lane C is the zero-time-point (minus RT) control. The sizes of the RNA bands, shown on the left, were estimated by comparison with an RNA ladder generated by alkaline hydrolysis of the 20-nt RNA, run on the same gel as the samples. The 19-nt band in all samples is a contaminant and represents ∼3% of the total 20-nt RNA preparation. (B) PhosphorImager analysis of the gel data. Total cleavage was calculated by dividing the “volume” of all of the cleavage products in each lane by the total volume and then multiplying by 100. The values for total cleavage were plotted as a function of the time of incubation. Circles, HIV-1 RT; squares, HIV-2 RT. (C) Schematic diagram illustrating the 32 P-labeled substrate. The 20-nt RNA primer is shown as an open rectangle; the 32 P label at the 5′ end of the RNA is indicated by an asterisk. The 55-nt minus-strand DNA is shown as a hatched rectangle.

    Techniques Used: Activity Assay, Generated, Incubation, Labeling

    26) Product Images from "Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs"

    Article Title: Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1901165116

    AUF1 expression is essential for myoblast differentiation and myotube formation. ( A ) Expression of AUBP mRNAs during differentiation at 96 h determined by qRT-PCR ( n = 3), normalized to invariant GAPDH mRNA. ( B ) Expression of AUBP proteins during differentiation corresponding to A , n = 4. Numbers below immunoblot: Twist1 fold increase normalized to WT cells. ( C ) Immunoblot of CTCF, AUF1, MyoD, and β-tubulin control during C2C12 myoblast differentiation. CTCF was silenced in C2C12 cells by lentiviral-mediated shRNA. Numbers under immunoblot correspond to AUF1 fold change relative to β-tubulin levels, normalized to 0-h Nsi time point. n = 4. ( D ) Immunoblot of CTCF and AUF1 with increasing transfection of a CTCF cDNA expression plasmid in HEK 293 cells. Overexpression of CTCF was performed using pMy-CTCF (Addgene), n = 3. CTCF Chromatin-IP (ChIP) analysis using Auf1 promoter in C2C12 cells at 48 h after induction of differentiation. DNA enrichment in fragmented ChIP assay with anti-CTCF antibody relative to anti-rabbit IgG IP control, normalized to intron signal measured by qRT-PCR. n = 3. * P
    Figure Legend Snippet: AUF1 expression is essential for myoblast differentiation and myotube formation. ( A ) Expression of AUBP mRNAs during differentiation at 96 h determined by qRT-PCR ( n = 3), normalized to invariant GAPDH mRNA. ( B ) Expression of AUBP proteins during differentiation corresponding to A , n = 4. Numbers below immunoblot: Twist1 fold increase normalized to WT cells. ( C ) Immunoblot of CTCF, AUF1, MyoD, and β-tubulin control during C2C12 myoblast differentiation. CTCF was silenced in C2C12 cells by lentiviral-mediated shRNA. Numbers under immunoblot correspond to AUF1 fold change relative to β-tubulin levels, normalized to 0-h Nsi time point. n = 4. ( D ) Immunoblot of CTCF and AUF1 with increasing transfection of a CTCF cDNA expression plasmid in HEK 293 cells. Overexpression of CTCF was performed using pMy-CTCF (Addgene), n = 3. CTCF Chromatin-IP (ChIP) analysis using Auf1 promoter in C2C12 cells at 48 h after induction of differentiation. DNA enrichment in fragmented ChIP assay with anti-CTCF antibody relative to anti-rabbit IgG IP control, normalized to intron signal measured by qRT-PCR. n = 3. * P

    Techniques Used: Expressing, Quantitative RT-PCR, shRNA, Transfection, Plasmid Preparation, Over Expression, Chromatin Immunoprecipitation

    Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)
    Figure Legend Snippet: Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)

    Techniques Used: Cell Culture, Mouse Assay, Staining, Isolation, Transduction, Expressing

    AUF1 targeted decay of Twist1 mRNA partially restores myogenesis. Relative expression of TWIST1 mRNA ( A ) and protein levels in WT C2C12 myoblasts and AUF1 KO C2C12 myoblasts ( B ), n = 3. Numbers under blot refer to fold increase in AUF1 normalized to GAPDH protein. ** P
    Figure Legend Snippet: AUF1 targeted decay of Twist1 mRNA partially restores myogenesis. Relative expression of TWIST1 mRNA ( A ) and protein levels in WT C2C12 myoblasts and AUF1 KO C2C12 myoblasts ( B ), n = 3. Numbers under blot refer to fold increase in AUF1 normalized to GAPDH protein. ** P

    Techniques Used: Expressing

    Targeted decay of RGS5 and Twist1 mRNAs restores myotube differentiation and maturation in the absence of AUF1. ( A ) Immunoblot of AUF1 and RGS5 and RGS5 quantification relative to GAPDH in WT and ( Left ) AUF1 KO ( Right ) C2C12 myoblasts 96 h after differentiation, n = 3. ( B ) RGS5 mRNA decay rate. WT C2C12 cells, dotted line; AUF1 KO C2C12 cells, solid line. n = 3, ±SEM, ** P
    Figure Legend Snippet: Targeted decay of RGS5 and Twist1 mRNAs restores myotube differentiation and maturation in the absence of AUF1. ( A ) Immunoblot of AUF1 and RGS5 and RGS5 quantification relative to GAPDH in WT and ( Left ) AUF1 KO ( Right ) C2C12 myoblasts 96 h after differentiation, n = 3. ( B ) RGS5 mRNA decay rate. WT C2C12 cells, dotted line; AUF1 KO C2C12 cells, solid line. n = 3, ±SEM, ** P

    Techniques Used:

    27) Product Images from "Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect"

    Article Title: Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect

    Journal: Journal of Virology

    doi: 10.1128/JVI.02680-05

    RNA binding of APO3G or HIV-1 NC in the presence of HIV-1 NC or APO3G, respectively. The EMSAs were performed with a 40-nt ssRNA (JL654) or a 33-nt SL-3 RNA (JL765). (A and C) The RNA (JL654 [A] or JL765 [C]) was preincubated for 15 min at 37°C
    Figure Legend Snippet: RNA binding of APO3G or HIV-1 NC in the presence of HIV-1 NC or APO3G, respectively. The EMSAs were performed with a 40-nt ssRNA (JL654) or a 33-nt SL-3 RNA (JL765). (A and C) The RNA (JL654 [A] or JL765 [C]) was preincubated for 15 min at 37°C

    Techniques Used: RNA Binding Assay

    Effect of zinc finger mutations on enzymatic and anti-HIV-1 activities of APO3G. (A) The effect of zinc finger mutations on the deaminase activity of APO3G was analyzed by the UDG-dependent deaminase assay. Substrate JL653 (containing the TCCCA motif)
    Figure Legend Snippet: Effect of zinc finger mutations on enzymatic and anti-HIV-1 activities of APO3G. (A) The effect of zinc finger mutations on the deaminase activity of APO3G was analyzed by the UDG-dependent deaminase assay. Substrate JL653 (containing the TCCCA motif)

    Techniques Used: Activity Assay

    Related Articles

    Concentration Assay:

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency
    Article Snippet: .. Reactions with HIV-1 Reverse Transcriptase from Worthington Biochemical Corporations (cat. LS05003) were performed as human RNase H1, with the final concentration of the enzyme 0.1 U/μl. .. Control hydrolysis reactions labeled ‘EDTA’ were performed using identical conditions, but in the presence of 10 mM EDTA, whereas control reactions performed in ‘cold’ conditions were incubated for 30 min at 16°C.

    Recombinant:

    Article Title: 3-O-Methylfunicone, a Selective Inhibitor of Mammalian Y-Family DNA Polymerases from an Australian Sea Salt Fungal Strain
    Article Snippet: .. HIV-1 reverse transcriptase (recombinant) and the Klenow fragment of pol I from E. coli were purchased from Worthington Biochemical Corp. (Freehold, NJ, USA). .. T4 pol, Taq pol, T7 RNA polymerase and T4 polynucleotide kinase were purchased from Takara Bio (Tokyo, Japan).

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *
    Article Snippet: .. Enzymes and reagents —Recombinant HIV-1 reverse transcriptase was obtained from Worthington Biochemicals. .. Recombinant wild-type M-MuLV reverse transcriptase, T7 DNA polymerase, and calf intestinal alkaline phosphatase were purchased from Amersham Pharmacia Biotech.

    Article Title: SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES
    Article Snippet: .. Recombinant HIV-1 RT was purchased from Worthington Biochemical Corporation (Lakewood, NJ), and phi29 DNA polymerase was purchased from New England Biolabs. .. We confirmed purity of the HIV-1 RT stock by running SDS-polyacrylamide gel electrophoresis.

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice
    Article Snippet: .. All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ). .. Statistics All descriptive statistics, one-way ANOVA to determine significance in CD4 surface down-modulation experiments to test Nef functionality, and Pearson correlation calculations to determine an association between p24+ cells and bioluminescent signal during longitudinal imaging of Hu-PBL mice were performed using GraphPad Prism software, version 7 (San Diego, CA).

    Activity Assay:

    Article Title: Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection
    Article Snippet: .. The number of particles in the original stock was determined as the average of the calculated particles at each dilution based upon a standard curve using HIV-1 RT enzyme (Worthington Biochemical Corporation Lakewood, NJ, USA, cat. No. LS05006; lot No. X1H2839), which was determined to have 262 pU of RT activity per particle [ ]. .. The STF-PERT one-step assay [ ] was modified into a two-step assay that no longer requires the use of AmpliWax® PCR Gem 50, a product that has been discontinued.

    other:

    Article Title: Enzymatic synthesis of DNA strands containing ?-L-LNA (?-L-configured locked nucleic acid) thymine nucleotides
    Article Snippet: HIV RT (Worthington biochemical company, LS05003) 4.00 μl 5X HIV RT buffer (250 mM TRIS-HCl, 300 mM KCl, 12.5 mM MgCl2 ) 1.50 μl Triphosphate mixture (2.50 mM each) 0.60 μl Primer:template complex (5.0:10.0 μM) 0.60 μl HIV RT (2.7 U/μl) 13.3 μl Twice distilled water

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    Worthington Biochemical reagents recombinant hiv 1 reverse transcriptase
    Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by <t>HIV-1</t> RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.
    Reagents Recombinant Hiv 1 Reverse Transcriptase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical cd45 congenic recipient mice 1 d
    Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into <t>CD45</t> <t>congenic</t> recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P
    Cd45 Congenic Recipient Mice 1 D, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical t1l t3d m2
    ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of <t>T1L/T3D</t> M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).
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    Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.

    Journal: The Journal of biological chemistry

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *

    doi: 10.1074/jbc.M510504200

    Figure Lengend Snippet: Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.

    Article Snippet: Enzymes and reagents —Recombinant HIV-1 reverse transcriptase was obtained from Worthington Biochemicals.

    Techniques: Sequencing

    Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.

    Journal: The Journal of biological chemistry

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *

    doi: 10.1074/jbc.M510504200

    Figure Lengend Snippet: Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.

    Article Snippet: Enzymes and reagents —Recombinant HIV-1 reverse transcriptase was obtained from Worthington Biochemicals.

    Techniques: Sequencing

    Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.

    Journal: The Journal of biological chemistry

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *

    doi: 10.1074/jbc.M510504200

    Figure Lengend Snippet: Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.

    Article Snippet: Enzymes and reagents —Recombinant HIV-1 reverse transcriptase was obtained from Worthington Biochemicals.

    Techniques: Generated, Labeling

    Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    doi: 10.1084/jem.20132267

    Figure Lengend Snippet: Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P

    Article Snippet: For in vivo cell transfer experiments, 5 × 105 CD8-depleted thymocytes from OTII or OTII Cul3cKO mice were i.v. transferred into CD45 congenic recipient mice 1 d before i.p. injection of 50 µg OVA (Worthington Biochemical Corporation) mixed 1:1 with alum (Thermo Fisher Scientific) or s.c. injection in the left and right hock of a total of 50 µg OVA mixed 1:1 with CFA (Sigma-Aldrich).

    Techniques: Mouse Assay, Transduction, Expressing, Injection, Staining

    Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    doi: 10.1084/jem.20132267

    Figure Lengend Snippet: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P

    Article Snippet: For in vivo cell transfer experiments, 5 × 105 CD8-depleted thymocytes from OTII or OTII Cul3cKO mice were i.v. transferred into CD45 congenic recipient mice 1 d before i.p. injection of 50 µg OVA (Worthington Biochemical Corporation) mixed 1:1 with alum (Thermo Fisher Scientific) or s.c. injection in the left and right hock of a total of 50 µg OVA mixed 1:1 with CFA (Sigma-Aldrich).

    Techniques: Expressing, Quantitative RT-PCR, Genome Wide, Generated, Magnetic Cell Separation, FACS, Irradiation, Purification

    Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    doi: 10.1084/jem.20132267

    Figure Lengend Snippet: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P

    Article Snippet: For in vivo cell transfer experiments, 5 × 105 CD8-depleted thymocytes from OTII or OTII Cul3cKO mice were i.v. transferred into CD45 congenic recipient mice 1 d before i.p. injection of 50 µg OVA (Worthington Biochemical Corporation) mixed 1:1 with alum (Thermo Fisher Scientific) or s.c. injection in the left and right hock of a total of 50 µg OVA mixed 1:1 with CFA (Sigma-Aldrich).

    Techniques: Injection, Mouse Assay, FACS, Staining, Fluorescence, In Vivo

    Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.

    Journal: The Journal of Experimental Medicine

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    doi: 10.1084/jem.20132267

    Figure Lengend Snippet: Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.

    Article Snippet: For in vivo cell transfer experiments, 5 × 105 CD8-depleted thymocytes from OTII or OTII Cul3cKO mice were i.v. transferred into CD45 congenic recipient mice 1 d before i.p. injection of 50 µg OVA (Worthington Biochemical Corporation) mixed 1:1 with alum (Thermo Fisher Scientific) or s.c. injection in the left and right hock of a total of 50 µg OVA mixed 1:1 with CFA (Sigma-Aldrich).

    Techniques: Injection, Expressing, Staining, Mouse Assay

    ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: ) ( n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs ( n = 3 independent replicates).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Cell Attachment Assay, Labeling, Staining, Binding Assay

    The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis

    The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, Incubation, SDS Page, Western Blot

    The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, Incubation, Infection, Plaque Assay, SDS Page, Staining

    The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs ( n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 ) ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, Quantitative RT-PCR

    ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: ). μ1N and Φ are too small to resolve on the gel ( n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Generated

    The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs ( n = 3 independent replicates).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, Incubation, Fluorescence

    The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 10 12 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 10 12 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained ( n = 3 independent replicates; results from 1 representative experiment are shown).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, Activity Assay, Incubation, Infection, Plaque Assay, Western Blot, SDS Page, Staining

    The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).

    Journal: Journal of Virology

    Article Title: Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

    doi: 10.1128/JVI.01848-17

    Figure Lengend Snippet: The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting ( n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 10 3 particles/cell) or T1L/T3D M2 Y581A (0.4 × 10 3 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 ( n = 3 independent replicates).

    Article Snippet: T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK ( N α- p -tosyl- l -lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C ( , ).

    Techniques: Mutagenesis, SDS Page, Western Blot, Infection, Immunofluorescence