hiv 1 rt enzyme  (Worthington Biochemical)


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    Structured Review

    Worthington Biochemical hiv 1 rt enzyme
    Sequence alignment of amino acids 101 to 240 in the RTs of <t>HIV-1</t> 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.
    Hiv 1 Rt Enzyme, 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
    https://www.bioz.com/result/hiv 1 rt enzyme/product/Worthington Biochemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiv 1 rt enzyme - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) 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

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    Worthington Biochemical hiv 1 rt enzyme
    Sequence alignment of amino acids 101 to 240 in the RTs of <t>HIV-1</t> 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.
    Hiv 1 Rt Enzyme, 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
    https://www.bioz.com/result/hiv 1 rt enzyme/product/Worthington Biochemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiv 1 rt enzyme - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    93
    Worthington Biochemical recombinant hiv 1 reverse transcriptase standard curve
    Longitudinal non-invasive bioluminescent imaging of <t>HIV-1</t> acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. 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 (dotted line).
    Recombinant Hiv 1 Reverse Transcriptase Standard Curve, 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
    https://www.bioz.com/result/recombinant hiv 1 reverse transcriptase standard curve/product/Worthington Biochemical
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant hiv 1 reverse transcriptase standard curve - by Bioz Stars, 2022-07
    93/100 stars
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    86
    Worthington Biochemical hiv 1 rt
    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 <t>HIV-1</t> 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
    Hiv 1 Rt, 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
    https://www.bioz.com/result/hiv 1 rt/product/Worthington Biochemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiv 1 rt - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    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.

    Journal: Journal of Virology

    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

    doi: 10.1128/JVI.78.24.13553-13561.2004

    Figure Lengend 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.

    Article Snippet: A standard curve was created using HIV-1 RT enzyme (Worthington, Lakewood, N.J.) diluted between 10−2 and 10−10 U/μl in the same buffer used for the samples and assayed in triplicate.

    Techniques: 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.

    Journal: Journal of Virology

    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

    doi: 10.1128/JVI.78.24.13553-13561.2004

    Figure Lengend 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.

    Article Snippet: A standard curve was created using HIV-1 RT enzyme (Worthington, Lakewood, N.J.) diluted between 10−2 and 10−10 U/μl in the same buffer used for the samples and assayed in triplicate.

    Techniques: In Vitro, Inhibition, Infection, Luciferase

    Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. 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 (dotted line).

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend Snippet: Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. 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 (dotted line).

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Ex Vivo, Activity Assay

    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.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend 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.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend Snippet: RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: RNA Viral-load Assay, RT Activity Assay, Derivative Assay, Quantitative RT-PCR, Viral-load Assay, Activity Assay, Real-time Polymerase Chain Reaction

    Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* 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-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend Snippet: Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* 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-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    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 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 ART cessation.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend 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 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 ART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 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 HIV-1 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 correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend Snippet: Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 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 HIV-1 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 correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    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 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.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend 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 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.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    Longitudinal imaging of HIV-1 acute infection, 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 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 ART cessation.

    Journal: bioRxiv

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

    doi: 10.1101/745125

    Figure Lengend Snippet: Longitudinal imaging of HIV-1 acute infection, 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 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 ART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

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

    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

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

    Techniques: 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.

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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.

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

    Techniques: 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.

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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.

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

    Techniques: 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.

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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.

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

    Techniques: 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

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

    Techniques: 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

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0063-9

    Figure Lengend 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

    Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

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

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm750

    Figure Lengend 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.

    Article Snippet: HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ).

    Techniques: 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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm750

    Figure Lengend 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).

    Article Snippet: HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm750

    Figure Lengend 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.

    Article Snippet: HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ).

    Techniques:

    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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm750

    Figure Lengend 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).

    Article Snippet: HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ).

    Techniques: DNA Synthesis, Labeling