baby hamster kidney bhk 21 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher baby hamster kidney bhk 21 cells
    Generation of MuHV-4 expressing luciferase. (a) A 2 kb luciferase-polyA cassette was placed downstream of a 500 bp M3 promoter, in a Mfe I restriction site between ORFs 57 and 58. Relevant restrictions sites are shown. (b) Viral DNA was digested with Eco RI or Hin dIII and probed with the 75 338–78 717 Bgl II clone shown in (a). The luciferase expression cassette changes a 14.9 kb Eco RI band to 5.5 kb+12.0 kb, and a 14.5 kb Hin dIII band to 6.8 kb+10.1 kb. M3-LUC1.6 and M3-LUC2.1 are independently generated recombinant viruses. (c) <t>BHK-21</t> cells were left uninfected or infected overnight (1 p.f.u. cell −1 ), then lysed and assayed for luciferase expression. Each bar shows the mean± sd of triplicate cultures. (d) BHK-21 cells were infected with wild-type or M3-LUC MuHV-4 (0.01 p.f.u. cell −1 , 2 h), washed with PBS to remove unbound virions, then incubated at 37 °C. The infectious virus in each culture was measured by plaque assay. (e) BHK-21 cells were infected with wild-type MuHV-4, the M3-LUC2.1 recombinant or its ORF50 − derivative. Luciferase expression was assayed 18 h later by luminometry. Each bar shows mean± sd of five replicate infections. The ORF50 − cultures contained no replication-competent virus by plaque assay.
    Baby Hamster Kidney Bhk 21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "In vivo imaging of murid herpesvirus-4 infection"

    Article Title: In vivo imaging of murid herpesvirus-4 infection

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.006569-0

    Generation of MuHV-4 expressing luciferase. (a) A 2 kb luciferase-polyA cassette was placed downstream of a 500 bp M3 promoter, in a Mfe I restriction site between ORFs 57 and 58. Relevant restrictions sites are shown. (b) Viral DNA was digested with Eco RI or Hin dIII and probed with the 75 338–78 717 Bgl II clone shown in (a). The luciferase expression cassette changes a 14.9 kb Eco RI band to 5.5 kb+12.0 kb, and a 14.5 kb Hin dIII band to 6.8 kb+10.1 kb. M3-LUC1.6 and M3-LUC2.1 are independently generated recombinant viruses. (c) BHK-21 cells were left uninfected or infected overnight (1 p.f.u. cell −1 ), then lysed and assayed for luciferase expression. Each bar shows the mean± sd of triplicate cultures. (d) BHK-21 cells were infected with wild-type or M3-LUC MuHV-4 (0.01 p.f.u. cell −1 , 2 h), washed with PBS to remove unbound virions, then incubated at 37 °C. The infectious virus in each culture was measured by plaque assay. (e) BHK-21 cells were infected with wild-type MuHV-4, the M3-LUC2.1 recombinant or its ORF50 − derivative. Luciferase expression was assayed 18 h later by luminometry. Each bar shows mean± sd of five replicate infections. The ORF50 − cultures contained no replication-competent virus by plaque assay.
    Figure Legend Snippet: Generation of MuHV-4 expressing luciferase. (a) A 2 kb luciferase-polyA cassette was placed downstream of a 500 bp M3 promoter, in a Mfe I restriction site between ORFs 57 and 58. Relevant restrictions sites are shown. (b) Viral DNA was digested with Eco RI or Hin dIII and probed with the 75 338–78 717 Bgl II clone shown in (a). The luciferase expression cassette changes a 14.9 kb Eco RI band to 5.5 kb+12.0 kb, and a 14.5 kb Hin dIII band to 6.8 kb+10.1 kb. M3-LUC1.6 and M3-LUC2.1 are independently generated recombinant viruses. (c) BHK-21 cells were left uninfected or infected overnight (1 p.f.u. cell −1 ), then lysed and assayed for luciferase expression. Each bar shows the mean± sd of triplicate cultures. (d) BHK-21 cells were infected with wild-type or M3-LUC MuHV-4 (0.01 p.f.u. cell −1 , 2 h), washed with PBS to remove unbound virions, then incubated at 37 °C. The infectious virus in each culture was measured by plaque assay. (e) BHK-21 cells were infected with wild-type MuHV-4, the M3-LUC2.1 recombinant or its ORF50 − derivative. Luciferase expression was assayed 18 h later by luminometry. Each bar shows mean± sd of five replicate infections. The ORF50 − cultures contained no replication-competent virus by plaque assay.

    Techniques Used: Expressing, Luciferase, Generated, Recombinant, Infection, Incubation, Plaque Assay

    2) Product Images from "Membrane Topology and Function of Dengue Virus NS2A Protein"

    Article Title: Membrane Topology and Function of Dengue Virus NS2A Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.02424-12

    In situ fluorescence protease protection assay. (A) E 24 -NS1-NS2A (truncated)-eGFP fusion constructs. Each NS2A fragment was N-terminally fused with E 24 -NS1 (representing a signal peptide derived from the last 24 residues of E protein [E 24 ] and NS1 protein), and C-terminally fused with eGFP. pTMS are shown as gray boxes. (B) In situ fluorescence protease protection assay. BHK-21 cells were transfected with the indicated NS2A constructs. At 24 h p.t., the cells were permeabilized with digitonin followed by protease K treatment. Once the protease K was added, the fluorescence intensities were continuously quantified for 300 s (see Materials and Methods). Relative intensities were calculated using the fluorescence intensity at every 8 s divided by that at time zero when protease K was added. The means of relative intensities derived from 5 to 7 fields (each field containing 4 to 6 positive cells) are presented. The positions of NS2A truncates are indicated in the corresponding panels. (C) Summary of relative fluorescence intensity and initial rate of fluorescence degradation. Average results from at least two independent experiments are presented. See Materials and Methods for calculations.
    Figure Legend Snippet: In situ fluorescence protease protection assay. (A) E 24 -NS1-NS2A (truncated)-eGFP fusion constructs. Each NS2A fragment was N-terminally fused with E 24 -NS1 (representing a signal peptide derived from the last 24 residues of E protein [E 24 ] and NS1 protein), and C-terminally fused with eGFP. pTMS are shown as gray boxes. (B) In situ fluorescence protease protection assay. BHK-21 cells were transfected with the indicated NS2A constructs. At 24 h p.t., the cells were permeabilized with digitonin followed by protease K treatment. Once the protease K was added, the fluorescence intensities were continuously quantified for 300 s (see Materials and Methods). Relative intensities were calculated using the fluorescence intensity at every 8 s divided by that at time zero when protease K was added. The means of relative intensities derived from 5 to 7 fields (each field containing 4 to 6 positive cells) are presented. The positions of NS2A truncates are indicated in the corresponding panels. (C) Summary of relative fluorescence intensity and initial rate of fluorescence degradation. Average results from at least two independent experiments are presented. See Materials and Methods for calculations.

    Techniques Used: In Situ, Fluorescence, Construct, Derivative Assay, Transfection

    Analysis of NS2A R84 and P85 in DENV-2 genome-length RNA. (A) IFA analysis. Equal amounts of WT or mutant genome-length RNA were electroporated into BHK-21 cells. The transfected cells were monitored for E protein expression at indicated time points. An anti-E monoclonal antibody 4G2 and a goat anti-mouse IgG conjugated with Alexa Fluor 488 were used as primary and secondary antibodies, respectively. Green and blue indicate E protein and nucleus staining, respectively. (B) Plaque morphology of WT and mutant viruses. The viruses were derived from the media collected on day 5 posttransfection. (C) Virus production. Culture media from cells transfected with genome-length RNAs from (A) were collected at the indicated time points; viral titers were quantified by plaque assay. Average results with standard deviations are presented. The dashed line indicates the limit of detection (L.O.D; 10 PFU/ml). (D) Plaque morphology of revertant viruses after 5 rounds of passaging on Vero cells. Amino acid substitutions identified at position 84 of NS2A are indicated.
    Figure Legend Snippet: Analysis of NS2A R84 and P85 in DENV-2 genome-length RNA. (A) IFA analysis. Equal amounts of WT or mutant genome-length RNA were electroporated into BHK-21 cells. The transfected cells were monitored for E protein expression at indicated time points. An anti-E monoclonal antibody 4G2 and a goat anti-mouse IgG conjugated with Alexa Fluor 488 were used as primary and secondary antibodies, respectively. Green and blue indicate E protein and nucleus staining, respectively. (B) Plaque morphology of WT and mutant viruses. The viruses were derived from the media collected on day 5 posttransfection. (C) Virus production. Culture media from cells transfected with genome-length RNAs from (A) were collected at the indicated time points; viral titers were quantified by plaque assay. Average results with standard deviations are presented. The dashed line indicates the limit of detection (L.O.D; 10 PFU/ml). (D) Plaque morphology of revertant viruses after 5 rounds of passaging on Vero cells. Amino acid substitutions identified at position 84 of NS2A are indicated.

    Techniques Used: Immunofluorescence, Mutagenesis, Transfection, Expressing, Staining, Derivative Assay, Plaque Assay, Passaging

    Comparison of intra- and extracellular infectious viral particles and viral RNA between the WT and the R84A mutant. BHK-21 cells were transfected with equal amounts of WT and mutant genome-length RNA. At the indicated time points, intra- and extracellular infectious viral particles were quantified by plaque assay (A); viral RNAs were measured by quantitative RT-PCR (B) (for details, see Materials and Methods). The intracellular viral particles were normalized to the cell number and presented as PFU per 10 6 cells. The relative RNA was calculated using formula 100% × 2 ( CT i − CT o) , where C T i is the C T value for the individual virus sample and C T o is the C T value derived from the intracellular WT viral RNA collected at 0 h p.t. for intracellular RNA calculation or at 12 h p.t. for extracellular RNA calculation. The intracellular viral RNAs were normalized to the C T values derived from GAPDH mRNA. An asterisk indicates statistical significance based on Student's t test ( P
    Figure Legend Snippet: Comparison of intra- and extracellular infectious viral particles and viral RNA between the WT and the R84A mutant. BHK-21 cells were transfected with equal amounts of WT and mutant genome-length RNA. At the indicated time points, intra- and extracellular infectious viral particles were quantified by plaque assay (A); viral RNAs were measured by quantitative RT-PCR (B) (for details, see Materials and Methods). The intracellular viral particles were normalized to the cell number and presented as PFU per 10 6 cells. The relative RNA was calculated using formula 100% × 2 ( CT i − CT o) , where C T i is the C T value for the individual virus sample and C T o is the C T value derived from the intracellular WT viral RNA collected at 0 h p.t. for intracellular RNA calculation or at 12 h p.t. for extracellular RNA calculation. The intracellular viral RNAs were normalized to the C T values derived from GAPDH mRNA. An asterisk indicates statistical significance based on Student's t test ( P

    Techniques Used: Mutagenesis, Transfection, Plaque Assay, Quantitative RT-PCR, Derivative Assay

    Prediction of membrane topology and analysis of membrane-associated activity of DENV-2 NS2A. (A) Schematic representation of DENV-2 NS2A transmembrane segments predicted by HMMTOP, TMHMM2, SOSUI, DAS, TOPCONS, Split, and MEMSAT3. The gray boxes indicate predicted transmembrane segments (pTMS). The positions of the first and last amino acid of pTMS are indicated. (B) A reference model of DENV-2 NS2A topology. Different fragments covering the entire NS2A were C-terminally fused with eGFP. The amino acid positions of each NS2A fragment are indicated on the left. (C) IFA analysis of BHK-21 cells transfected with various NS2A-eGFP constructs. At 24 h p.t., the expression of eGFP was monitored by a mouse monoclonal antibody against eGFP and a goat anti-mouse IgG conjugated with Alexa Fluor 488. The eGFP signal is in white. (D) Membrane flotation analysis of 293T cells transfected with plasmids expressing NS2A fragment-eGFP fusion proteins. NS2A fragment-eGFP proteins in each fraction were detected using an antibody against eGFP. Calnexin, probed with rabbit IgG against calnexin (Sigma), was used as an integral membrane protein control. The percentages of signals detected in the low-density (LD) fractions (1 to 4) and high-density (HD) fractions (5 to 8) were calculated by ImageJ software and are indicated below the panels.
    Figure Legend Snippet: Prediction of membrane topology and analysis of membrane-associated activity of DENV-2 NS2A. (A) Schematic representation of DENV-2 NS2A transmembrane segments predicted by HMMTOP, TMHMM2, SOSUI, DAS, TOPCONS, Split, and MEMSAT3. The gray boxes indicate predicted transmembrane segments (pTMS). The positions of the first and last amino acid of pTMS are indicated. (B) A reference model of DENV-2 NS2A topology. Different fragments covering the entire NS2A were C-terminally fused with eGFP. The amino acid positions of each NS2A fragment are indicated on the left. (C) IFA analysis of BHK-21 cells transfected with various NS2A-eGFP constructs. At 24 h p.t., the expression of eGFP was monitored by a mouse monoclonal antibody against eGFP and a goat anti-mouse IgG conjugated with Alexa Fluor 488. The eGFP signal is in white. (D) Membrane flotation analysis of 293T cells transfected with plasmids expressing NS2A fragment-eGFP fusion proteins. NS2A fragment-eGFP proteins in each fraction were detected using an antibody against eGFP. Calnexin, probed with rabbit IgG against calnexin (Sigma), was used as an integral membrane protein control. The percentages of signals detected in the low-density (LD) fractions (1 to 4) and high-density (HD) fractions (5 to 8) were calculated by ImageJ software and are indicated below the panels.

    Techniques Used: Activity Assay, Immunofluorescence, Transfection, Construct, Expressing, Software

    Replicon analysis of NS2A R84 and P85. (A) Schematic diagram of a luciferase replicon of DENV-2. Rluc2A, Renilla luciferase gene followed by the foot-and-mouth disease virus 2A peptide; C 38 , nucleotides encoding the first 38 amino acids of C protein; E 31 , nucleotides encoding the last 31 amino acids of E protein; HDVr, hepatitis delta virus ribozyme sequence. (B) Transient replicon assay. Equal amounts of replicon RNA (WT or mutant R84A, R84E, R84S, or P85A) were electroporated into BHK-21 cells. At the indicated time points, the transfected cells were lysed and assayed for luciferase activities. The y axis shows the log 10 value of Renilla luciferase activity (RLU). Each data point is the average for three replicates, and error bars show the standard deviations.
    Figure Legend Snippet: Replicon analysis of NS2A R84 and P85. (A) Schematic diagram of a luciferase replicon of DENV-2. Rluc2A, Renilla luciferase gene followed by the foot-and-mouth disease virus 2A peptide; C 38 , nucleotides encoding the first 38 amino acids of C protein; E 31 , nucleotides encoding the last 31 amino acids of E protein; HDVr, hepatitis delta virus ribozyme sequence. (B) Transient replicon assay. Equal amounts of replicon RNA (WT or mutant R84A, R84E, R84S, or P85A) were electroporated into BHK-21 cells. At the indicated time points, the transfected cells were lysed and assayed for luciferase activities. The y axis shows the log 10 value of Renilla luciferase activity (RLU). Each data point is the average for three replicates, and error bars show the standard deviations.

    Techniques Used: Luciferase, Sequencing, Mutagenesis, Transfection, Activity Assay

    3) Product Images from "Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function"

    Article Title: Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

    Journal: Journal of Virology

    doi:

    New SFV SHQL variants as helpers. Recombinant RNA encoding LacZ and helper RNAs were cotransfected into BHK-21 cells, pulse-labelled for 15 min, and chased for 3 h. Lysates were either loaded directly on the gel (lanes 1 to 4) or immunoprecipitated with anti-E1 and anti-E2 antibodies (lanes 5 to 8). Packaged virus was pelleted from the growth medium by ultracentrifugation through a 20% sucrose cushion (lanes 9 to 12). Lanes: 1, 5, and 9, SHQL helper (original helper 2); 2, 6, and 10, helper SHQL S64; 3, 7, and 11, helper SHQL S62; 4, 8, and 12, helper SHQL G59. The C protein of purified virus often smears in SDS-PAGE and is not a good indicator of quantitation (lanes 9 to 12). ppt, precipitate.
    Figure Legend Snippet: New SFV SHQL variants as helpers. Recombinant RNA encoding LacZ and helper RNAs were cotransfected into BHK-21 cells, pulse-labelled for 15 min, and chased for 3 h. Lysates were either loaded directly on the gel (lanes 1 to 4) or immunoprecipitated with anti-E1 and anti-E2 antibodies (lanes 5 to 8). Packaged virus was pelleted from the growth medium by ultracentrifugation through a 20% sucrose cushion (lanes 9 to 12). Lanes: 1, 5, and 9, SHQL helper (original helper 2); 2, 6, and 10, helper SHQL S64; 3, 7, and 11, helper SHQL S62; 4, 8, and 12, helper SHQL G59. The C protein of purified virus often smears in SDS-PAGE and is not a good indicator of quantitation (lanes 9 to 12). ppt, precipitate.

    Techniques Used: Recombinant, Immunoprecipitation, Purification, SDS Page, Quantitation Assay

    Characterization of SFV isolates by SDS-PAGE. BHK-21 cells transfected with SFV RNA were pulse-labelled for 15 min and chased for 3 h. Cell lysates were immunoprecipitated with anti-E2 and anti-E1 polyclonal antibodies. (A) Lanes: 1, wild type; 2, SHQL; 3 and 4, two independent revertant isolates, which later turned out to be of type SHQL R64. (B) Reconstructed mutants with the SHQL background. Lanes: 1, SHQL R64; 2, SHQL R64/R4; 3, SHQL RRR; 4, SHQL RRR/R1; 5, SHQL R62.
    Figure Legend Snippet: Characterization of SFV isolates by SDS-PAGE. BHK-21 cells transfected with SFV RNA were pulse-labelled for 15 min and chased for 3 h. Cell lysates were immunoprecipitated with anti-E2 and anti-E1 polyclonal antibodies. (A) Lanes: 1, wild type; 2, SHQL; 3 and 4, two independent revertant isolates, which later turned out to be of type SHQL R64. (B) Reconstructed mutants with the SHQL background. Lanes: 1, SHQL R64; 2, SHQL R64/R4; 3, SHQL RRR; 4, SHQL RRR/R1; 5, SHQL R62.

    Techniques Used: SDS Page, Transfection, Immunoprecipitation

    4) Product Images from "Detection of a Novel Intraneuronal Pool of Insoluble Amyloid ? Protein that Accumulates with Time in Culture "

    Article Title: Detection of a Novel Intraneuronal Pool of Insoluble Amyloid ? Protein that Accumulates with Time in Culture

    Journal: The Journal of Cell Biology

    doi:

    FA extraction of a variety of cell lines reveals the presence of varying levels of insoluble intracellular Aβ. ( A ) Uninfected and SFV-APPwt-infected NT2 cells, NT2N cells (replate 2, 4 wk old), CHO Pro5 cells, CHO-695 cells (uninfected only), and BHK-21 cells were sequentially extracted in RIPA followed by FA. Aβ 1-40 and 1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Means and standard errors (four separate experiments done in triplicate) of Aβ levels are shown. ( B ) Samples from RIPA cell lysates of each of these cell lines (both uninfected and SFV-APPwt infected) were resolved on a 7.5% Tris-glycine acrylamide gel, and immunoblotted with Karen antibody; bands were detected by PhosphorImager after using an I 125 -labeled secondary antibody.
    Figure Legend Snippet: FA extraction of a variety of cell lines reveals the presence of varying levels of insoluble intracellular Aβ. ( A ) Uninfected and SFV-APPwt-infected NT2 cells, NT2N cells (replate 2, 4 wk old), CHO Pro5 cells, CHO-695 cells (uninfected only), and BHK-21 cells were sequentially extracted in RIPA followed by FA. Aβ 1-40 and 1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Means and standard errors (four separate experiments done in triplicate) of Aβ levels are shown. ( B ) Samples from RIPA cell lysates of each of these cell lines (both uninfected and SFV-APPwt infected) were resolved on a 7.5% Tris-glycine acrylamide gel, and immunoblotted with Karen antibody; bands were detected by PhosphorImager after using an I 125 -labeled secondary antibody.

    Techniques Used: Infection, Sandwich ELISA, Acrylamide Gel Assay, Labeling

    5) Product Images from "The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion"

    Article Title: The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.23.12300-12311.2002

    Three-color fusion assay. BHK-21 cells were infected with wild-type (wt) VSV (panels A and B), GS HA virus (panel C), or ΔG virus (panel D) at a multiplicity of infection of 10. After 8 h of infection, the cells were labeled with DiI (red) and calcein-AM (green) and then removed from the plates. The cells were then overlaid on BHK-21 cells that had been labeled with CMAC (blue). After the virus-infected donor cells had attached to the plate or settled onto the target cells, the medium was replaced with fusion medium buffered to pH 5.9 or 7.0. After 1 min, the fusion buffer was removed, and the cells were incubated for 20 min in growth medium at 37 o C and then placed on ice to prevent any further lipid or content mixing. Phase-contrast and fluorescence images were obtained with filters for 4′,6′-diamidino-2-phenylindole (DAPI) (blue), rhodamine (red), and fluorescein isothiocyanate (FITC) (green). The DAPI filter set did not eliminate bleedthrough of the FITC fluorescence, and therefore the FITC-labeled cells appear greenish-blue in the micrographs. The target cells labeled with CMAC appear as larger, flat blue cells, while the donor cells appear as small, round cells and are labeled both red and green. Relevant target cells are highlighted by a thin white outline in panels A, B, and D. (A) Fusion mediated by wild-type G protein at pH 5.9. The phase-contrast image shows one small donor cell adjacent to two target cells. Transfer of both DiI and calcein-AM from the donor cell to the target cell (blue) can be seen (arrows). (B) A second plate identical to the one shown in panel A but maintained at pH 7.0 shows a lack of transfer of DiI or calcein-AM to the outlined target cell, consistent with the requirement for low pH in initiating VSV G-mediated membrane fusion. (C) Fusion mediated by GS HA at pH 7.0. Several donor cells in contact with target cells are shown. Only the transfer of DiI from the donor to the target cells was seen (arrows). There was no transfer of calcein-AM, showing that complete fusion had not occurred. (D) Multiple donor cells infected with ΔG-VSV which are in contact with an outlined blue target cell are shown, but no transfer of either DiI or calcein-AM occurred.
    Figure Legend Snippet: Three-color fusion assay. BHK-21 cells were infected with wild-type (wt) VSV (panels A and B), GS HA virus (panel C), or ΔG virus (panel D) at a multiplicity of infection of 10. After 8 h of infection, the cells were labeled with DiI (red) and calcein-AM (green) and then removed from the plates. The cells were then overlaid on BHK-21 cells that had been labeled with CMAC (blue). After the virus-infected donor cells had attached to the plate or settled onto the target cells, the medium was replaced with fusion medium buffered to pH 5.9 or 7.0. After 1 min, the fusion buffer was removed, and the cells were incubated for 20 min in growth medium at 37 o C and then placed on ice to prevent any further lipid or content mixing. Phase-contrast and fluorescence images were obtained with filters for 4′,6′-diamidino-2-phenylindole (DAPI) (blue), rhodamine (red), and fluorescein isothiocyanate (FITC) (green). The DAPI filter set did not eliminate bleedthrough of the FITC fluorescence, and therefore the FITC-labeled cells appear greenish-blue in the micrographs. The target cells labeled with CMAC appear as larger, flat blue cells, while the donor cells appear as small, round cells and are labeled both red and green. Relevant target cells are highlighted by a thin white outline in panels A, B, and D. (A) Fusion mediated by wild-type G protein at pH 5.9. The phase-contrast image shows one small donor cell adjacent to two target cells. Transfer of both DiI and calcein-AM from the donor cell to the target cell (blue) can be seen (arrows). (B) A second plate identical to the one shown in panel A but maintained at pH 7.0 shows a lack of transfer of DiI or calcein-AM to the outlined target cell, consistent with the requirement for low pH in initiating VSV G-mediated membrane fusion. (C) Fusion mediated by GS HA at pH 7.0. Several donor cells in contact with target cells are shown. Only the transfer of DiI from the donor to the target cells was seen (arrows). There was no transfer of calcein-AM, showing that complete fusion had not occurred. (D) Multiple donor cells infected with ΔG-VSV which are in contact with an outlined blue target cell are shown, but no transfer of either DiI or calcein-AM occurred.

    Techniques Used: Single Vesicle Fusion Assay, Infection, Labeling, Incubation, Fluorescence

    GS enhances membrane fusion induced by G NJ . BHK-21 cells were transfected with plasmids encoding GS HA (A), GS HA and G NJ (B), or G NJ alone (C). At 36 h posttransfection, cells were fixed with 3% paraformaldehyde and stained. The cells were not pretreated with fusion medium, and therefore, the pH was maintained at 7.2 to 7.4 throughout the incubation period. Cells expressing G NJ and G NJ plus GS HA were probed with monoclonal antibody VIII, which is specific for the G NJ glycoprotein. Cells expressing GS HA were stained with the 12CA5 monoclonal antibody, which is specific for the HA epitope. Representative epifluorescence and phase-contrast micrographs of the cells are shown.
    Figure Legend Snippet: GS enhances membrane fusion induced by G NJ . BHK-21 cells were transfected with plasmids encoding GS HA (A), GS HA and G NJ (B), or G NJ alone (C). At 36 h posttransfection, cells were fixed with 3% paraformaldehyde and stained. The cells were not pretreated with fusion medium, and therefore, the pH was maintained at 7.2 to 7.4 throughout the incubation period. Cells expressing G NJ and G NJ plus GS HA were probed with monoclonal antibody VIII, which is specific for the G NJ glycoprotein. Cells expressing GS HA were stained with the 12CA5 monoclonal antibody, which is specific for the HA epitope. Representative epifluorescence and phase-contrast micrographs of the cells are shown.

    Techniques Used: Transfection, Staining, Incubation, Expressing

    Fusion potentiation by GS deletion mutants. BHK-21 cells were cotransfected with plasmids encoding SV5 F and either GFP or the GS truncation mutants that were tagged with the Flag epitope. At 36 h posttransfection, the cells were fixed and probed with the F1a monoclonal antibody to detect SV5 F protein. Rhodamine-labeled goat anti-mouse immunoglobulin antibody was used to visualize the cells. Phase-contrast and epifluorescence images were obtained with a 10× water immersion lens with a Zeiss Axiophot microscope. At least 10 images were obtained for each sample. Representative photomicrographs of cells expressing GS with either 14 amino acids (N449) or 9 amino acids (V454) are shown.
    Figure Legend Snippet: Fusion potentiation by GS deletion mutants. BHK-21 cells were cotransfected with plasmids encoding SV5 F and either GFP or the GS truncation mutants that were tagged with the Flag epitope. At 36 h posttransfection, the cells were fixed and probed with the F1a monoclonal antibody to detect SV5 F protein. Rhodamine-labeled goat anti-mouse immunoglobulin antibody was used to visualize the cells. Phase-contrast and epifluorescence images were obtained with a 10× water immersion lens with a Zeiss Axiophot microscope. At least 10 images were obtained for each sample. Representative photomicrographs of cells expressing GS with either 14 amino acids (N449) or 9 amino acids (V454) are shown.

    Techniques Used: FLAG-tag, Labeling, Microscopy, Expressing

    Effect of GS on SV5 F-mediated membrane fusion. BHK-21 cells were transfected with plasmids encoding SV5 F alone or cotransfected with plasmids encoding SV5 F and GS HA . Cells were fixed with 3% paraformaldehyde at 36 h after transfection and stained for surface expression of SV5 F with the monoclonal antibody F1a, which is specific for F protein. (A) Fusion activity of SV5 F alone. (B) Syncytium formation when SV5 F and GS HA were coexpressed.
    Figure Legend Snippet: Effect of GS on SV5 F-mediated membrane fusion. BHK-21 cells were transfected with plasmids encoding SV5 F alone or cotransfected with plasmids encoding SV5 F and GS HA . Cells were fixed with 3% paraformaldehyde at 36 h after transfection and stained for surface expression of SV5 F with the monoclonal antibody F1a, which is specific for F protein. (A) Fusion activity of SV5 F alone. (B) Syncytium formation when SV5 F and GS HA were coexpressed.

    Techniques Used: Transfection, Staining, Expressing, Activity Assay

    Chlorpromazine rescues GS HA virus infectivity. Equivalent amounts of GS HA -GFP virus, ΔG (DelG)-GFP virus, and VSV-GFP virus were resuspended in binding medium buffered to pH 7.0 and incubated at room temperature for 30 min. Following the incubation, the virus suspension was cooled on ice for 10 min and then added to BHK-21 cells on ice. Following 3 h of binding on ice, the inoculum was removed, and the cells were washed with fresh medium to remove the unbound virus. Cells were then treated with medium containing 0.4 mM CPZ for 1 min. The drug-containing medium was removed immediately, and the cells were incubated in drug-free medium overnight. Twelve hours later, the number of cells expressing GFP was counted. The open boxes show the number of GFP-positive cells in the absence of CPZ, while the hatched boxes indicate the number of GFP-positive cells after CPZ treatment. VSV-GFP infected more than 90% of the cells in the presence or absence of CPZ, and the GFP cell count is not shown.
    Figure Legend Snippet: Chlorpromazine rescues GS HA virus infectivity. Equivalent amounts of GS HA -GFP virus, ΔG (DelG)-GFP virus, and VSV-GFP virus were resuspended in binding medium buffered to pH 7.0 and incubated at room temperature for 30 min. Following the incubation, the virus suspension was cooled on ice for 10 min and then added to BHK-21 cells on ice. Following 3 h of binding on ice, the inoculum was removed, and the cells were washed with fresh medium to remove the unbound virus. Cells were then treated with medium containing 0.4 mM CPZ for 1 min. The drug-containing medium was removed immediately, and the cells were incubated in drug-free medium overnight. Twelve hours later, the number of cells expressing GFP was counted. The open boxes show the number of GFP-positive cells in the absence of CPZ, while the hatched boxes indicate the number of GFP-positive cells after CPZ treatment. VSV-GFP infected more than 90% of the cells in the presence or absence of CPZ, and the GFP cell count is not shown.

    Techniques Used: Infection, Binding Assay, Incubation, Expressing, Cell Counting

    6) Product Images from "Both E Protein Glycans Adversely Affect Dengue Virus Infectivity but Are Beneficial for Virion Release ▿"

    Article Title: Both E Protein Glycans Adversely Affect Dengue Virus Infectivity but Are Beneficial for Virion Release ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01900-09

    Growth properties of loss-of-glycan variants relative to rPUO parent virus in mammalian cells. (A) FACS histograms showing BHK-21 cells electroporated with 1 μg wt or variant RNA transcripts (solid lines) or left untreated (dotted line) and stained
    Figure Legend Snippet: Growth properties of loss-of-glycan variants relative to rPUO parent virus in mammalian cells. (A) FACS histograms showing BHK-21 cells electroporated with 1 μg wt or variant RNA transcripts (solid lines) or left untreated (dotted line) and stained

    Techniques Used: FACS, Variant Assay, Staining

    7) Product Images from "Identification of a Novel Structural Protein of Arteriviruses"

    Article Title: Identification of a Novel Structural Protein of Arteriviruses

    Journal: Journal of Virology

    doi:

    Viral protein synthesis in BHK-21 cells transfected with EAV030-2aKO (2a − ), EAV030-2bKO (2b − ), and wild-type (wt) EAV030 RNA. 35 ), the EAV E protein (see the text), the EAV G S ). The positions of the different structural proteins and nsp2 are displayed at the left. Numbers on the right indicate sizes in kilodaltons. Ab, antibody.
    Figure Legend Snippet: Viral protein synthesis in BHK-21 cells transfected with EAV030-2aKO (2a − ), EAV030-2bKO (2b − ), and wild-type (wt) EAV030 RNA. 35 ), the EAV E protein (see the text), the EAV G S ). The positions of the different structural proteins and nsp2 are displayed at the left. Numbers on the right indicate sizes in kilodaltons. Ab, antibody.

    Techniques Used: Transfection

    Subcellular localization of the EAV E protein. (A) Immunofluorescence analysis of the localization of the EAV E protein in infected BHK-21 cells (8 h p.i.) (left panel) and upon independent expression in the same cells, using the MVA-T7 system and expression vector pAVI02a (7 h p.i.) (right panel). Cells were double labeled for the EAV E protein and either an ER marker (PDI) or a marker of the Golgi complex (the EAV G L glycoprotein in infected cells, or the protein recognized by MAb F20/65-1-4 in transfected cells). In both systems, part of the E protein was seen in the Golgi complex, but most of it colocalized with PDI. Staining of mock-infected cells and MVA-T7-infected, pAVI16-transfected cells with the E protein-specific antiserum did not yield a detectable signal (data not shown). Bar, 25 μm. (B) Immunogold labeling of cryosections of EAV-infected RK-13 cells (8 h p.i.) with the E protein-specific antiserum and protein A coupled to 10-nm-diameter gold particles. A specific but not very abundant labeling of the membranes of the Golgi complex and ER (not shown) was observed. Bar, 100 nm.
    Figure Legend Snippet: Subcellular localization of the EAV E protein. (A) Immunofluorescence analysis of the localization of the EAV E protein in infected BHK-21 cells (8 h p.i.) (left panel) and upon independent expression in the same cells, using the MVA-T7 system and expression vector pAVI02a (7 h p.i.) (right panel). Cells were double labeled for the EAV E protein and either an ER marker (PDI) or a marker of the Golgi complex (the EAV G L glycoprotein in infected cells, or the protein recognized by MAb F20/65-1-4 in transfected cells). In both systems, part of the E protein was seen in the Golgi complex, but most of it colocalized with PDI. Staining of mock-infected cells and MVA-T7-infected, pAVI16-transfected cells with the E protein-specific antiserum did not yield a detectable signal (data not shown). Bar, 25 μm. (B) Immunogold labeling of cryosections of EAV-infected RK-13 cells (8 h p.i.) with the E protein-specific antiserum and protein A coupled to 10-nm-diameter gold particles. A specific but not very abundant labeling of the membranes of the Golgi complex and ER (not shown) was observed. Bar, 100 nm.

    Techniques Used: Immunofluorescence, Infection, Expressing, Plasmid Preparation, Labeling, Marker, Transfection, Staining

    Identification of the E protein in EAV particles. (A) Analysis of pellets obtained after ultracentrifugation through a 20% (wt/wt) sucrose cushion of supernatants from mock- or EAV-infected BHK-21 cells that were labeled with 35 S[Met]- 35 S[Cys]. Pellets were analyzed directly (−) or resuspended and subjected to immunoprecipitation analysis with E protein-specific antiserum (E) or the preimmunization serum (pE). The positions of the EAV E protein (8 kDa), N protein (apparent molecular mass, 14 kDa), M protein (16 kDa), and the small (G S ) and large (G L ) glycoproteins (25 kDa and 30 to 42 kDa, respectively) are shown at the left. The positions and sizes (in kilodaltons) of marker proteins (M r ) analyzed in the same gel are indicated at the right. Ab, antibody. (B) Sucrose density gradient centrifugation of [ 35 S]Met- or [ 35 S]Cys-labeled EAV preparations. The numbers of the gradient fractions and the position of each sample relative to the top and bottom of the centrifuge tube are indicated, as are the positions of the EAV structural proteins E, N, M, G S , and G L . Note that the N protein does not contain any Cys residues.
    Figure Legend Snippet: Identification of the E protein in EAV particles. (A) Analysis of pellets obtained after ultracentrifugation through a 20% (wt/wt) sucrose cushion of supernatants from mock- or EAV-infected BHK-21 cells that were labeled with 35 S[Met]- 35 S[Cys]. Pellets were analyzed directly (−) or resuspended and subjected to immunoprecipitation analysis with E protein-specific antiserum (E) or the preimmunization serum (pE). The positions of the EAV E protein (8 kDa), N protein (apparent molecular mass, 14 kDa), M protein (16 kDa), and the small (G S ) and large (G L ) glycoproteins (25 kDa and 30 to 42 kDa, respectively) are shown at the left. The positions and sizes (in kilodaltons) of marker proteins (M r ) analyzed in the same gel are indicated at the right. Ab, antibody. (B) Sucrose density gradient centrifugation of [ 35 S]Met- or [ 35 S]Cys-labeled EAV preparations. The numbers of the gradient fractions and the position of each sample relative to the top and bottom of the centrifuge tube are indicated, as are the positions of the EAV structural proteins E, N, M, G S , and G L . Note that the N protein does not contain any Cys residues.

    Techniques Used: Infection, Labeling, Immunoprecipitation, Marker, Gradient Centrifugation

    Identification and stability of the EAV E protein in infected cells and upon independent expression in the MVA-T7 system. (A) The left panel shows an immunoprecipitation analysis of lysates of EAV (V)- or mock (M)-infected BHK-21 cells that were 35 S labeled for 60 min at 7 h p.i. Immunoprecipitations were performed with the E protein-specific antiserum (E) or the corresponding preimmunization serum (pE). The positions of the EAV nucleocapsid protein (N), the full-length E protein (E), and a truncated version of the E protein (E i ), which was only observed after a long exposure of the gel, are indicated. The right panel shows the results of a pulse-chase experiment. Cells were pulse labeled for 15 min and subsequently chased for the time periods (in minutes) indicated above the lanes. Protein synthesis during the chase was inhibited by the addition of cycloheximide. The numbers on the right represent the sizes (in kilodaltons) of marker proteins (M r ) run in the same SDS–20% polyacrylamide gel. (B) Corresponding analysis of the independently expressed EAV E protein, using lysates of MVA-T7-infected and pAVI02a (2a)- or pAVI16 (6)-transfected OST-7.1 cells that were labeled at 6 h p.i., as described above for the EAV-infected cells. Ab, antibody.
    Figure Legend Snippet: Identification and stability of the EAV E protein in infected cells and upon independent expression in the MVA-T7 system. (A) The left panel shows an immunoprecipitation analysis of lysates of EAV (V)- or mock (M)-infected BHK-21 cells that were 35 S labeled for 60 min at 7 h p.i. Immunoprecipitations were performed with the E protein-specific antiserum (E) or the corresponding preimmunization serum (pE). The positions of the EAV nucleocapsid protein (N), the full-length E protein (E), and a truncated version of the E protein (E i ), which was only observed after a long exposure of the gel, are indicated. The right panel shows the results of a pulse-chase experiment. Cells were pulse labeled for 15 min and subsequently chased for the time periods (in minutes) indicated above the lanes. Protein synthesis during the chase was inhibited by the addition of cycloheximide. The numbers on the right represent the sizes (in kilodaltons) of marker proteins (M r ) run in the same SDS–20% polyacrylamide gel. (B) Corresponding analysis of the independently expressed EAV E protein, using lysates of MVA-T7-infected and pAVI02a (2a)- or pAVI16 (6)-transfected OST-7.1 cells that were labeled at 6 h p.i., as described above for the EAV-infected cells. Ab, antibody.

    Techniques Used: Infection, Expressing, Immunoprecipitation, Labeling, Pulse Chase, Marker, Transfection

    8) Product Images from "TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection"

    Article Title: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection

    Journal: Viruses

    doi: 10.3390/v10110630

    JEV infection and TIM-1 expression in cell lines. BHK, A549, and 293T cells were infected with JEV NJ2008 (MOI of 0.5) for 40 h. ( A ) Supernatants were collected and the production of progeny virions were determined by a plaque assay titrated on BHK-21 cells. Data are presented as mean ± standard deviation (SD) from three independent experiments; ( B ) Cell lysates were harvested to detect the level of JEV NS5 and TIM-1 protein by Western blot analysis using specific antibodies. One representative experiment out of three is shown. Endogenous TIM-1 expression in A549 and 293T cells was detected by ( C ) Western blot analysis and ( D ) confocal microscopy using an anti-TIM-1 monoclonal antibody. Scale bars, 10 µm.
    Figure Legend Snippet: JEV infection and TIM-1 expression in cell lines. BHK, A549, and 293T cells were infected with JEV NJ2008 (MOI of 0.5) for 40 h. ( A ) Supernatants were collected and the production of progeny virions were determined by a plaque assay titrated on BHK-21 cells. Data are presented as mean ± standard deviation (SD) from three independent experiments; ( B ) Cell lysates were harvested to detect the level of JEV NS5 and TIM-1 protein by Western blot analysis using specific antibodies. One representative experiment out of three is shown. Endogenous TIM-1 expression in A549 and 293T cells was detected by ( C ) Western blot analysis and ( D ) confocal microscopy using an anti-TIM-1 monoclonal antibody. Scale bars, 10 µm.

    Techniques Used: Infection, Expressing, Plaque Assay, Standard Deviation, Western Blot, Confocal Microscopy

    Polymorphisms of TIM-1 are associated with JEV susceptibility to host cells. 293T cells were transfected with an empty vector and plasmids encoding three TIM-1 variants for 24 h. ( A ) Cells were challenged with JEV NJ2008 (MOI of 1), and cell lysates were harvest at 24 h.p.i. JEV NS1 protein and TIM-1 expression were determined by Western blot analysis using a specific monoclonal antibody. One representative experiment out of three is shown; ( B ) cells were fixed and detected by confocal microscopy under permeabilized and unpermeabilized condition. Scale bars, 5 µm; ( C ) supernatants were collected at 24 h.p.i. and 36 h.p.i. and the production of progeny virions was determined by a plaque assay titered on BHK-21 cells. Data are presented as mean ± SD from three independent experiments using t-test. * p
    Figure Legend Snippet: Polymorphisms of TIM-1 are associated with JEV susceptibility to host cells. 293T cells were transfected with an empty vector and plasmids encoding three TIM-1 variants for 24 h. ( A ) Cells were challenged with JEV NJ2008 (MOI of 1), and cell lysates were harvest at 24 h.p.i. JEV NS1 protein and TIM-1 expression were determined by Western blot analysis using a specific monoclonal antibody. One representative experiment out of three is shown; ( B ) cells were fixed and detected by confocal microscopy under permeabilized and unpermeabilized condition. Scale bars, 5 µm; ( C ) supernatants were collected at 24 h.p.i. and 36 h.p.i. and the production of progeny virions was determined by a plaque assay titered on BHK-21 cells. Data are presented as mean ± SD from three independent experiments using t-test. * p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Western Blot, Confocal Microscopy, Plaque Assay

    Knockdown of TIM-1 expression impairs JEV entry and infection. A549 cells were seeded in the 12-well plate, and they were transfected with TIM-1-specific siRNA of different concentrations or control siRNA at 50 nM using lipofectamine RNAiMAX Reagent. At 48 h post-transfection, cells were infected with JEV NJ2008 (MOI = 1) for 24 h. ( A ) Cell lysates were harvested to detect the level of JEV NS1 protein and cellular TIM-1 expression by Western blot analysis, using GAPDH as an internal control. One representative experiment out of three is shown; ( B ) production of progeny virions were determined by titering the supernatants of indicated cells in BHK-21 cells. Data are presented as mean ± SD from three independent experiments; ( C ) cells were transfected with TIM-1 siRNA or control siRNA for 48 h before being challenged with JEV NJ2008 (MOI of 10). At 24 h post-infection, cells were fixed and stained for E glycoprotein (red) and TIM-1 (green), followed by confocal microscopy analysis. Scale bars, 20 µm. A549 cells were transfected with TIM-1 siRNA or control siRNA for 48 h; ( D ) cells were incubated with JEV NJ2008 (MOI of 5) at 4 °C for 30 min and then washed with PBS three times; ( E ) cells were incubated with JEV NJ2008 with a MOI of 5 at 4 °C for 1 h and washed with PBS three times, then shifted to 37 °C for 15 min to allow JEV entry. Cells were treated with proteinase K (1 mg/mL) to remove non-internalized virions. Total RNA was extracted and used for quantification of JEV RNA, the efficiency of TIM-1 silencing and JEV attachment and entry were detected by qRT-PCR. Data are presented as mean ± SD from three independent experiments using t-test. ** p
    Figure Legend Snippet: Knockdown of TIM-1 expression impairs JEV entry and infection. A549 cells were seeded in the 12-well plate, and they were transfected with TIM-1-specific siRNA of different concentrations or control siRNA at 50 nM using lipofectamine RNAiMAX Reagent. At 48 h post-transfection, cells were infected with JEV NJ2008 (MOI = 1) for 24 h. ( A ) Cell lysates were harvested to detect the level of JEV NS1 protein and cellular TIM-1 expression by Western blot analysis, using GAPDH as an internal control. One representative experiment out of three is shown; ( B ) production of progeny virions were determined by titering the supernatants of indicated cells in BHK-21 cells. Data are presented as mean ± SD from three independent experiments; ( C ) cells were transfected with TIM-1 siRNA or control siRNA for 48 h before being challenged with JEV NJ2008 (MOI of 10). At 24 h post-infection, cells were fixed and stained for E glycoprotein (red) and TIM-1 (green), followed by confocal microscopy analysis. Scale bars, 20 µm. A549 cells were transfected with TIM-1 siRNA or control siRNA for 48 h; ( D ) cells were incubated with JEV NJ2008 (MOI of 5) at 4 °C for 30 min and then washed with PBS three times; ( E ) cells were incubated with JEV NJ2008 with a MOI of 5 at 4 °C for 1 h and washed with PBS three times, then shifted to 37 °C for 15 min to allow JEV entry. Cells were treated with proteinase K (1 mg/mL) to remove non-internalized virions. Total RNA was extracted and used for quantification of JEV RNA, the efficiency of TIM-1 silencing and JEV attachment and entry were detected by qRT-PCR. Data are presented as mean ± SD from three independent experiments using t-test. ** p

    Techniques Used: Expressing, Infection, Transfection, Western Blot, Staining, Confocal Microscopy, Incubation, Quantitative RT-PCR

    9) Product Images from "NP and L Proteins of Lymphocytic Choriomeningitis Virus (LCMV) Are Sufficient for Efficient Transcription and Replication of LCMV Genomic RNA Analogs †"

    Article Title: NP and L Proteins of Lymphocytic Choriomeningitis Virus (LCMV) Are Sufficient for Efficient Transcription and Replication of LCMV Genomic RNA Analogs †

    Journal: Journal of Virology

    doi:

    Plasmid-mediated expression of LCMV trans -acting factors. BHK-21 cells (4 × 10 5 ) were infected with vTF7-3 at an MOI of 3 and then transfected with pCITE-NP, pUCIRES-Z, and pGEM-L. Transfected cells were harvested at 24 h p.i. BHK-21 cells (2 × 10 5 ) were infected with LCMV at an MOI of 0.1 PFU/cell and harvested at 48 h p.i. Whole cell extracts were separated by 10, 16, and 6% SDS-PAGE for NP, Z, and L (top, middle, and bottom, respectively). Monoclonal antibody to NP and polyclonal antibody to Z and L were used for detection of LCMV proteins by Western blotting using chemiluminescence. Lanes: 1, cells infected with vTF7-3, 2, cells infected with vTF7-3 and transfected with 0.4 μg of either pCITE-NP, pUCIRES-Z, or pGEM-L; 3, BHK-21 cells infected with vTF7-3 and transfected with 1.2 μg of either pCITE-NP, pUCIRES-Z, or pGEM-L; 4, BHK-21 cells infected with vTF7-3 and cotransfected with 0.4 μg of each of plasmids pCITE-NP, pUCIRES-Z, and pGEM-L; 5, cells infected with LCMV at an MOI of 0.1. Antibodies used are indicated at the right.
    Figure Legend Snippet: Plasmid-mediated expression of LCMV trans -acting factors. BHK-21 cells (4 × 10 5 ) were infected with vTF7-3 at an MOI of 3 and then transfected with pCITE-NP, pUCIRES-Z, and pGEM-L. Transfected cells were harvested at 24 h p.i. BHK-21 cells (2 × 10 5 ) were infected with LCMV at an MOI of 0.1 PFU/cell and harvested at 48 h p.i. Whole cell extracts were separated by 10, 16, and 6% SDS-PAGE for NP, Z, and L (top, middle, and bottom, respectively). Monoclonal antibody to NP and polyclonal antibody to Z and L were used for detection of LCMV proteins by Western blotting using chemiluminescence. Lanes: 1, cells infected with vTF7-3, 2, cells infected with vTF7-3 and transfected with 0.4 μg of either pCITE-NP, pUCIRES-Z, or pGEM-L; 3, BHK-21 cells infected with vTF7-3 and transfected with 1.2 μg of either pCITE-NP, pUCIRES-Z, or pGEM-L; 4, BHK-21 cells infected with vTF7-3 and cotransfected with 0.4 μg of each of plasmids pCITE-NP, pUCIRES-Z, and pGEM-L; 5, cells infected with LCMV at an MOI of 0.1. Antibodies used are indicated at the right.

    Techniques Used: Plasmid Preparation, Expressing, Infection, Transfection, SDS Page, Western Blot

    Rescue of CAT activity by LCMV helper virus correlates with expression of viral NP. (A) Helper virus-dependent rescue of CAT expression. BHK-21 cells were infected with LCMV at an MOI of either 0.1 or 3 and then transfected with 5 μg of in vitro-transcribed LCMVSCAT1 RNA at 0, 12, and 24 p.i. Cells were harvested at 48 h p.i., and extracts for CAT assay were prepared. CAT assay was performed as described in Materials and Methods. Shown is a representative result of four independent experiments. (B) Kinetics of NP expression in LCMV-infected BHK-21 cells. BHK-21 cells were infected with LCMV at an MOI of either 0.1 or 3. Cells were harvested at 12, 24, 36, and 48 h p.i. Uninfected BHK-21 control cells were harvested at 48 h p.i. Similar amounts of whole cell extracts from each sample were analyzed by Western blotting with a monoclonal antibody to NP. Immunoreactivity was detected by chemiluminescence. U, uninfected cells. Numbers at the bottom correspond to time of harvest. Molecular weight standards are indicated on the right. The position of NP is indicated by an arrow on the left. Consistent with previous reports, additional bands corresponding to specific NP fragments could be seen in extracts from LCMV-infected cells.
    Figure Legend Snippet: Rescue of CAT activity by LCMV helper virus correlates with expression of viral NP. (A) Helper virus-dependent rescue of CAT expression. BHK-21 cells were infected with LCMV at an MOI of either 0.1 or 3 and then transfected with 5 μg of in vitro-transcribed LCMVSCAT1 RNA at 0, 12, and 24 p.i. Cells were harvested at 48 h p.i., and extracts for CAT assay were prepared. CAT assay was performed as described in Materials and Methods. Shown is a representative result of four independent experiments. (B) Kinetics of NP expression in LCMV-infected BHK-21 cells. BHK-21 cells were infected with LCMV at an MOI of either 0.1 or 3. Cells were harvested at 12, 24, 36, and 48 h p.i. Uninfected BHK-21 control cells were harvested at 48 h p.i. Similar amounts of whole cell extracts from each sample were analyzed by Western blotting with a monoclonal antibody to NP. Immunoreactivity was detected by chemiluminescence. U, uninfected cells. Numbers at the bottom correspond to time of harvest. Molecular weight standards are indicated on the right. The position of NP is indicated by an arrow on the left. Consistent with previous reports, additional bands corresponding to specific NP fragments could be seen in extracts from LCMV-infected cells.

    Techniques Used: Activity Assay, Expressing, Infection, Transfection, In Vitro, Western Blot, Molecular Weight

    Schematic diagrams and characterization of LCMV genomic RNA analogs. (A) Plasmid pLCMVSCAT1 was constructed by combining, in the order indicated in the schematic, the following elements: the minimum T7 RNA polymerase promoter (T7 P) followed by three extra G's, the 5′ UTR of the LCMV S RNA (nt 1 to 78), the IGR of the LCMV S RNA (nt 1484 to 1694), a DNA encoding the full-length CAT ORF in antisense orientation with respect to the T7 promoter, and the 3′ UTR of the LCMV S RNA (nt 3316 to 3376). (B) Plasmid pLCMVSCAT2 was made by inserting an LCMV-specific 3′ HR followed by T7T into pLCMVSCAT1. Viral sequences were derived from LCMV Arm 5. Nucleotide numbers correspond to those of the S RNA. Numbers in parentheses indicate lengths of cDNA fragments. Restriction enzymes used for cloning are indicated at the bottom. Hatched and striped boxes represent the 3′ ends of the GP and NP, respectively. (C) Northern blot analysis of in vitro and intracellularly synthesized LCMV RNA minigenomes. BHK-21 cells were infected with vTF7-3 at an MOI of 3 PFU/cell and subsequently transfected with 2 μg of pLCMVSCAT2. Total cellular RNA was prepared by using TRI reagent at 24 h after infection with vTF7-3. RNA (5 μg) was analyzed by Northern blotting using a CAT sense riboprobe. LCMVSCAT1 RNA was prepared in vitro transcription with T7 RNA polymerase of Mvn I-digested template pLCMVSCAT1 DNA. Unprocessed and ribozyme-processed RNA species are indicated by arrowheads at the left.
    Figure Legend Snippet: Schematic diagrams and characterization of LCMV genomic RNA analogs. (A) Plasmid pLCMVSCAT1 was constructed by combining, in the order indicated in the schematic, the following elements: the minimum T7 RNA polymerase promoter (T7 P) followed by three extra G's, the 5′ UTR of the LCMV S RNA (nt 1 to 78), the IGR of the LCMV S RNA (nt 1484 to 1694), a DNA encoding the full-length CAT ORF in antisense orientation with respect to the T7 promoter, and the 3′ UTR of the LCMV S RNA (nt 3316 to 3376). (B) Plasmid pLCMVSCAT2 was made by inserting an LCMV-specific 3′ HR followed by T7T into pLCMVSCAT1. Viral sequences were derived from LCMV Arm 5. Nucleotide numbers correspond to those of the S RNA. Numbers in parentheses indicate lengths of cDNA fragments. Restriction enzymes used for cloning are indicated at the bottom. Hatched and striped boxes represent the 3′ ends of the GP and NP, respectively. (C) Northern blot analysis of in vitro and intracellularly synthesized LCMV RNA minigenomes. BHK-21 cells were infected with vTF7-3 at an MOI of 3 PFU/cell and subsequently transfected with 2 μg of pLCMVSCAT2. Total cellular RNA was prepared by using TRI reagent at 24 h after infection with vTF7-3. RNA (5 μg) was analyzed by Northern blotting using a CAT sense riboprobe. LCMVSCAT1 RNA was prepared in vitro transcription with T7 RNA polymerase of Mvn I-digested template pLCMVSCAT1 DNA. Unprocessed and ribozyme-processed RNA species are indicated by arrowheads at the left.

    Techniques Used: Plasmid Preparation, Construct, Derivative Assay, Clone Assay, Northern Blot, In Vitro, Synthesized, Infection, Transfection

    Expression of LCMV minigenome in cells infected with vTF7-3 and transfected with pLCMVSCAT2 and LCMV NP, Z, and L protein expression plasmids. (A) Analysis of CAT activity. BHK-21 cells (4 × 10 5 ) were infected with vTF7-3 at an MOI of 3 PFU/cell and then transfected with various combination of plasmids pLCMVSCAT2, pCITE-NP, pUCIRES-Z, and pGEM-L as indicated in Materials and Methods. Cells were harvested at 24 h p.i. and analyzed for CAT activity by TLC as described in Materials and Methods. + and − indicate presence and the absence of plasmid or vTF7-3. NAc, MAc, and DAc indicate the positions of nonacetylated, monoacetylated, and diacetylated forms, respectively, of chloramphenicol in the TLC; O indicates the origin of the TLC. (B) CAT activities were quantified as described in Materials and Methods and presented as percent conversion from nonacetylated to acetylated forms of chloramphenicol. Values correspond to average ± standard deviation of three independent experiments.
    Figure Legend Snippet: Expression of LCMV minigenome in cells infected with vTF7-3 and transfected with pLCMVSCAT2 and LCMV NP, Z, and L protein expression plasmids. (A) Analysis of CAT activity. BHK-21 cells (4 × 10 5 ) were infected with vTF7-3 at an MOI of 3 PFU/cell and then transfected with various combination of plasmids pLCMVSCAT2, pCITE-NP, pUCIRES-Z, and pGEM-L as indicated in Materials and Methods. Cells were harvested at 24 h p.i. and analyzed for CAT activity by TLC as described in Materials and Methods. + and − indicate presence and the absence of plasmid or vTF7-3. NAc, MAc, and DAc indicate the positions of nonacetylated, monoacetylated, and diacetylated forms, respectively, of chloramphenicol in the TLC; O indicates the origin of the TLC. (B) CAT activities were quantified as described in Materials and Methods and presented as percent conversion from nonacetylated to acetylated forms of chloramphenicol. Values correspond to average ± standard deviation of three independent experiments.

    Techniques Used: Expressing, Infection, Transfection, Activity Assay, Thin Layer Chromatography, Plasmid Preparation, Standard Deviation

    10) Product Images from "Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase ▿"

    Article Title: Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05152-11

    WNK4 and Syk have antagonistic effects on the cell surface expression of CFTR. BHK-21-CFTR cells were transiently transfected with GFP-tagged expression vectors encoding YFP alone (vector), WNK4-wt, Syk-wt, or Syk-kd and then analyzed by different techniques
    Figure Legend Snippet: WNK4 and Syk have antagonistic effects on the cell surface expression of CFTR. BHK-21-CFTR cells were transiently transfected with GFP-tagged expression vectors encoding YFP alone (vector), WNK4-wt, Syk-wt, or Syk-kd and then analyzed by different techniques

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The presence of phosphomimetic mutants of CFTR tyrosine 512 is sufficient to change CFTR cell surface expression. BHK-21 cells were stably transfected with CFTR mutant constructs carrying point mutations in tyrosine 512 so that either a phosphomimetic
    Figure Legend Snippet: The presence of phosphomimetic mutants of CFTR tyrosine 512 is sufficient to change CFTR cell surface expression. BHK-21 cells were stably transfected with CFTR mutant constructs carrying point mutations in tyrosine 512 so that either a phosphomimetic

    Techniques Used: Expressing, Stable Transfection, Transfection, Mutagenesis, Construct

    Expression of WNK4 in mammalian cells increases cell surface expression of CFTR. BHK-21 cells stably expressing human CFTR protein were transfected with GFP-tagged expression vectors encoding GFP alone (vector) or WNK4 wild type (wt) or its kinase-dead
    Figure Legend Snippet: Expression of WNK4 in mammalian cells increases cell surface expression of CFTR. BHK-21 cells stably expressing human CFTR protein were transfected with GFP-tagged expression vectors encoding GFP alone (vector) or WNK4 wild type (wt) or its kinase-dead

    Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation

    CFTR is a substrate for Syk protein kinase and contains a Syk substrate motif in the NBD1 domain. (A) CFTR coimmunoprecipitates with Syk. YFP vector or YFP-Syk-wt was transfected into CFTR-expressing BHK-21 cells and immunoprecipitated with anti-GFP antibodies.
    Figure Legend Snippet: CFTR is a substrate for Syk protein kinase and contains a Syk substrate motif in the NBD1 domain. (A) CFTR coimmunoprecipitates with Syk. YFP vector or YFP-Syk-wt was transfected into CFTR-expressing BHK-21 cells and immunoprecipitated with anti-GFP antibodies.

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Immunoprecipitation

    11) Product Images from "Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells"

    Article Title: Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01113-17

    JEV entry depends on dynamin. (A) Dynasore inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection, cells were lysed to determine viral RNA copy number by RT-qPCR. (B) Dynasore inhibited JEV infection. Cells were pretreated with increasing concentrations of dynasore at 37°C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 0.05). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of dynasore on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by dynasore. Cells were treated with 100 μM dynasore or dimethyl sulfoxide (DMSO) for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (E and F) The inhibitory effect of the dynamin DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the GFP-tagged dynamin WT or DN construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 37°C. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Transfected BHK-21 cells were infected with JEV (MOI of 0.1); at 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were screened for JEV infection, and values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P
    Figure Legend Snippet: JEV entry depends on dynamin. (A) Dynasore inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection, cells were lysed to determine viral RNA copy number by RT-qPCR. (B) Dynasore inhibited JEV infection. Cells were pretreated with increasing concentrations of dynasore at 37°C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 0.05). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of dynasore on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by dynasore. Cells were treated with 100 μM dynasore or dimethyl sulfoxide (DMSO) for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (E and F) The inhibitory effect of the dynamin DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the GFP-tagged dynamin WT or DN construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 37°C. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Transfected BHK-21 cells were infected with JEV (MOI of 0.1); at 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were screened for JEV infection, and values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P

    Techniques Used: Binding Assay, Quantitative RT-PCR, Infection, Viability Assay, Incubation, Labeling, Staining, Fluorescence, Software, Construct, Confocal Microscopy, Transfection

    Effects of EPS15 Δ95/295 overexpression and chlorpromazine on JEV infection. (A) Chlorpromazine (CPZ) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection, cells were lysed to determine viral RNA copy number by RT-qPCR. (B) Chlorpromazine (CPZ) inhibited JEV infection. Cells were pretreated with increasing concentrations of CPZ for 1 h at 37°C, the medium was replaced, and then cells were infected with JEV (MOI of 0.05). At 24 hpi, cells were lysed to quantitate viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of CPZ drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by CPZ. Cells were treated with CPZ or DMSO for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (E and F) The inhibitory effect of the EPS15 DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the EPS15 WT or DN plasmid construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C and transferred to 37°C for 30 min. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Cells transfected with the EPS15 WT or DN plasmid construct were infected with JEV (MOI of 0.1). At 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were counted and scored as positive or negative for JEV infection. Values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P
    Figure Legend Snippet: Effects of EPS15 Δ95/295 overexpression and chlorpromazine on JEV infection. (A) Chlorpromazine (CPZ) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection, cells were lysed to determine viral RNA copy number by RT-qPCR. (B) Chlorpromazine (CPZ) inhibited JEV infection. Cells were pretreated with increasing concentrations of CPZ for 1 h at 37°C, the medium was replaced, and then cells were infected with JEV (MOI of 0.05). At 24 hpi, cells were lysed to quantitate viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of CPZ drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by CPZ. Cells were treated with CPZ or DMSO for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (E and F) The inhibitory effect of the EPS15 DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the EPS15 WT or DN plasmid construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C and transferred to 37°C for 30 min. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Cells transfected with the EPS15 WT or DN plasmid construct were infected with JEV (MOI of 0.1). At 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were counted and scored as positive or negative for JEV infection. Values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P

    Techniques Used: Over Expression, Infection, Binding Assay, Quantitative RT-PCR, Viability Assay, Incubation, Labeling, Staining, Fluorescence, Software, Construct, Confocal Microscopy, Transfection, Plasmid Preparation

    Effects of microtubules and actin cytoskeleton on JEV infection. (A and C) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide at 37°C for 1 h, the medium was replaced, and then cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection at 37°C, cells were lysed to determine viral RNA copy number by RT-qPCR. (B and D) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide at 37°C for 1 h and then inoculated with JEV (MOI of 0.05) at 37°C for 1 h. At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. The horizontal lines show results of subtoxic doses of the two drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of data from three independent experiments. **, P
    Figure Legend Snippet: Effects of microtubules and actin cytoskeleton on JEV infection. (A and C) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide at 37°C for 1 h, the medium was replaced, and then cells were inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection at 37°C, cells were lysed to determine viral RNA copy number by RT-qPCR. (B and D) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide at 37°C for 1 h and then inoculated with JEV (MOI of 0.05) at 37°C for 1 h. At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. The horizontal lines show results of subtoxic doses of the two drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of data from three independent experiments. **, P

    Techniques Used: Infection, Binding Assay, Quantitative RT-PCR, Viability Assay

    Role of macropinocytosis on JEV binding, entry, and infection. Cells were pretreated with subtoxic doses of EIPA or wortmannin, as indicated, for 1 h at 37°C. Drugs were present in the medium during the adsorption period. BHK-21 cells were infected with JEV (MOI of 5) (A and D) or DF-1 cells were infected with NDV (MOI = 5) (C and F) at 4°C for 1 h and then at 37°C for 0 h (binding) or 1 h (entry); the infected cells were lysed to determine viral RNA copy number by RT-qPCR. (B and E) cells were infected with JEV (MOI of 0.05) at 37°C; at 24 hpi, viral RNA copy number was determined by RT-qPCR. The horizontal lines show results of subtoxic doses of EIPA and wortmannin on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of three independent experiments. **, P
    Figure Legend Snippet: Role of macropinocytosis on JEV binding, entry, and infection. Cells were pretreated with subtoxic doses of EIPA or wortmannin, as indicated, for 1 h at 37°C. Drugs were present in the medium during the adsorption period. BHK-21 cells were infected with JEV (MOI of 5) (A and D) or DF-1 cells were infected with NDV (MOI = 5) (C and F) at 4°C for 1 h and then at 37°C for 0 h (binding) or 1 h (entry); the infected cells were lysed to determine viral RNA copy number by RT-qPCR. (B and E) cells were infected with JEV (MOI of 0.05) at 37°C; at 24 hpi, viral RNA copy number was determined by RT-qPCR. The horizontal lines show results of subtoxic doses of EIPA and wortmannin on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of three independent experiments. **, P

    Techniques Used: Binding Assay, Infection, Adsorption, Quantitative RT-PCR, Viability Assay

    Colocalization of Rab with JEV during the early stage of infection. BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals, monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4), rabbit anti-Rab5, rabbit anti-Rab7, rabbit anti-Rab9, or rabbit anti-Rab11 antibody and examined by confocal microscopy. Bar, 10 μm.
    Figure Legend Snippet: Colocalization of Rab with JEV during the early stage of infection. BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals, monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4), rabbit anti-Rab5, rabbit anti-Rab7, rabbit anti-Rab9, or rabbit anti-Rab11 antibody and examined by confocal microscopy. Bar, 10 μm.

    Techniques Used: Infection, Staining, Confocal Microscopy

    JEV entry and infection require acidic endosomal pH. (A, C, and E) Chloroquine, NH 4 Cl, and bafilomycin A1 (Baf A1) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h and inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection at 37°C, cells were lysed to determine viral RNA copy number by RT-qPCR. (B, D, and F) Chloroquine, NH 4 Cl, and bafilomycin A1 inhibit JEV infection. Cells were pretreated with subtoxic doses at 37°C for 1 h and then inoculated with JEV (MOI of 0.05) at 37°C for 1 h. At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Horizontal lines show results of subtoxic doses of these three drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (G) Effect of V-ATPase knockdown on JEV infectivity was determined by Western blotting. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05). At 24 hpi the expression of V-ATPase or JEV NS5 was probed with anti-V-ATPase or anti-JEV NS5 antibody as indicated. (H) V-ATPase knockdown inhibited JEV infection. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05 or 0.01). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. *, P
    Figure Legend Snippet: JEV entry and infection require acidic endosomal pH. (A, C, and E) Chloroquine, NH 4 Cl, and bafilomycin A1 (Baf A1) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h and inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection at 37°C, cells were lysed to determine viral RNA copy number by RT-qPCR. (B, D, and F) Chloroquine, NH 4 Cl, and bafilomycin A1 inhibit JEV infection. Cells were pretreated with subtoxic doses at 37°C for 1 h and then inoculated with JEV (MOI of 0.05) at 37°C for 1 h. At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Horizontal lines show results of subtoxic doses of these three drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (G) Effect of V-ATPase knockdown on JEV infectivity was determined by Western blotting. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05). At 24 hpi the expression of V-ATPase or JEV NS5 was probed with anti-V-ATPase or anti-JEV NS5 antibody as indicated. (H) V-ATPase knockdown inhibited JEV infection. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05 or 0.01). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. *, P

    Techniques Used: Infection, Binding Assay, Quantitative RT-PCR, Viability Assay, Western Blot, Transfection, Expressing

    Effects of MβCD on JEV infection. (A) Cell viability upon MβCD treatment was assessed as described previously. (B) Cells were treated with 5 mM MβCD for 1 h and then infected with JEV (MOI of 0.5) in the presence of the inhibitor. At 24 hpi, cells were fixed and stained with an anti-JEV E antibody. Bar, 10 μm. (C)For cholesterol depletion of cell membrane, cells were pretreated with the indicated concentrations of MβCD for 1 h at 37°C, washed twice with medium, infected with JEV (MOI of 0.05) at 37°C for 1 h, washed twice with medium, and then incubated with fresh medium at 37°C for 8 or 24 h. (D) MβCD was mixed with JEV (MOI of 0.05) and incubated at 37°C for 1 h. BHK-21 cells were infected with the treated JEV at 37°C for 1 h. The treated virus was subjected to ultracentrifugation through a 20% sucrose cushion at 70,000 × g for 2 h to remove MβCD, resuspended in medium containing 10% FCS, and filtered before being used to infect cells at 37°C for 8 or 24 h. (E) Cells were infected with JEV (MOI of 0.05) for 8 and 24 h and then treated with the indicated concentrations of MβCD. The medium was replaced, and cells were then incubated for 12 h at 37°C. All infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. **, P
    Figure Legend Snippet: Effects of MβCD on JEV infection. (A) Cell viability upon MβCD treatment was assessed as described previously. (B) Cells were treated with 5 mM MβCD for 1 h and then infected with JEV (MOI of 0.5) in the presence of the inhibitor. At 24 hpi, cells were fixed and stained with an anti-JEV E antibody. Bar, 10 μm. (C)For cholesterol depletion of cell membrane, cells were pretreated with the indicated concentrations of MβCD for 1 h at 37°C, washed twice with medium, infected with JEV (MOI of 0.05) at 37°C for 1 h, washed twice with medium, and then incubated with fresh medium at 37°C for 8 or 24 h. (D) MβCD was mixed with JEV (MOI of 0.05) and incubated at 37°C for 1 h. BHK-21 cells were infected with the treated JEV at 37°C for 1 h. The treated virus was subjected to ultracentrifugation through a 20% sucrose cushion at 70,000 × g for 2 h to remove MβCD, resuspended in medium containing 10% FCS, and filtered before being used to infect cells at 37°C for 8 or 24 h. (E) Cells were infected with JEV (MOI of 0.05) for 8 and 24 h and then treated with the indicated concentrations of MβCD. The medium was replaced, and cells were then incubated for 12 h at 37°C. All infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. **, P

    Techniques Used: Infection, Staining, Incubation, Quantitative RT-PCR

    Caveolae are not required for JEV entry. The effect of dominant negative caveolin on CTB uptake or JEV infection was determined by confocal microscopy. (A and B) BHK-21 cells transfected with plasmid constructs expressing GFP-tagged WT and DN caveolin were incubated with 10 μg/ml Alexa Fluor 568-labeled CTB for 30 min at 4°C and then transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. (B) Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. CTB uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (C) Transfected cells were infected with JEV (MOI of 0.1). At 24 hpi, cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (D) At least 300 transfected cells were scored as positive or negative for JEV infection; values are expressed as percentages of the number of infected cells observed in the control experiment. (E and F) Effect of caveolin depletion on JEV propagation. Cells transfected with the indicated siRNAs were infected with JEV (MOI of 0.05) and incubated for 24 h to allow virus propagation. (E) The silencing efficiency of siCav and the level of JEV NS5 were analyzed using anti-caveolin-1 antibody or anti-JEV NS5 monoclonal antibody. (F) Infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. (G) BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4) and anti-caveolin-1 antibodies and examined by confocal microscopy. Bar, 10 μm. **, P
    Figure Legend Snippet: Caveolae are not required for JEV entry. The effect of dominant negative caveolin on CTB uptake or JEV infection was determined by confocal microscopy. (A and B) BHK-21 cells transfected with plasmid constructs expressing GFP-tagged WT and DN caveolin were incubated with 10 μg/ml Alexa Fluor 568-labeled CTB for 30 min at 4°C and then transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. (B) Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. CTB uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (C) Transfected cells were infected with JEV (MOI of 0.1). At 24 hpi, cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (D) At least 300 transfected cells were scored as positive or negative for JEV infection; values are expressed as percentages of the number of infected cells observed in the control experiment. (E and F) Effect of caveolin depletion on JEV propagation. Cells transfected with the indicated siRNAs were infected with JEV (MOI of 0.05) and incubated for 24 h to allow virus propagation. (E) The silencing efficiency of siCav and the level of JEV NS5 were analyzed using anti-caveolin-1 antibody or anti-JEV NS5 monoclonal antibody. (F) Infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. (G) BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4) and anti-caveolin-1 antibodies and examined by confocal microscopy. Bar, 10 μm. **, P

    Techniques Used: Dominant Negative Mutation, CtB Assay, Infection, Confocal Microscopy, Transfection, Plasmid Preparation, Construct, Expressing, Incubation, Labeling, Staining, Fluorescence, Software, Quantitative RT-PCR

    12) Product Images from "Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology"

    Article Title: Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.16.8011-8018.2002

    Cell-rounding phenotype of the M33,51A mutant. BHK-21 cells were infected with either WT virus or the M33,51A mutant at an MOI of 10. At each time point indicated, the medium was removed and cells were fixed with 3% paraformaldehyde. Cells were observed by phase-contrast microscopy using a Zeiss Axiophot microscope with a 10× objective, and images were captured using a Zeiss Axiocam digital camera and Axiovision software.
    Figure Legend Snippet: Cell-rounding phenotype of the M33,51A mutant. BHK-21 cells were infected with either WT virus or the M33,51A mutant at an MOI of 10. At each time point indicated, the medium was removed and cells were fixed with 3% paraformaldehyde. Cells were observed by phase-contrast microscopy using a Zeiss Axiophot microscope with a 10× objective, and images were captured using a Zeiss Axiocam digital camera and Axiovision software.

    Techniques Used: Mutagenesis, Infection, Microscopy, Software

    M2 and M3 proteins are made independently of M1 protein. (A) Schematic diagram showing mutations in the M gene. The positions of the first three methionines are shown. XXX represents three consecutive stop codons, which were introduced downstream of the first AUG (M1SC) or the first and second AUGs (M2SC). (B) Transient expression of M1, M2, and M3. Approximately 5 × 10 5 BHK-21 cells were first infected with a recombinant vaccinia virus expressing T7 polymerase and then transfected with 2.5 μg of either pBS-M, containing the WT M cDNA, or one of the mutant constructs. At 24 h p.t., cells were lysed in a detergent buffer. M-specific proteins in the cell lysates were detected by Western blotting. Cell extracts from a WT virus-infected cell were used as a positive control (lane 5).
    Figure Legend Snippet: M2 and M3 proteins are made independently of M1 protein. (A) Schematic diagram showing mutations in the M gene. The positions of the first three methionines are shown. XXX represents three consecutive stop codons, which were introduced downstream of the first AUG (M1SC) or the first and second AUGs (M2SC). (B) Transient expression of M1, M2, and M3. Approximately 5 × 10 5 BHK-21 cells were first infected with a recombinant vaccinia virus expressing T7 polymerase and then transfected with 2.5 μg of either pBS-M, containing the WT M cDNA, or one of the mutant constructs. At 24 h p.t., cells were lysed in a detergent buffer. M-specific proteins in the cell lysates were detected by Western blotting. Cell extracts from a WT virus-infected cell were used as a positive control (lane 5).

    Techniques Used: Expressing, Infection, Recombinant, Transfection, Mutagenesis, Construct, Western Blot, Positive Control

    Cell-rounding phenotypes of mutant viruses. BHK-21 cells were infected with either WT or mutant viruses at an MOI of 10. At the indicated time points, cells were fixed and observed by phase-contrast microscopy (magnification, ×125).
    Figure Legend Snippet: Cell-rounding phenotypes of mutant viruses. BHK-21 cells were infected with either WT or mutant viruses at an MOI of 10. At the indicated time points, cells were fixed and observed by phase-contrast microscopy (magnification, ×125).

    Techniques Used: Mutagenesis, Infection, Microscopy

    Expression of M2 and M3 proteins during WT VSV infection. (A) Cells were infected with WT virus (rVSV-GFP) at an MOI of 10. At 8 h postinfection, the supernatant was harvested and the virus was concentrated by centrifugation. The cells were then lysed in a detergent buffer. An aliquot of pelleted virus and cell lysate was separated on an SDS-10% polyacrylamide gel, and the M proteins were detected by Western blotting using an M-specific monoclonal antibody (23H12). (B) BHK-21, D17, HeLa, and QT6 cells were infected with WT virus at an MOI of 10. At 8 h postinfection, cells were radioactively labeled with [ 35 S]methionine for 1 h. Cell extracts were made, and proteins were immunoprecipitated by using monoclonal antibody 23H12. Immunoprecipitated proteins were analyzed on an SDS-10% polyacrylamide gel followed by autoradiography.
    Figure Legend Snippet: Expression of M2 and M3 proteins during WT VSV infection. (A) Cells were infected with WT virus (rVSV-GFP) at an MOI of 10. At 8 h postinfection, the supernatant was harvested and the virus was concentrated by centrifugation. The cells were then lysed in a detergent buffer. An aliquot of pelleted virus and cell lysate was separated on an SDS-10% polyacrylamide gel, and the M proteins were detected by Western blotting using an M-specific monoclonal antibody (23H12). (B) BHK-21, D17, HeLa, and QT6 cells were infected with WT virus at an MOI of 10. At 8 h postinfection, cells were radioactively labeled with [ 35 S]methionine for 1 h. Cell extracts were made, and proteins were immunoprecipitated by using monoclonal antibody 23H12. Immunoprecipitated proteins were analyzed on an SDS-10% polyacrylamide gel followed by autoradiography.

    Techniques Used: Expressing, Infection, Centrifugation, Western Blot, Labeling, Immunoprecipitation, Autoradiography

    Virus yield and growth kinetics of the M33,51A mutant. (A) BHK-21 cells were infected with either the WT or M33,51A virus at an MOI of 10. Cells were continuously labeled from 7 to 15 h postinfection with [ 35 S]methionine. At 15 h postinfection, viruses were harvested from the supernatants by centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The positions of VSV proteins are indicated on the left. (B) The growth kinetics of the M33,51A mutant was compared with that of WT virus by taking aliquots of the supernatant at various times postinfection and determining the virus titer by a standard plaque assay on BHK cells.
    Figure Legend Snippet: Virus yield and growth kinetics of the M33,51A mutant. (A) BHK-21 cells were infected with either the WT or M33,51A virus at an MOI of 10. Cells were continuously labeled from 7 to 15 h postinfection with [ 35 S]methionine. At 15 h postinfection, viruses were harvested from the supernatants by centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The positions of VSV proteins are indicated on the left. (B) The growth kinetics of the M33,51A mutant was compared with that of WT virus by taking aliquots of the supernatant at various times postinfection and determining the virus titer by a standard plaque assay on BHK cells.

    Techniques Used: Mutagenesis, Infection, Labeling, Centrifugation, Polyacrylamide Gel Electrophoresis, Autoradiography, Plaque Assay

    Transient expression of M2 and M3 causes cell rounding. BHK-21 cells were transfected with plasmids encoding either M1 (WT M) (A), M2 and M3 (M1SC) (B), M3 (M2SC) (C and D), or VSV nucleocapsid (N) protein (E and F). Cells were stained with either the M-specific monoclonal antibody 23H12 (A, B, and C), or an N-specific antibody (E). Fluorescence (A, B, C, and E) and phase-contrast (D and F) images were captured using a Zeiss Axiophot microscope with a 40× objective and a Zeiss Axiocam digital camera and associated Axiovision software.
    Figure Legend Snippet: Transient expression of M2 and M3 causes cell rounding. BHK-21 cells were transfected with plasmids encoding either M1 (WT M) (A), M2 and M3 (M1SC) (B), M3 (M2SC) (C and D), or VSV nucleocapsid (N) protein (E and F). Cells were stained with either the M-specific monoclonal antibody 23H12 (A, B, and C), or an N-specific antibody (E). Fluorescence (A, B, C, and E) and phase-contrast (D and F) images were captured using a Zeiss Axiophot microscope with a 40× objective and a Zeiss Axiocam digital camera and associated Axiovision software.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence, Microscopy, Software

    13) Product Images from "Two Distinct Sets of NS2A Molecules Are Responsible for Dengue Virus RNA Synthesis and Virion Assembly"

    Article Title: Two Distinct Sets of NS2A Molecules Are Responsible for Dengue Virus RNA Synthesis and Virion Assembly

    Journal: Journal of Virology

    doi: 10.1128/JVI.02882-14

    Characterization of class I mutants. (A) IFA. BHK-21 cells were electroporated with DENV-2 WT strain New Guinea C or individual NS2A mutant genome-length RNAs. From days 1 to 4 p.t., E protein was probed with the 4G2 antibody. Blue and green, nucleus
    Figure Legend Snippet: Characterization of class I mutants. (A) IFA. BHK-21 cells were electroporated with DENV-2 WT strain New Guinea C or individual NS2A mutant genome-length RNAs. From days 1 to 4 p.t., E protein was probed with the 4G2 antibody. Blue and green, nucleus

    Techniques Used: Immunofluorescence, Mutagenesis

    Characterization of class II mutants. (A) IFA. BHK-21 cells were electroporated with WT, D125A, or G200A genome-length RNAs. E-protein expression was monitored by the use of MAb 4G2 (green). (B) Plaque morphologies. (C) Chromatography of viral genome
    Figure Legend Snippet: Characterization of class II mutants. (A) IFA. BHK-21 cells were electroporated with WT, D125A, or G200A genome-length RNAs. E-protein expression was monitored by the use of MAb 4G2 (green). (B) Plaque morphologies. (C) Chromatography of viral genome

    Techniques Used: Immunofluorescence, Expressing, Chromatography

    trans -Complementation analysis using NS2A (G200A)-EGFP BHK-21 cells. (A) Characterization of NS2A (G200A)-EGFP BHK-21 cells by IFA. EGFP fusion proteins probed by a mouse anti-EGFP IgG (green) are shown. (B) Western blot analysis. EGFP fusion proteins
    Figure Legend Snippet: trans -Complementation analysis using NS2A (G200A)-EGFP BHK-21 cells. (A) Characterization of NS2A (G200A)-EGFP BHK-21 cells by IFA. EGFP fusion proteins probed by a mouse anti-EGFP IgG (green) are shown. (B) Western blot analysis. EGFP fusion proteins

    Techniques Used: Immunofluorescence, Western Blot

    trans -Complementation analysis using BHK-21 Rep cells. (A) Flowchart of trans -complementation analysis. (B) IFA analysis of transfected cells. BHK-21 Rep cells were electroporated with equal amounts (10 μg) of WT or individual mutant genome-length
    Figure Legend Snippet: trans -Complementation analysis using BHK-21 Rep cells. (A) Flowchart of trans -complementation analysis. (B) IFA analysis of transfected cells. BHK-21 Rep cells were electroporated with equal amounts (10 μg) of WT or individual mutant genome-length

    Techniques Used: Immunofluorescence, Transfection, Mutagenesis

    Characterization of class III mutants. (A) After electroporation of BHK-21 cells with WT or individual class III mutant genome-length RNAs on days 1 to 4 p.t., E protein was monitored by use of the 4G2 antibody (green). Representative images are shown.
    Figure Legend Snippet: Characterization of class III mutants. (A) After electroporation of BHK-21 cells with WT or individual class III mutant genome-length RNAs on days 1 to 4 p.t., E protein was monitored by use of the 4G2 antibody (green). Representative images are shown.

    Techniques Used: Electroporation, Mutagenesis

    trans -Complementation analysis using NS2A-EGFP BHK-21 cells. (A) NS2A-EGFP BHK-21 cells were electroporated with equal amounts (10 μg) of WT or individual mutant genome-length RNAs. From days 1 to 4 p.t., E proteins were detected using MAb 4G2,
    Figure Legend Snippet: trans -Complementation analysis using NS2A-EGFP BHK-21 cells. (A) NS2A-EGFP BHK-21 cells were electroporated with equal amounts (10 μg) of WT or individual mutant genome-length RNAs. From days 1 to 4 p.t., E proteins were detected using MAb 4G2,

    Techniques Used: Mutagenesis

    Selection of the NS2A-EGFP BHK-21 cell line. (A) Schematic diagram for selection of the NS2A-EGFP BHK-21 cell line. (B) Fluorescence microscopy analysis. EGFP signals were observed under a fluorescence microscope with a ×20 or a ×63 objective
    Figure Legend Snippet: Selection of the NS2A-EGFP BHK-21 cell line. (A) Schematic diagram for selection of the NS2A-EGFP BHK-21 cell line. (B) Fluorescence microscopy analysis. EGFP signals were observed under a fluorescence microscope with a ×20 or a ×63 objective

    Techniques Used: Selection, Fluorescence, Microscopy

    14) Product Images from "Mx Is Not Responsible for the Antiviral Activity of Interferon-α against Japanese Encephalitis Virus"

    Article Title: Mx Is Not Responsible for the Antiviral Activity of Interferon-α against Japanese Encephalitis Virus

    Journal: Viruses

    doi: 10.3390/v9010005

    Mx1 or Mx2 overexpression has no impact on JEV replication in cells treated with Brefeldin A (BFA). GFP-poMx1 (or poMx2)-overexpressing BHK-21 cells and control cells were infected with JEV at an MOI of 0.05. At 12 hpi, BFA (5 μg/mL) was added to the culture medium for an additional 12 h. The lysates of cell culture were used to determine the viral protein levels by Western blot analysis, and cell supernatants were used to determine the levels of infectious virus by plaque assay. Western blot analysis ( A ) and virus titer ( B ) of infectious virus of GFP-poMx1-overexpressing infected cells treated with or without BFA. Alternatively, Western blot analysis ( C ) and virus titer ( D ) of infectious virus of the GFP-poMx2-overexpressing infected cells treated with or without BFA. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ns ( p > 0.05) and ** ( p
    Figure Legend Snippet: Mx1 or Mx2 overexpression has no impact on JEV replication in cells treated with Brefeldin A (BFA). GFP-poMx1 (or poMx2)-overexpressing BHK-21 cells and control cells were infected with JEV at an MOI of 0.05. At 12 hpi, BFA (5 μg/mL) was added to the culture medium for an additional 12 h. The lysates of cell culture were used to determine the viral protein levels by Western blot analysis, and cell supernatants were used to determine the levels of infectious virus by plaque assay. Western blot analysis ( A ) and virus titer ( B ) of infectious virus of GFP-poMx1-overexpressing infected cells treated with or without BFA. Alternatively, Western blot analysis ( C ) and virus titer ( D ) of infectious virus of the GFP-poMx2-overexpressing infected cells treated with or without BFA. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ns ( p > 0.05) and ** ( p

    Techniques Used: Over Expression, Infection, Cell Culture, Western Blot, Plaque Assay, Standard Deviation

    JEV replication in pEGFP-poMx1 or pEGFP-poMx2-transfected BHK-21 cells. Transfected cells grown on glass coverslips were infected with JEV at an MOI of 0.05. At 24 hpi, cells were washed with PBS and subjected to immunofluorescence assay. The cell nucleus was counterstained with DAPI (blue). White arrows indicate cells with high GFP-poMx1 or GFP-poMx2 expression levels along with high JEV viral proteins (NS1, NS5, or E).
    Figure Legend Snippet: JEV replication in pEGFP-poMx1 or pEGFP-poMx2-transfected BHK-21 cells. Transfected cells grown on glass coverslips were infected with JEV at an MOI of 0.05. At 24 hpi, cells were washed with PBS and subjected to immunofluorescence assay. The cell nucleus was counterstained with DAPI (blue). White arrows indicate cells with high GFP-poMx1 or GFP-poMx2 expression levels along with high JEV viral proteins (NS1, NS5, or E).

    Techniques Used: Transfection, Infection, Immunofluorescence, Expressing

    IFNα inhibits JEV replication in Mx-knockdown BHK-21 cells. Cells were transfected with siRNA targeting Mx1, Mx2, and a siRNA control (siCtrl). Six hours after transfection cells were treated with huIFNα for 12 h, then infected with JEV at an MOI of 0.05. At 24 hpi, cell supernatants were used to determine the levels of infectious virus by plaque assay, and the cell culture lysates were used to determine the viral protein levels by Western blot analysis. Percent knockdown of ( A ) Mx1 and ( C ) Mx2. JEV replication determined by plaque assay and Western blot analysis in ( B ) siMx1- or ( D ) siMx2-transfected and control cells. Quantification of the blotted proteins was performed using Image J software. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ns ( p > 0.05) and ** ( p
    Figure Legend Snippet: IFNα inhibits JEV replication in Mx-knockdown BHK-21 cells. Cells were transfected with siRNA targeting Mx1, Mx2, and a siRNA control (siCtrl). Six hours after transfection cells were treated with huIFNα for 12 h, then infected with JEV at an MOI of 0.05. At 24 hpi, cell supernatants were used to determine the levels of infectious virus by plaque assay, and the cell culture lysates were used to determine the viral protein levels by Western blot analysis. Percent knockdown of ( A ) Mx1 and ( C ) Mx2. JEV replication determined by plaque assay and Western blot analysis in ( B ) siMx1- or ( D ) siMx2-transfected and control cells. Quantification of the blotted proteins was performed using Image J software. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ns ( p > 0.05) and ** ( p

    Techniques Used: Transfection, Infection, Plaque Assay, Cell Culture, Western Blot, Software, Standard Deviation

    Antiviral activity of BFA. ( A ) Cytotoxic effect of BFA. BHK-21 cells at 80% confluence in 24-well plates were treated with various concentrations of BFA for 24 h. After treatment, a cell proliferation reagent was added to each well, and 2 h later, the absorbance at 490 nm was recorded. ( B – E ) BHK-21 cells were seeded into six-well plates and infected with JEV at an MOI of 0.05. After virus adsorption and washing, cells were maintained in medium containing BFA at various concentrations or an equivalent volume of DMSO. At 24 hpi, cell culture lysates were used to determine viral protein levels by Western blot analysis, cell supernatants were used to determine levels of infectious virus by plaque assay ( B ); Infected cells were maintained in medium containing BFA at 5 μg/mL or an equivalent volume of DMSO. At 13, 15, 18, and 24 hpi, cell culture lysates were used to determine viral protein levels by Western blot analysis ( C ); Cell supernatants were used to determine levels of infectious virus by plaque assay ( D ); The inhibitory effect of BFA was detected using anti-JEV NS5 or E mAbs by confocal microscopy ( E ). JEV was strained with red fluorescence, and nucleus was strained with DAPI. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ** ( p
    Figure Legend Snippet: Antiviral activity of BFA. ( A ) Cytotoxic effect of BFA. BHK-21 cells at 80% confluence in 24-well plates were treated with various concentrations of BFA for 24 h. After treatment, a cell proliferation reagent was added to each well, and 2 h later, the absorbance at 490 nm was recorded. ( B – E ) BHK-21 cells were seeded into six-well plates and infected with JEV at an MOI of 0.05. After virus adsorption and washing, cells were maintained in medium containing BFA at various concentrations or an equivalent volume of DMSO. At 24 hpi, cell culture lysates were used to determine viral protein levels by Western blot analysis, cell supernatants were used to determine levels of infectious virus by plaque assay ( B ); Infected cells were maintained in medium containing BFA at 5 μg/mL or an equivalent volume of DMSO. At 13, 15, 18, and 24 hpi, cell culture lysates were used to determine viral protein levels by Western blot analysis ( C ); Cell supernatants were used to determine levels of infectious virus by plaque assay ( D ); The inhibitory effect of BFA was detected using anti-JEV NS5 or E mAbs by confocal microscopy ( E ). JEV was strained with red fluorescence, and nucleus was strained with DAPI. All data are presented as means ± standard deviation (S.D.) as indicated. Statistical significance is indicated as ** ( p

    Techniques Used: Activity Assay, Infection, Adsorption, Cell Culture, Western Blot, Plaque Assay, Confocal Microscopy, Fluorescence, Standard Deviation

    15) Product Images from "Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation"

    Article Title: Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.17.7828-7839.2001

    NaSal and aspirin inhibit flavivirus replication in BHK-21 and N18 cells. (A) BHK-21 cells were infected by JEV at an MO1 of 5, and at 1 h postinfection, the infected cells were treated with varying concentrations of NaSal (SA) or aspirin (ASA). After a 48-h incubation at 37°C, the virus titers (expressed as PFU per milliliter) in the culture media were determined by a plaque forming assay as described in Materials and Methods. Values of virus titers are shown as means ± standard errors of the means for three independent experiments. (B) MTT assay of salicylate-treated BHK-21 cells. BHK-21 cells were treated with SA or ASA at the indicated concentrations for 48 h at 37°C, and the viabilities of the resulting cells were determined by an MTT assay. (C) LDH release assay. At 32 h postinfection, amounts of LDH released from the JEV-infected BHK-21 cells treated with varying amounts of salicylates were measured by an ELISA plate reader at 490 nm. Optical densities at 490 mm are shown as means for representative experiments performed in triplicate. (D) BHK-21 cells were infected by DEN at an MO1 of 5 and were treated with varying concentrations of SA or ASA as indicated. After a 48-h incubation at 37°C, the virus titers (in PFU per milliliter) in the culture media were determined as described for panel A). (E) N18 cells were infected by DEN and treated with SA or ASA. After a 48-h incubation at 37°C, the virus titers (in PFU per milliliter) in the culture media were determined as described for panel A.
    Figure Legend Snippet: NaSal and aspirin inhibit flavivirus replication in BHK-21 and N18 cells. (A) BHK-21 cells were infected by JEV at an MO1 of 5, and at 1 h postinfection, the infected cells were treated with varying concentrations of NaSal (SA) or aspirin (ASA). After a 48-h incubation at 37°C, the virus titers (expressed as PFU per milliliter) in the culture media were determined by a plaque forming assay as described in Materials and Methods. Values of virus titers are shown as means ± standard errors of the means for three independent experiments. (B) MTT assay of salicylate-treated BHK-21 cells. BHK-21 cells were treated with SA or ASA at the indicated concentrations for 48 h at 37°C, and the viabilities of the resulting cells were determined by an MTT assay. (C) LDH release assay. At 32 h postinfection, amounts of LDH released from the JEV-infected BHK-21 cells treated with varying amounts of salicylates were measured by an ELISA plate reader at 490 nm. Optical densities at 490 mm are shown as means for representative experiments performed in triplicate. (D) BHK-21 cells were infected by DEN at an MO1 of 5 and were treated with varying concentrations of SA or ASA as indicated. After a 48-h incubation at 37°C, the virus titers (in PFU per milliliter) in the culture media were determined as described for panel A). (E) N18 cells were infected by DEN and treated with SA or ASA. After a 48-h incubation at 37°C, the virus titers (in PFU per milliliter) in the culture media were determined as described for panel A.

    Techniques Used: Infection, Incubation, MTT Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay

    Salicylates suppress flavivirus-induced apoptosis in BHK-21 and N18 cells. (A) Agarose gel electrophoresis of DNA fragmentation. BHK-21 cells were either mock infected (lanes 2 and 6) or infected with JEV at an MOI of 5 (lanes 1, 3 to 5, and 7 to 9), and cells were then left untreated (lane 1) or treated with NaSal (SA) (lanes 2 to 5) or aspirin (ASA) (lanes 6 to 9) at the indicated concentrations. Low-molecular-weight DNA was isolated from cells at 32 h postinfection and analyzed by 2% agarose gel in the presence of EtBr. Lane M, 100-bp ladders as DNA markers. (B) Gel analysis of DNA ladders from JEV-infected N18 cells that were left untreated (lane 1) or treated with 5 mM SA (lane 2) at 32 h postinfection. (C) Kinetics of DNA fragmentation from JEV-infected N18 cells that were left untreated (filled squares) or treated (open circles) with 5 mM SA, determined by ELISA. Prior to virus infection, cells were labeled with BrdU overnight. At the indicated time points following infection, the cells were permeabilized to release the cytoplasmic DNA fragments into the supernatants. The amounts of BrdU-labeled DNA released were measured by ELISA as optical densities (O.D.) at 450 nm using antibodies against DNA and BrdU (see Materials and Methods).
    Figure Legend Snippet: Salicylates suppress flavivirus-induced apoptosis in BHK-21 and N18 cells. (A) Agarose gel electrophoresis of DNA fragmentation. BHK-21 cells were either mock infected (lanes 2 and 6) or infected with JEV at an MOI of 5 (lanes 1, 3 to 5, and 7 to 9), and cells were then left untreated (lane 1) or treated with NaSal (SA) (lanes 2 to 5) or aspirin (ASA) (lanes 6 to 9) at the indicated concentrations. Low-molecular-weight DNA was isolated from cells at 32 h postinfection and analyzed by 2% agarose gel in the presence of EtBr. Lane M, 100-bp ladders as DNA markers. (B) Gel analysis of DNA ladders from JEV-infected N18 cells that were left untreated (lane 1) or treated with 5 mM SA (lane 2) at 32 h postinfection. (C) Kinetics of DNA fragmentation from JEV-infected N18 cells that were left untreated (filled squares) or treated (open circles) with 5 mM SA, determined by ELISA. Prior to virus infection, cells were labeled with BrdU overnight. At the indicated time points following infection, the cells were permeabilized to release the cytoplasmic DNA fragments into the supernatants. The amounts of BrdU-labeled DNA released were measured by ELISA as optical densities (O.D.) at 450 nm using antibodies against DNA and BrdU (see Materials and Methods).

    Techniques Used: Agarose Gel Electrophoresis, Infection, Molecular Weight, Isolation, Enzyme-linked Immunosorbent Assay, Labeling

    Effects of constitutive IκBα-ΔN expression on antiflavivirus capability of salicylates. The ability of JEV (A) or DEN (B) to replicate in IκBα-ΔN-expressing cell clones (IκB#1, #8, #12, #17, and #18) was compared to that in wild-type BHK-21 cells by measurement of titers of virus released into the culture medium. (C) The anti-JEV effects of salicylates were determined for the IκBα-ΔN-expressing cell clones (IκB#12 and #17) and the wild-type BHK-21 cells. After 1 h of JEV infection, cells were left untreated or treated with 5 mM NaSal (SA) or aspirin (ASA) and then incubated at 37°C for another 24 h. Virus production in the culture medium was determined by a plaque assay. Data are mean virus titers for two independent experiments.
    Figure Legend Snippet: Effects of constitutive IκBα-ΔN expression on antiflavivirus capability of salicylates. The ability of JEV (A) or DEN (B) to replicate in IκBα-ΔN-expressing cell clones (IκB#1, #8, #12, #17, and #18) was compared to that in wild-type BHK-21 cells by measurement of titers of virus released into the culture medium. (C) The anti-JEV effects of salicylates were determined for the IκBα-ΔN-expressing cell clones (IκB#12 and #17) and the wild-type BHK-21 cells. After 1 h of JEV infection, cells were left untreated or treated with 5 mM NaSal (SA) or aspirin (ASA) and then incubated at 37°C for another 24 h. Virus production in the culture medium was determined by a plaque assay. Data are mean virus titers for two independent experiments.

    Techniques Used: Expressing, Clone Assay, Infection, Incubation, Plaque Assay

    Effect of NaSal or the dominant-negative mutant IκB-ΔN on JEV-induced NF-κB activation. (A) NaSal effect. A total of 8 × 10 4 BHK-21 cells transfected with pNFκB-Luc were either left untreated or treated with 5 mM NaSa1 or for 18 h. The resulting cells were then either infected with JEV (at an MOI of 5) or mock infected, and at 6 h postinfection, cell lysates were prepared for determination of luciferase activity. (B) IκB-ΔN effect. A total of 8 × 10 4 BHK-21 cells were transfected either with pNFκB-Luc together with plκB-ΔN or with pNFκB-Luc plus the pCR3.1 vector, and these cells were then infected with JEV at an MOI of 5 for another 6 h. The resulting cell lysates were prepared and assessed for luciferase activity. Values shown are representative of the results from three independent experiments. Luciferase activity is expressed as relative light units.
    Figure Legend Snippet: Effect of NaSal or the dominant-negative mutant IκB-ΔN on JEV-induced NF-κB activation. (A) NaSal effect. A total of 8 × 10 4 BHK-21 cells transfected with pNFκB-Luc were either left untreated or treated with 5 mM NaSa1 or for 18 h. The resulting cells were then either infected with JEV (at an MOI of 5) or mock infected, and at 6 h postinfection, cell lysates were prepared for determination of luciferase activity. (B) IκB-ΔN effect. A total of 8 × 10 4 BHK-21 cells were transfected either with pNFκB-Luc together with plκB-ΔN or with pNFκB-Luc plus the pCR3.1 vector, and these cells were then infected with JEV at an MOI of 5 for another 6 h. The resulting cell lysates were prepared and assessed for luciferase activity. Values shown are representative of the results from three independent experiments. Luciferase activity is expressed as relative light units.

    Techniques Used: Dominant Negative Mutation, Activation Assay, Transfection, Infection, Luciferase, Activity Assay, Plasmid Preparation

    16) Product Images from "Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection"

    Article Title: Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection

    Journal: Virus Research

    doi: 10.1016/j.virusres.2016.01.016

    Evaluation of specific interaction of GRFT with E and prM proteins in vitro by Pull-down assay: IMAC-beads were premixed with GRFT for 2 h and incubated with the lysate of BHK-21 cells expressing E protein (A) or prM protein (B) for another 2 h (Lane 2). The IMAC beads without GRFT was also incubated with the lysate of BHK-21 cells expressing E protein (A) or prM protein (B) for 2 h (Lane 3). The beads were washed and analyzed by Western blot using anti-MYC monoclonal antibody. The E and prM proteins in the cell lysates detected with anti-MYC monoclonal antibody served as a molecular mass marker (Lane 1).
    Figure Legend Snippet: Evaluation of specific interaction of GRFT with E and prM proteins in vitro by Pull-down assay: IMAC-beads were premixed with GRFT for 2 h and incubated with the lysate of BHK-21 cells expressing E protein (A) or prM protein (B) for another 2 h (Lane 2). The IMAC beads without GRFT was also incubated with the lysate of BHK-21 cells expressing E protein (A) or prM protein (B) for 2 h (Lane 3). The beads were washed and analyzed by Western blot using anti-MYC monoclonal antibody. The E and prM proteins in the cell lysates detected with anti-MYC monoclonal antibody served as a molecular mass marker (Lane 1).

    Techniques Used: In Vitro, Pull Down Assay, Incubation, Expressing, Western Blot, Marker

    Evaluation of specific interaction of GRFT with E and prM viral proteins in vivo by co-immunoprecipitation: (A and B) BHK-21 cells were co-transfected with pcDNA3- G -HA and pcDNA3- E -MYC or pcDNA3- G -HA and pcDNA3- M -MYC using Polyethylenimine (PEI). Cells transfected with empty vector used as control. At 24 h post-transfection, co-immunoprecipitation was performed form cell lysate using anti-HA monoclonal antibody. The immunoprecipitated protein was detected with anti-MYC monoclonal antibody (first panel in A and B). Cell lysates were evaluated using anti-MYC monoclonal antibody (second panel in A and B) and anti-HA monoclonal antibody (third panel in A and B) to detect the expression of E, prM and GRFT.
    Figure Legend Snippet: Evaluation of specific interaction of GRFT with E and prM viral proteins in vivo by co-immunoprecipitation: (A and B) BHK-21 cells were co-transfected with pcDNA3- G -HA and pcDNA3- E -MYC or pcDNA3- G -HA and pcDNA3- M -MYC using Polyethylenimine (PEI). Cells transfected with empty vector used as control. At 24 h post-transfection, co-immunoprecipitation was performed form cell lysate using anti-HA monoclonal antibody. The immunoprecipitated protein was detected with anti-MYC monoclonal antibody (first panel in A and B). Cell lysates were evaluated using anti-MYC monoclonal antibody (second panel in A and B) and anti-HA monoclonal antibody (third panel in A and B) to detect the expression of E, prM and GRFT.

    Techniques Used: In Vivo, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing

    Competitive inhibition of GRFT binding to the JEV by Mannose and its impact in viral infectivity: (A) mannose (5–100 μg/ml) was preincubated with 5 μg of His-GRFT ((Mannose + His-GRFT) or mannose (100 μg/ml) with PBS as a control (Mannose + PBS) for 30 min on a rocker and then added to 50 μl IMAC beads (Mannose + His-GRFT + IMAC or Mannose + PBS + IMAC) for 30 min. The aliquots in both experiment and control, were washed five times by centrifugation at 2000 g for 2 min in 0.5 ml of washing buffer, mixed with JEV at MOI = 1 (Mannose + His-GRFT + IMAC + JEV or Mannose + PBS + IMAC+ JEV) for 1 hr and incubated at a room temperature. The mixtures were then washed five times as above and the competitive binding of GRFT to JEV was measured by Pull-down assay using anti-JEV-EDIII monoclonal antibody. Binding of GRFT to the JEV was shown to be inhibited by increasing concentrations of mannose. (B and C) GRFT (5 μg) or PBS (control), were preincubated with different concentration of mannose and then with JEV at MOI = 1. BHK-21 cells were then infected with the mixture. Inhibition of the virus infection was analyzed by plaque forming assay (B) and Western blot using anti-JEV-EDIII monoclonal antibody (C). Data presented as Means ± SD ( n = 3). ** P
    Figure Legend Snippet: Competitive inhibition of GRFT binding to the JEV by Mannose and its impact in viral infectivity: (A) mannose (5–100 μg/ml) was preincubated with 5 μg of His-GRFT ((Mannose + His-GRFT) or mannose (100 μg/ml) with PBS as a control (Mannose + PBS) for 30 min on a rocker and then added to 50 μl IMAC beads (Mannose + His-GRFT + IMAC or Mannose + PBS + IMAC) for 30 min. The aliquots in both experiment and control, were washed five times by centrifugation at 2000 g for 2 min in 0.5 ml of washing buffer, mixed with JEV at MOI = 1 (Mannose + His-GRFT + IMAC + JEV or Mannose + PBS + IMAC+ JEV) for 1 hr and incubated at a room temperature. The mixtures were then washed five times as above and the competitive binding of GRFT to JEV was measured by Pull-down assay using anti-JEV-EDIII monoclonal antibody. Binding of GRFT to the JEV was shown to be inhibited by increasing concentrations of mannose. (B and C) GRFT (5 μg) or PBS (control), were preincubated with different concentration of mannose and then with JEV at MOI = 1. BHK-21 cells were then infected with the mixture. Inhibition of the virus infection was analyzed by plaque forming assay (B) and Western blot using anti-JEV-EDIII monoclonal antibody (C). Data presented as Means ± SD ( n = 3). ** P

    Techniques Used: Inhibition, Binding Assay, Infection, Centrifugation, Incubation, Pull Down Assay, Concentration Assay, Western Blot

    17) Product Images from "Amino Acid Residues within Conserved Domain VI of the Vesicular Stomatitis Virus Large Polymerase Protein Essential for mRNA Cap Methyltransferase Activity"

    Article Title: Amino Acid Residues within Conserved Domain VI of the Vesicular Stomatitis Virus Large Polymerase Protein Essential for mRNA Cap Methyltransferase Activity

    Journal:

    doi: 10.1128/JVI.79.21.13373-13384.2005

    Single-step growth assay of recombinant VSV in BHK-21 cells. Confluent BHK-21 cells were infected with individual viruses at an MOI of 3. After a 1-h adsorption, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2%
    Figure Legend Snippet: Single-step growth assay of recombinant VSV in BHK-21 cells. Confluent BHK-21 cells were infected with individual viruses at an MOI of 3. After a 1-h adsorption, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2%

    Techniques Used: Growth Assay, Recombinant, Infection, Adsorption

    Effect of L gene mutations on viral protein synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Proteins were labeled by incorporation of [ 35 S]methionine-cysteine in the presence of actinomycin
    Figure Legend Snippet: Effect of L gene mutations on viral protein synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Proteins were labeled by incorporation of [ 35 S]methionine-cysteine in the presence of actinomycin

    Techniques Used: Infection, Mutagenesis, Labeling

    Effect of L gene mutations on viral RNA synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Viral RNAs were labeled with [ 3 H]uridine as described in Materials and Methods, resolved by electrophoresis
    Figure Legend Snippet: Effect of L gene mutations on viral RNA synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Viral RNAs were labeled with [ 3 H]uridine as described in Materials and Methods, resolved by electrophoresis

    Techniques Used: Infection, Mutagenesis, Labeling, Electrophoresis

    18) Product Images from "Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry ▿"

    Article Title: Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry ▿

    Journal:

    doi: 10.1128/JVI.01864-08

    Electron microscopy of VSV associated with the plasma membranes of BHK-21 cells. Virus was bound to cells at 4°C for 1 h. The cells were fixed and analyzed in thin sections. Some of the membrane-bound viruses were associated with electron-dense,
    Figure Legend Snippet: Electron microscopy of VSV associated with the plasma membranes of BHK-21 cells. Virus was bound to cells at 4°C for 1 h. The cells were fixed and analyzed in thin sections. Some of the membrane-bound viruses were associated with electron-dense,

    Techniques Used: Electron Microscopy

    Electron microscopy of VSV in endocytic vesicles. Virus was bound to BHK-21 cells at 4°C for 1 h, and the cells were shifted to a 37°C incubator to allow internalization. Viruses were seen either in coated or partly coated endocytic vesicles
    Figure Legend Snippet: Electron microscopy of VSV in endocytic vesicles. Virus was bound to BHK-21 cells at 4°C for 1 h, and the cells were shifted to a 37°C incubator to allow internalization. Viruses were seen either in coated or partly coated endocytic vesicles

    Techniques Used: Electron Microscopy

    19) Product Images from "Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus"

    Article Title: Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01657-19

    Construction and rescue of four recombinant viruses. (A) Schematic representation showing the constructs of prO, pr-D70, pr-L10, and pr-D70-L10. (B) Plaque assays of rO, rO-D70, rO-L10, and rO-D70-L10 on BHK-21 cells. (C) Alignment of the 5′-UTR region sequences of the four recombinant viruses, O/Mya98/JX/2010, and O/BY/CHA/2010, as well as another O/SEA/Mya-98 lineage FMDV strain, O/GZ/CHA/2010. (D) Alignment of the polyprotein sequences of the four recombinant viruses and O/Mya98/JX/2010, O/BY/CHA/2010, and O/GZ/CHA/2010. A solid black rectangle indicates the leucine insertion in L pro .
    Figure Legend Snippet: Construction and rescue of four recombinant viruses. (A) Schematic representation showing the constructs of prO, pr-D70, pr-L10, and pr-D70-L10. (B) Plaque assays of rO, rO-D70, rO-L10, and rO-D70-L10 on BHK-21 cells. (C) Alignment of the 5′-UTR region sequences of the four recombinant viruses, O/Mya98/JX/2010, and O/BY/CHA/2010, as well as another O/SEA/Mya-98 lineage FMDV strain, O/GZ/CHA/2010. (D) Alignment of the polyprotein sequences of the four recombinant viruses and O/Mya98/JX/2010, O/BY/CHA/2010, and O/GZ/CHA/2010. A solid black rectangle indicates the leucine insertion in L pro .

    Techniques Used: Recombinant, Construct

    rO-D70-L10 showed significantly decreased infective ability for bovine cells compared to that of rO. One-step growth curves are shown for the four recombinant viruses in BHK-21 (A), IBRS-2 (B), PK-15 (C), and BTY (D) cells. The cells were infected with each virus at a multiplicity of infection (MOI) of 0.5 and maintained at 37°C. Samples of supernatants were harvested at the indicated times, and viral titers were measured.
    Figure Legend Snippet: rO-D70-L10 showed significantly decreased infective ability for bovine cells compared to that of rO. One-step growth curves are shown for the four recombinant viruses in BHK-21 (A), IBRS-2 (B), PK-15 (C), and BTY (D) cells. The cells were infected with each virus at a multiplicity of infection (MOI) of 0.5 and maintained at 37°C. Samples of supernatants were harvested at the indicated times, and viral titers were measured.

    Techniques Used: Recombinant, Infection

    Related Articles

    Modification:

    Article Title: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection
    Article Snippet: .. Baby hamster kidney (BHK-21) cells and human embryonic kidney 293T (HEK-293T) cells were grown in Dulbecco’s modified essential medium (DMEM, GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO), 100 U/mL penicillin–streptomycin, at 37 °C in 5% CO2 . .. A549 cells and C6/36 cells were cultured in RPMI-1640 (GIBCO) supplemented with 10% FBS, 100 U/mL penicillin–streptomycin, at 37 °C and 28 °C, respectively, in 5% CO2 .

    Article Title: In vivo imaging of murid herpesvirus-4 infection
    Article Snippet: .. Baby hamster kidney (BHK-21) cells, NIH-3T3 cells, NIH-3T3-CRE cells ( ) and NIH-3T3-TET50 cells were propagated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM glutamine, 100 U penicillin ml−1 , 100 μg streptomycin ml−1 and 10 % fetal calf serum. ..

    Article Title: Membrane Topology and Function of Dengue Virus NS2A Protein
    Article Snippet: .. Baby hamster kidney (BHK-21) cells were maintained in high-glucose Dulbecco modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 4 mM l -glutamine, 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). .. HEK293T (human embryo kidney 293T) cells were grown in low-glucose DMEM (Invitrogen) with 10% FBS and 1% penicillin/streptomycin.

    Transfection:

    Article Title: The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion
    Article Snippet: .. Approximately 7 × 105 baby hamster kidney (BHK-21) cells were transfected with a DNA-liposome suspension containing 2 μg of the appropriate plasmid and 10 μl of Lipofectamine (Gibco-BRL) according to the manufacturer's instructions. .. Three hours posttransfection, the medium was replaced with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and the cells were incubated for 36 h at 37°C.

    other:

    Article Title: Both E Protein Glycans Adversely Affect Dengue Virus Infectivity but Are Beneficial for Virion Release ▿
    Article Snippet: African green monkey kidney (Vero) and baby hamster kidney (BHK-21) cells were maintained at 37°C in medium M199 (Gibco) plus 5% fetal calf serum (FCS) and Glasgow's minimum essential medium (Gibco) supplemented with tryptose phosphate broth (Sigma-Aldrich) and 5% fetal calf serum, respectively.

    Plasmid Preparation:

    Article Title: The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion
    Article Snippet: .. Approximately 7 × 105 baby hamster kidney (BHK-21) cells were transfected with a DNA-liposome suspension containing 2 μg of the appropriate plasmid and 10 μl of Lipofectamine (Gibco-BRL) according to the manufacturer's instructions. .. Three hours posttransfection, the medium was replaced with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and the cells were incubated for 36 h at 37°C.

    Cell Culture:

    Article Title: Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function
    Article Snippet: .. Baby hamster kidney (BHK-21) cells were cultured in either Glasgow minimal essential medium (MEM) supplemented with 5% fetal calf serum, 10% tryptose phosphate broth, 2 mM glutamine, and 20 mM HEPES (Life Technologies, Inc., Paisley, Scotland) or MEM with Earl’s salts supplemented with 0.2% bovine serum albumin, 2 mM glutamine, and 20 mM HEPES. .. SFV was derived from the pSFV4 infectious clone plasmid ( ).

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    Thermo Fisher bhk 21 cells
    More than 2 PKs provides a replicative advantage in co-transfection competition experiments. <t>BHK-21</t> cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).
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    More than 2 PKs provides a replicative advantage in co-transfection competition experiments. BHK-21 cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: More than 2 PKs provides a replicative advantage in co-transfection competition experiments. BHK-21 cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).

    Article Snippet: Passaging in competition assays was performed by co-transfecting BHK-21 cells with in vitro transcribed replicon RNA and harvesting total cell RNA at 8 hours post transfection using TRIzol reagent (Thermo Fisher Scientific).

    Techniques: Cotransfection, Transfection

    The poly-C-tract is dispensable and only one PK is required for wt replication. Wt , 3D-GNN and a replicon with a truncated poly-C-tract (C11) were transfected into BHK-21 and MDBK cell lines, replication was monitored using the Incucyte Zoom (A). A replicon with entire poly-C-tract removed (C0) was transfected alongside wt , 3D-GNN and C11 replicons into BHK-21 cells (B). Replicons with sequentially deleted PKs (ΔPK 34, ΔPK 234 and C11 ΔPK 1234) were assayed for replication in BHK-21 cells (C) and MDBK cells (D). Replication of replicon with PK 4 as the sole remaining PK (C11 PK 4), transfected into MDBK and BHK-21 cells (E). All replication assays were measured by counting the number of GFP positive cells per well using the Incucyte Zoom with data shown at 8 hours post-transection. Error bars shown are calculated by SEM, n = 3. Significance is shown compared to the wt (A, B) or the wt and 3D-GNN (C, D, E) * P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: The poly-C-tract is dispensable and only one PK is required for wt replication. Wt , 3D-GNN and a replicon with a truncated poly-C-tract (C11) were transfected into BHK-21 and MDBK cell lines, replication was monitored using the Incucyte Zoom (A). A replicon with entire poly-C-tract removed (C0) was transfected alongside wt , 3D-GNN and C11 replicons into BHK-21 cells (B). Replicons with sequentially deleted PKs (ΔPK 34, ΔPK 234 and C11 ΔPK 1234) were assayed for replication in BHK-21 cells (C) and MDBK cells (D). Replication of replicon with PK 4 as the sole remaining PK (C11 PK 4), transfected into MDBK and BHK-21 cells (E). All replication assays were measured by counting the number of GFP positive cells per well using the Incucyte Zoom with data shown at 8 hours post-transection. Error bars shown are calculated by SEM, n = 3. Significance is shown compared to the wt (A, B) or the wt and 3D-GNN (C, D, E) * P

    Article Snippet: Passaging in competition assays was performed by co-transfecting BHK-21 cells with in vitro transcribed replicon RNA and harvesting total cell RNA at 8 hours post transfection using TRIzol reagent (Thermo Fisher Scientific).

    Techniques: Transfection

    Disrupting the PK structure and reversing the orientation of a PK reduces replication. Cartoon representations of disrupting and restoring mutations made to PK 1, where nucleotides in the bulge of the stem loop and interacting region downstream were mutated to disrupt structure formation ‘PK disrupt’, or mutated to maintain bulge and downstream interaction but with different nucleotides ‘PK restore’ (A i ). Replication of PK disrupt and restore mutants were measured by transfection of RNA into BHK-21 cells and shown here at 8 hours post-transfection alongside wt , 3D-GNN and C11 ΔPK 1234 controls. Significance is shown comparing the replication of C11 PK disrupt and C11 PK restore (A ii ). Visual representation of the reversing of the nucleotide sequence of PK1 creating the C11 PK Rvs construct (B i ). Replication of PK Rvs at 8 hours post transfection of BHK-21 cells (B ii ). Significance shown is compared to wt replicon. Error bars are calculated by SEM, n = 3, * P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: Disrupting the PK structure and reversing the orientation of a PK reduces replication. Cartoon representations of disrupting and restoring mutations made to PK 1, where nucleotides in the bulge of the stem loop and interacting region downstream were mutated to disrupt structure formation ‘PK disrupt’, or mutated to maintain bulge and downstream interaction but with different nucleotides ‘PK restore’ (A i ). Replication of PK disrupt and restore mutants were measured by transfection of RNA into BHK-21 cells and shown here at 8 hours post-transfection alongside wt , 3D-GNN and C11 ΔPK 1234 controls. Significance is shown comparing the replication of C11 PK disrupt and C11 PK restore (A ii ). Visual representation of the reversing of the nucleotide sequence of PK1 creating the C11 PK Rvs construct (B i ). Replication of PK Rvs at 8 hours post transfection of BHK-21 cells (B ii ). Significance shown is compared to wt replicon. Error bars are calculated by SEM, n = 3, * P

    Article Snippet: Passaging in competition assays was performed by co-transfecting BHK-21 cells with in vitro transcribed replicon RNA and harvesting total cell RNA at 8 hours post transfection using TRIzol reagent (Thermo Fisher Scientific).

    Techniques: Transfection, Sequencing, Construct

    Recovered virus showed a delay in rate of CPE and a small plaque phenotype. BHK-21 cells were infected with wt , ΔPK 34 and ΔPK 234 virus at a MOI of 0.01, alongside a mock infected control and cell confluency (shown as phase object confluence %) monitored every half an hour for 62 hours using an Incucyte Zoom (A). Representative plaque assay of BHK-21 cells infected with wt , ΔPK 34 and ΔPK 234 viruses. Recovered virus from passage 5 of blind passaging was infected onto BHK-21 cells, cells were fixed and stained 48 hours post infection (B). Virus plaques were imaged and size of plaques measured using Image J, all plaques per well were counted, additional wells were used until a minimum plaque count of 40 was reached. Significance is shown when compared to the wt , box and whisker plots were made using the Tukey method.

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: Recovered virus showed a delay in rate of CPE and a small plaque phenotype. BHK-21 cells were infected with wt , ΔPK 34 and ΔPK 234 virus at a MOI of 0.01, alongside a mock infected control and cell confluency (shown as phase object confluence %) monitored every half an hour for 62 hours using an Incucyte Zoom (A). Representative plaque assay of BHK-21 cells infected with wt , ΔPK 34 and ΔPK 234 viruses. Recovered virus from passage 5 of blind passaging was infected onto BHK-21 cells, cells were fixed and stained 48 hours post infection (B). Virus plaques were imaged and size of plaques measured using Image J, all plaques per well were counted, additional wells were used until a minimum plaque count of 40 was reached. Significance is shown when compared to the wt , box and whisker plots were made using the Tukey method.

    Article Snippet: Passaging in competition assays was performed by co-transfecting BHK-21 cells with in vitro transcribed replicon RNA and harvesting total cell RNA at 8 hours post transfection using TRIzol reagent (Thermo Fisher Scientific).

    Techniques: Infection, Plaque Assay, Passaging, Staining, Whisker Assay

    PKs are not essential for viral genome replication. BHK-21 cells were transfected with RNA transcribed from infectious clones of wt , ΔPK 34, ΔPK 234 and C11 ΔPK 1234. Non-transfected cells and a wt transfection treated with 3 mM GuHCl were used as negative controls. Cells were harvested, fixed and labelled using an anti-3A antibody and fluorescent anti-mouse secondary before separation by flow cytometry. Gates were used to select for live cell and single cell populations, and virus positive/negative populations were identified based on levels of Alexa-488 fluorescence. Where distinct virus positive and negative populations exist, gates were drawn to separate these in order to determine the mean fluorescent intensity (MFI) of the virus positive cells. Where no clear separate populations exist ( wt with GuHCl, mock treatment and untreated cells) gates could not be drawn and therefore the total MFI has been reported (denoted by #). Representative images have been shown here for the live cell gate from the wt virus transfection (A), the single cell gate from the wt virus transfection (B) and the relative fluorescence of the cells with mock antibody treatment (C), wt virus (D), C11 ΔPK 1234 virus (E) and wt virus transfection with 3 mM GuHCl (F). The experiment was performed in triplicate and the MFI values for each condition were calculated (G). The error bars represent the SEM. Significance is shown compared to the wt plus 3 mM GuHCl control (**** P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: PKs are not essential for viral genome replication. BHK-21 cells were transfected with RNA transcribed from infectious clones of wt , ΔPK 34, ΔPK 234 and C11 ΔPK 1234. Non-transfected cells and a wt transfection treated with 3 mM GuHCl were used as negative controls. Cells were harvested, fixed and labelled using an anti-3A antibody and fluorescent anti-mouse secondary before separation by flow cytometry. Gates were used to select for live cell and single cell populations, and virus positive/negative populations were identified based on levels of Alexa-488 fluorescence. Where distinct virus positive and negative populations exist, gates were drawn to separate these in order to determine the mean fluorescent intensity (MFI) of the virus positive cells. Where no clear separate populations exist ( wt with GuHCl, mock treatment and untreated cells) gates could not be drawn and therefore the total MFI has been reported (denoted by #). Representative images have been shown here for the live cell gate from the wt virus transfection (A), the single cell gate from the wt virus transfection (B) and the relative fluorescence of the cells with mock antibody treatment (C), wt virus (D), C11 ΔPK 1234 virus (E) and wt virus transfection with 3 mM GuHCl (F). The experiment was performed in triplicate and the MFI values for each condition were calculated (G). The error bars represent the SEM. Significance is shown compared to the wt plus 3 mM GuHCl control (**** P

    Article Snippet: Passaging in competition assays was performed by co-transfecting BHK-21 cells with in vitro transcribed replicon RNA and harvesting total cell RNA at 8 hours post transfection using TRIzol reagent (Thermo Fisher Scientific).

    Techniques: Transfection, Clone Assay, Flow Cytometry, Fluorescence

    Anti-GRP78 antibody inhibits TMUV infection in BHK-21 cells. BHK-21 cells were pre-incubated with 100 μg/ml rabbit IgG, anti-GRP78 N-terminal or anti-GRP78 C-terminal antibodies at 4°C or 1 h followed by TMUV infection. The level of TMUV RNA in rabbit IgG incubated cells was taken as 1 for determining the relative RNA levels. The viral RNA was compared to those in rabbit IgG incubated cells. Data were presented from three independent experiments and statistic analysis was done with SPSS software. The asterisk designates statistically significant differences ( p

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Anti-GRP78 antibody inhibits TMUV infection in BHK-21 cells. BHK-21 cells were pre-incubated with 100 μg/ml rabbit IgG, anti-GRP78 N-terminal or anti-GRP78 C-terminal antibodies at 4°C or 1 h followed by TMUV infection. The level of TMUV RNA in rabbit IgG incubated cells was taken as 1 for determining the relative RNA levels. The viral RNA was compared to those in rabbit IgG incubated cells. Data were presented from three independent experiments and statistic analysis was done with SPSS software. The asterisk designates statistically significant differences ( p

    Article Snippet: Western Blot for Surface Expression of GRP78 The membrane proteins and cytosolic proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) as described above.

    Techniques: Infection, Incubation, Software

    Identification of GRP78 as TMUV-binding membrane protein. (A) Detection of protein involved in TMUV binding in BHK-21 cell membrane by VOPBA. The PVDF membrane containing BHK-21 cell membrane proteins were incubated without (Lane 1) or with 10 5 TCID 50 of TMUV (Lane 2). Virus binding bands were detected by monoclonal antibody against TMUV. The approximate 70 kDa band was observed in Lane 2 (black arrow). Lane 3, molecular weight marker. (B) Coomassie staining of the membrane protein extracted from BHK-21 cells. Lane 1, molecular weight marker; Lane 2, membrane protein extracted from BHK-21 cells. (C) Co-immunoprecipitation assay of TMUV binding membrane protein. The membrane protein extracted from BHK-21 cells immunoprecipitated with (Lane 1) or without (Lane 2) TMUV. The immunoprecipitated complexes were analyzed by SDS-PAGE and transferred to PVDF membrane. The membrane was then incubated with anti-GRP78 antibody. The approximate 70 kDa band was observed in Lane 1 (black arrow). Lane 3, molecular weight marker. (D) Identification of TMUV binding protein by mass spectrometry. The peptide sequences of GRP78 identified by mass spectrometry were underlined.

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Identification of GRP78 as TMUV-binding membrane protein. (A) Detection of protein involved in TMUV binding in BHK-21 cell membrane by VOPBA. The PVDF membrane containing BHK-21 cell membrane proteins were incubated without (Lane 1) or with 10 5 TCID 50 of TMUV (Lane 2). Virus binding bands were detected by monoclonal antibody against TMUV. The approximate 70 kDa band was observed in Lane 2 (black arrow). Lane 3, molecular weight marker. (B) Coomassie staining of the membrane protein extracted from BHK-21 cells. Lane 1, molecular weight marker; Lane 2, membrane protein extracted from BHK-21 cells. (C) Co-immunoprecipitation assay of TMUV binding membrane protein. The membrane protein extracted from BHK-21 cells immunoprecipitated with (Lane 1) or without (Lane 2) TMUV. The immunoprecipitated complexes were analyzed by SDS-PAGE and transferred to PVDF membrane. The membrane was then incubated with anti-GRP78 antibody. The approximate 70 kDa band was observed in Lane 1 (black arrow). Lane 3, molecular weight marker. (D) Identification of TMUV binding protein by mass spectrometry. The peptide sequences of GRP78 identified by mass spectrometry were underlined.

    Article Snippet: Western Blot for Surface Expression of GRP78 The membrane proteins and cytosolic proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) as described above.

    Techniques: Binding Assay, Incubation, Molecular Weight, Marker, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Mass Spectrometry

    Colocalization of GRP78 and TMUV on the surface of BHK-21 cells. (A) Membrane proteins and cytosolic proteins of BHK-21 cells were western blotted with GRP78 antibody. GAPDH was chosen as cytosolic marker. (B) Immunofluorescence detection of non-permeabilized cells infected with TMUV.

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Colocalization of GRP78 and TMUV on the surface of BHK-21 cells. (A) Membrane proteins and cytosolic proteins of BHK-21 cells were western blotted with GRP78 antibody. GAPDH was chosen as cytosolic marker. (B) Immunofluorescence detection of non-permeabilized cells infected with TMUV.

    Article Snippet: Western Blot for Surface Expression of GRP78 The membrane proteins and cytosolic proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) as described above.

    Techniques: Western Blot, Marker, Immunofluorescence, Infection

    Effect of GRP78 shRNA transfection on TMUV entry at 48 h post-transfection. (A) Relative GRP78 mRNA level in transfected BHK-21 cells. (B) Analysis of surface-expressed GRP78 protein in transfected BHK-21 cells by western blot (Na+/K+ ATPase was chosen as plasma membrane marker). (C) Relative TMUV RNA were detected by qRT-PCR. Data were presented from three independent experiments and the asterisk designates statistically significant differences ( p

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Effect of GRP78 shRNA transfection on TMUV entry at 48 h post-transfection. (A) Relative GRP78 mRNA level in transfected BHK-21 cells. (B) Analysis of surface-expressed GRP78 protein in transfected BHK-21 cells by western blot (Na+/K+ ATPase was chosen as plasma membrane marker). (C) Relative TMUV RNA were detected by qRT-PCR. Data were presented from three independent experiments and the asterisk designates statistically significant differences ( p

    Article Snippet: Western Blot for Surface Expression of GRP78 The membrane proteins and cytosolic proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) as described above.

    Techniques: shRNA, Transfection, Western Blot, Marker, Quantitative RT-PCR

    Effect of over-expression of GRP78 on TMUV entry at 48 h post-transfection. (A) qRT-PCR analysis of over-expression of GRP78 in transfected BHK-21 cells. (B) Surface-expressed GRP78 protein was determined by western blot (Na+/K+ ATPase was chosen as plasma membrane marker). (C) The TMUV entry in BHK-21 cells transfected with GRP78-pcDNA was measured by qRT-PCR. Data were presented from three independent experiments and the asterisk designates statistically significant differences ( p

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Effect of over-expression of GRP78 on TMUV entry at 48 h post-transfection. (A) qRT-PCR analysis of over-expression of GRP78 in transfected BHK-21 cells. (B) Surface-expressed GRP78 protein was determined by western blot (Na+/K+ ATPase was chosen as plasma membrane marker). (C) The TMUV entry in BHK-21 cells transfected with GRP78-pcDNA was measured by qRT-PCR. Data were presented from three independent experiments and the asterisk designates statistically significant differences ( p

    Article Snippet: Western Blot for Surface Expression of GRP78 The membrane proteins and cytosolic proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) as described above.

    Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Marker

    Improved MHBs expression and secretion using VLV dp vectors. ( A ) Schematic of the double subgenomic promoter (SGP) VLV vectors used in this study. ( B ) Immunoblot analyses of MHBs and VSV-G expression in BHK-21 cells at 24 h post-infection with MT2A or dpG IND -MHBs VLV (MOI = 1). Densitometry values are for MHBs signal (PreS2) relative to VSV-G or actin. ( C ) Immunofluorescence analyses of MHBs expression (green) in BHK-21 cells at 18 h post-infection with MT2A or dpG IND -MHBs VLV at MOI = 0.5. Nuclear counterstaining (DAPI) is shown in blue. ( D ) MHBs expression in BHK-21 cells at 24 h after infection with dp VLV with serotype switch of the envelope glycoprotein from Indiana to Chandipura (IND vs. CH) or position of MHBs (all at MOI = 1). ( E ) Representative plaques from VLV dpMHBs-G CH and VLV dpMHBs-G IND in BHK-21 cells. Plaques were stained with crystal violet and images were obtained using a ZOE fluorescent cell imager (Bio-Rad laboratories). ( F ) Percentage of body weight after intranasal infection with VLV dpMHBs-G CH and VLV dpMHBs-G IND . ( G ) Secretion of MHBs measured by ELISA using conditioned media from BHK-21 cells infected with the indicated VLV (all at MOI = 1).

    Journal: Vaccines

    Article Title: Modified Alphavirus-Vesiculovirus Hybrid Vaccine Vectors for Homologous Prime-Boost Immunotherapy of Chronic Hepatitis B

    doi: 10.3390/vaccines8020279

    Figure Lengend Snippet: Improved MHBs expression and secretion using VLV dp vectors. ( A ) Schematic of the double subgenomic promoter (SGP) VLV vectors used in this study. ( B ) Immunoblot analyses of MHBs and VSV-G expression in BHK-21 cells at 24 h post-infection with MT2A or dpG IND -MHBs VLV (MOI = 1). Densitometry values are for MHBs signal (PreS2) relative to VSV-G or actin. ( C ) Immunofluorescence analyses of MHBs expression (green) in BHK-21 cells at 18 h post-infection with MT2A or dpG IND -MHBs VLV at MOI = 0.5. Nuclear counterstaining (DAPI) is shown in blue. ( D ) MHBs expression in BHK-21 cells at 24 h after infection with dp VLV with serotype switch of the envelope glycoprotein from Indiana to Chandipura (IND vs. CH) or position of MHBs (all at MOI = 1). ( E ) Representative plaques from VLV dpMHBs-G CH and VLV dpMHBs-G IND in BHK-21 cells. Plaques were stained with crystal violet and images were obtained using a ZOE fluorescent cell imager (Bio-Rad laboratories). ( F ) Percentage of body weight after intranasal infection with VLV dpMHBs-G CH and VLV dpMHBs-G IND . ( G ) Secretion of MHBs measured by ELISA using conditioned media from BHK-21 cells infected with the indicated VLV (all at MOI = 1).

    Article Snippet: Propagation of the working stocks was performed by single passage of the master stock in BHK-21 cells cultured in Opti-MEM™ I Reduced Serum Medium (ThermoFisher, Waltham, MA, USA).

    Techniques: Expressing, Infection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

    Plaque phenotype and viral growth kinetics. (A) Plaque phenotypes for FMDV A12 WT, A12 LLV, and A12 LproW105A were evaluated in BHK-21 cells. Cell monolayers were infected for 48 h in semisolid medium, followed by staining with crystal violet. (B) Kinetics of growth in multiple cell lines; BHK-21, LFPKαVβ6, and SK6 cells were infected at an MOI of 5 with WT, LLV, or LproW105A FMDV, and at the indicated times, virus titer was measured in BHK-21 cells. FMDV yield was determined by the endpoint dilution method on BHK-21 cells 24 h after infection. Titers are expressed as log 10 50% tissue culture infective dose (TCID 50 ) per milliliter. The values are presented as the mean ± standard deviation of three independent experiments. (C) Fluorescent microscopy images from LFPKαVβ6 cells infected with WT or LproW105A FMDV at an MOI of 10 and fixed at 4 h postinfection. Samples were stained with anti-Lpro (green) and with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar represents 10 μm. Statistical analysis was performed using Student’s t test. *, P

    Journal: Journal of Virology

    Article Title: Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

    doi: 10.1128/JVI.00341-20

    Figure Lengend Snippet: Plaque phenotype and viral growth kinetics. (A) Plaque phenotypes for FMDV A12 WT, A12 LLV, and A12 LproW105A were evaluated in BHK-21 cells. Cell monolayers were infected for 48 h in semisolid medium, followed by staining with crystal violet. (B) Kinetics of growth in multiple cell lines; BHK-21, LFPKαVβ6, and SK6 cells were infected at an MOI of 5 with WT, LLV, or LproW105A FMDV, and at the indicated times, virus titer was measured in BHK-21 cells. FMDV yield was determined by the endpoint dilution method on BHK-21 cells 24 h after infection. Titers are expressed as log 10 50% tissue culture infective dose (TCID 50 ) per milliliter. The values are presented as the mean ± standard deviation of three independent experiments. (C) Fluorescent microscopy images from LFPKαVβ6 cells infected with WT or LproW105A FMDV at an MOI of 10 and fixed at 4 h postinfection. Samples were stained with anti-Lpro (green) and with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar represents 10 μm. Statistical analysis was performed using Student’s t test. *, P

    Article Snippet: All viruses were derived and propagated in BHK-21 cells, concentrated by polyethylene glycol precipitation, titrated on the same cells, and stored at –70°C.

    Techniques: Infection, Staining, Standard Deviation, Microscopy

    Virulence of A12-LproW105A in infected mice. Six- to 7-week-old female C57BL/6 mice ( n = 6/group) were subcutaneously inoculated in the footpad with the indicated doses of A12-WT or A12-LproW105A mutant FMDV. (A) Clinical disease was followed for 7 days after inoculation, and percent survival was calculated as (number of surviving animals/number of animals per group) × 100, daily. (B) Serum samples collected at the indicated times after inoculation were assayed for the presence of virus by plaque assay on BHK-21 cells. (C) FMDV serum neutralizing antibody titers were determined by endpoint dilution and expressed as log TCID 50 /ml ( 48 ). Statistical analysis was performed using Student’s t test. *, P

    Journal: Journal of Virology

    Article Title: Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

    doi: 10.1128/JVI.00341-20

    Figure Lengend Snippet: Virulence of A12-LproW105A in infected mice. Six- to 7-week-old female C57BL/6 mice ( n = 6/group) were subcutaneously inoculated in the footpad with the indicated doses of A12-WT or A12-LproW105A mutant FMDV. (A) Clinical disease was followed for 7 days after inoculation, and percent survival was calculated as (number of surviving animals/number of animals per group) × 100, daily. (B) Serum samples collected at the indicated times after inoculation were assayed for the presence of virus by plaque assay on BHK-21 cells. (C) FMDV serum neutralizing antibody titers were determined by endpoint dilution and expressed as log TCID 50 /ml ( 48 ). Statistical analysis was performed using Student’s t test. *, P

    Article Snippet: All viruses were derived and propagated in BHK-21 cells, concentrated by polyethylene glycol precipitation, titrated on the same cells, and stored at –70°C.

    Techniques: Infection, Mouse Assay, Mutagenesis, Plaque Assay

    FMDV replication in cells overexpressing ISG15. (A) Viral titers were examined in LFPKαVβ6 previously transfected with ISG15 and infected at an MOI of 0.1 with WT, LLV, or LproW105A. FMDV yield was determined by plaque assay in BHK-21 cells. Plaques were counted 24 hpi, and titers were expressed as plaque forming units (PFUs) per ml. Values are presented as the mean ± standard deviation from three independent experiments. (B) Schematic representation of the replication-defective Ad5-pISG15 plasmid. HA-tagged pISG15 was cloned in the Ad5-Blue plasmid using ClaI and XbaI restriction enzyme sites. (C) Western blot analysis of protein ISGylation in mock or Ad5-GFP/Ad5-HA-pISG15-transduced LFPKαVβ6 cells in the presence or absence of increasing amounts of porcine IFN-β (5 to 40 U). Eighteen hours after IFN-β treatment, protein ISGylation was detected by using anti-HA antibodies. (D) Viral titers were examined in LFPKαVβ6 previously transduced with Ad5-HA-pISG15 and infected at an MOI of 0.1 with WT virus. Virus yield were determined by plaque assay in BHK-21 cells. The values are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using Student’s t test. *, P

    Journal: Journal of Virology

    Article Title: Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

    doi: 10.1128/JVI.00341-20

    Figure Lengend Snippet: FMDV replication in cells overexpressing ISG15. (A) Viral titers were examined in LFPKαVβ6 previously transfected with ISG15 and infected at an MOI of 0.1 with WT, LLV, or LproW105A. FMDV yield was determined by plaque assay in BHK-21 cells. Plaques were counted 24 hpi, and titers were expressed as plaque forming units (PFUs) per ml. Values are presented as the mean ± standard deviation from three independent experiments. (B) Schematic representation of the replication-defective Ad5-pISG15 plasmid. HA-tagged pISG15 was cloned in the Ad5-Blue plasmid using ClaI and XbaI restriction enzyme sites. (C) Western blot analysis of protein ISGylation in mock or Ad5-GFP/Ad5-HA-pISG15-transduced LFPKαVβ6 cells in the presence or absence of increasing amounts of porcine IFN-β (5 to 40 U). Eighteen hours after IFN-β treatment, protein ISGylation was detected by using anti-HA antibodies. (D) Viral titers were examined in LFPKαVβ6 previously transduced with Ad5-HA-pISG15 and infected at an MOI of 0.1 with WT virus. Virus yield were determined by plaque assay in BHK-21 cells. The values are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using Student’s t test. *, P

    Article Snippet: All viruses were derived and propagated in BHK-21 cells, concentrated by polyethylene glycol precipitation, titrated on the same cells, and stored at –70°C.

    Techniques: Transfection, Infection, Plaque Assay, Standard Deviation, Plasmid Preparation, Clone Assay, Western Blot, Transduction