VSV-G antagonizes human and porcine tetherin.
2017-12-07T18:57:58Z (GMT) by
<p>(A) 293T cells were transiently transfected with plasmids encoding HIV-Gag, tetherin and the indicated viral proteins or empty plasmid as negative control (Mock). HIV-1 Vpu and EBOV-GP were employed as positive controls for tetherin antagonism. The presence of Gag in supernatants and cell lysates was determined by Western blot analysis using an anti-Gag antibody. Expression of tetherin was detected using anti-c-Myc antibody while rabbit sera were employed to detect expression of EBOV-GP, Vpu and VSV proteins. Detection of ß-actin in cell lysates served as loading control. Similar results were obtained in four separate experiments. (B) The average of eight independent experiments conducted as described for panel (A) and quantified via the ImageJ program is shown. Release of Gag from cells coexpressing Vpu and tetherin was set to 100%. One-way ANOVA with Bonferroni post-test analysis was performed to test whether signals measured in the presence of tetherin antagonists were statistically different from those detected in the absence of antagonists (Mock). (C) The same experiment was performed as in (A) but instead of human tetherin porcine tetherin was examined. The results of a representative experiment are shown and were confirmed in three separate experiments. (D) 293T cells were transfected as described for panel (A) and at 16 h post transfection the medium was replaced by fresh culture medium supplemented with ((+)Ab) or without ((-)Ab) hybridoma supernatant containing VSV neutralizing antibody at a final concentration of 1:1,000. (E) The average of four independent experiments conducted as described for panel (D) and quantified via the ImageJ program is presented. Release of Gag from cells coexpressing Vpu and tetherin was set to 100%. One-way ANOVA with Bonferroni post-test analysis was performed to test whether differences between signals obtained from cells expressing viral tetherin antagonists and cells expressing no antagonist (Mock), or between cells expressing VSV-G and treated with or without anti-VSV-G antibody (**, p ≤ 0.005; ***, p ≤ 0.001) were statistically significant. (F) 293T cells were transiently transfected with the indicated amounts of plasmid encoding VSV-G. At 48 h post transfection the expression of VSV-G was determined via Western blot using anti-VSV-G hybridoma supernatant (left panel). In parallel, 293T cells were infected with VSV using the indicated multiplicities of infection (MOI) and VSV-G expression was examined by Western blot at 24 h post infection using anti-VSV-G antibody (concentrated supernatants from hybridoma CL-2700). The results of a representative experiment are shown and were confirmed in three separate experiments.</p>