The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells

Abstract

Vesicular stomatitis virus (VSV) release from infected cells is inhibited by the interferon (IFN)-inducible antiviral host cell factor tetherin (BST-2, CD317). However, several viruses encode tetherin antagonists and it is at present unknown whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the viral glycoprotein (VSV-G) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the HIV-1 Vpu protein. Tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a GXXXG motif in the transmembrane domain of VSV-G. However, mutation of the GXXXG motif did not modulate tetherin sensitivity of infectious VSV. These results identify VSV-G as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread.

Fig 1. VSV-G antagonizes human and porcine tetherin.

(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.

Fig 2. VSV-G does not alter expression levels or cell surface localization of tetherin.

(A) Plasmids encoding tetherin and the indicated viral tetherin antagonists were transiently transfected into 293T cells. Transfection of empty plasmid (Mock) served as negative control. At 48 h post transfection, cells were harvested and stained for surface expression of tetherin using anti-tetherin antibody and Alexa-647 coupled secondary antibody. Staining was analyzed by flow cytometry. The graph shows relative surface expression values from three independent experiments, for which expression of tetherin on the surface of cells not expressing any antagonist (Mock) was set as 100%. Error bars indicate standard error of the mean (SEM). (B) 293T cells were transfected with plasmids encoding tetherin with an N-terminal c-Myc-tag and the indicated viral tetherin antagonists. At 48 h post transfection, tetherin expression in cell lysates was detected by Western blot analysis, using anti-c-Myc antibody. Detection of β-actin served as loading control. The results were confirmed in three separate experiments. Arrow heads indicate bands exhibiting the molecular weight expected for unglycosylated (white), partially (grey) and fully (black) N-glycosylated tetherin. (C) Quantification of four experiments performed as described for panel (B). The intensities measured for all tetherin signals with a molecular weight between 17 and 30 kDa were added to yield total tetherin expression. Tetherin expression in the absence of antagonist (mock) was set to 100%. Error bars indicate SEM. One-way ANOVA with Bonferroni post-test analyses were performed for panels A and C to test for statistically significant differences between samples with and without (Mock) antagonist (*, p ≤ 0.05).

Fig 3. Tetherin inhibits VSV spread.

(A) 293T cells were transiently transfected with rising amounts of plasmid encoding human tetherin. At 24 h post transfection cells were infected with VSV at an MOI of 0.001 washed and viral titers present in culture supernatants were determined at 8 h post infection. Titers measured in the absence of tetherin were set to 100%. The average of six independent experiments is shown, error bars indicate standard error of the mean (SEM). Unpaired two-tailed Student’s t-test was used to examine whether differences in titers obtained from cells transiently expressing tetherin versus cells transfected with empty expression vector are of statistical significance (*, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.001). (B) HeLa cells were transfected with the indicated siRNAs. At 48 h post transfection, cells were harvested and stained with anti-tetherin antibody. Cell staining was then analyzed by flow cytometry. The results of a single representative experiment carried out with triplicate samples are shown, in which tetherin surface expression levels in cells transfected with control siRNA (scrambled) were set as 100%. Error bars indicate standard deviation (SD). An unpaired two-tailed Student’s t-test was used to assess statistical significance (***, p ≤ 0.001). (C) HeLa cells were transfected with the indicated siRNAs and at 24 h post transfection cells were infected with VSV at an MOI of 0.005 At 12 h post infection, viral titers in culture supernatants were determined. The results of a single representative experiment conducted with triplicate samples are shown and were confirmed in two separate experiments. Error bars indicate standard deviation. To test whether differences in VSV titers measured for cells transfected with control (scrambled) and tetherin-specific siRNA were statistically significant, an unpaired two-tailed Student’s t-test was performed (**, p ≤ 0.05).

Fig 4. Mutagenesis of the GXXXG motif in the transmembrane domain of VSV-G.

Schematic representation of the VSV* genome in which the authentic viral open reading frames (ORFs, VSV-N, -P, -M, -G and -L; light grey) are surrounded by non-translated regulatory elements (white). The additional ORF for eGFP (dark grey) was inserted between the ORFs for VSV-G and -L (restriction sites used for cloning and further modification of the genome are highlighted). The transmembrane domain (underlined amino acids) of VSV-G wt contains a GXXXG motif (amino acid residues 473–477) that has been mutated to LXXXL.

Fig 5. The GXXXG motif in the transmembrane domain of VSV-G is required for tetherin antagonism in transfected cells.

(A) 293T cells were transfected with plasmids encoding the indicated glycoproteins or mock-transfected. Expression was determined by Western blot analysis using anti-VSV-G antibody (concentrated supernatants from hybridoma CL-2700). Detection of β-actin expression served as loading control. (B) The average of four independent experiments conducted as described for panel (A) and quantified via the ImageJ program is presented. Expression of VSV-G wt was set to 100%. (C) Incorporation of VSV-G wt and mutant LXXXL into VSV pseudotypes was investigated by Western blot analysis using an anti-VSV-G antibody (concentrated supernatants from hybridoma CL-2700). To ensure that similar amounts of pseudotypes were analyzed, levels of particle-associated M proteins were determined using an anti-VSV-M antibody. Pseudotypes harboring no glycoprotein (Mock) served as negative control. The results of a single immunoblot are shown from which irrelevant lanes were cut out. Similar results were obtained in two separate experiments. (D) 293T cells were transduced with equal volumes of the VSV pseudotypes described in panel (C). At 24 h post transduction, luciferase activity in cell lysates was measured. Transduction driven by VSV-G wt was set as 100%. The average of three independent experiments is shown. Error bars indicate SEM. (E) 293T cells were cotransfected with plasmids encoding Gag, the indicated glycoproteins and tetherin. Expression of Gag in supernatants and cell lysates was determined by Western blot. Detection of β-actin expression served as loading control. (F) The average of three independent experiments conducted as described for panel (E) and quantified via the ImageJ program is presented. Error bars indicate standard error of the mean (SEM). Release of Gag from cells coexpressing the highest amount of VSV-G and tetherin was set to 100%. For all graphs (B, D, F), paired two-tailed Students’ t-tests were performed to assess whether differences between VSV-G wt and LXXXL mutant were of statistical significance.

Fig 6. The GXXXG motif is dispensable for viral spread in tetherin-positive cells.

(A) HeLa cells were transfected with the indicated siRNAs and subsequently infected with VSV wt or LXXXL mutant at an MOI of 0.005 for 1 h. Virus titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (B) The experiment was carried out as described for panel (A) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers measured for untransfected control cells were set to 1. Error bars indicate standard error of the mean (SEM). (C) Vero cells stably expressing human tetherin (Vero-Tetherin) or stably containing empty vector (Vero) were infected with VSV wt or mutant LXXXL. Viral titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (D) The experiment was carried out as described for panel (C) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers obtained from the respective control Vero cells were set to 1. Error bars indicate SEM. One-way ANOVA with Bonferroni post-test analyses were performed (B and D) to test statistical significance between selected groups (***, p ≤ 0.001).