The antiviral protein Viperin suppresses T7 promoter dependent RNA synthesis–possible implications for its antiviral activity

Figures

• 

  Figure 1. Viperin inhibits T7-GFP expression. (a) Viperin is the only ISG specifically inhibiting expression from T7-GFP. HEK293-T7 cells were infected with ISG expressing lentiviruses. After 24 h the cells were co-transfected with T7-GFP and a CFP-YFP fusion protein expressed from a CMV promoter. The cells were analyzed 48 h post-transfection by Western blot with a GFP antibody. RFP expression was used to confirm lentivirus infection. Actin was used as a loading control. Bands showing the effect of viperin on T7-GFP are labeled with white arrow head. Quantification of the bands compared to luciferase control and normalized to actin is shown at the top. (b) Viperin inhibits T7-GFP expression in a dose dependent manner. HEK293-T7 cells were transfected with the indicated amounts of viperin-expressing plasmid. After 24 h the cells were transfected with a T7-GFP or CMV-GFP. The cells were lysed after 48 h and analyzed by Western blot using GFP, tubulin and viperin antibodies. Quantification was performed as described above and is shown at the bottom of each panel. Presented is a representative gel from at least 3 independent repeats. (c) Viperin inhibits T7-GFP expression. HEK293-T7 cells…

• 

  Figure 2. Mutations in the functional domains of viperin abolish its effect on T7-GFP expression. (a) Schematic representation of the viperin mutants used. (b) Subcellular Localization of the viperin mutants. Viperin mutants were co-transfected into Huh7 cells with a plasmid expressing calnexin-GFP as an ER marker. The cells were fixed 24 h post transfection and immunostained using a viperin antibody. (c) Western blot showing the effect of the various mutants on T7-GFP or CMV-GFP expression. HEK293-T7 cells were co-transfected with wild type viperin or the viperin mutants Δ42 N, Δ33 C and S1 and T7-GFP or CMV-GFP. The cells were lysed and analyzed by Western blot with GFP (green) and viperin (red) specific antibodies 24 h post-transfection. Tubulin (red, top) was used as a loading control. The effect of wild type viperin is marked with a white arrow head. Quantification of the bands compared to luciferase control is shown at the top of each panel. Full-length blots are presented in Supplementary Fig. 2.

• 

  Figure 3. The inhibition of T7-GFP expression by viperin is not at the translational level. (a) Viperin does not affect global protein synthesis. HEK293T cells were seeded in a 24-well plate. The next day the cells were transfected the indicated amounts of a viperin expressing plasmid and incubated for 48 h. The cells were then incubated at 37 °C in the presence of puromycin followed Western blotting with puromycin, viperin and tubulin specific antibodies. Cycloheximide (Chx) was used as a negative control. (b) Viperin does not inhibit cap or IRES dependent translation. HEK293-T7 cells were co-transfected with the indicated increasing amounts of viperin and a bicistronic plasmid expressing dsRed in a cap dependent manner and GFP from an IRES. The cells were lysed and analyzed by Western blot with viperin (red), GFP or RFP antibodies (green) 24 h post-transfection. (c) Viperin does not affect T7-polymerase protein levels. BHK-21 cells were seeded to 24-wells plate. The next day the cells were co-transfected with viperin or empty plasmid (50 ng/well) together with T7 polymerase and T7-GFP (400 ng/well). The cells we’re lysed and analyzed by Western blot 48 h post-transfection using T7…

• 

  Figure 4. Inhibition by viperin is not affected by T7-polymerase localization. (a) Intracellular localization of the NLS-T7-polymerase. Fluorescent microscopy images of BHK-21 cells co-transfected with T7-GFP and either T7-polymerase or NLS-T7-polymerase. Images were obtained 48 h post-transfection Bar = 10 µM. (b) The effect of viperin on NLS-T7 polymerase. BHK-21 cells were co-transfected with viperin, T7-GFP and either T7 polymerase or NLS-T7-polymerase. The cells were analyzed by Western blot using the indicated specific antibodies 48 h post transfection. (c) HEK293-T7 cells were transfected with viperin or a control plasmid as indicated. Cell lysates were subjected to immunoprecipitation 48 hours later, using A/G agarose beads and viperin specific antibody. Western blot analysis was then performed using anti-Viperin and anti-T7 antibodies. Full-length blots are presented in Supplementary Fig. 4.

• 

  Figure 5. Viperin inhibits T7 polymerase mediated RNA synthesis. (a) BSR-T7 cells were infected with bicistronic lentiviruses co-expressing either viperin or luciferase and RFP as a transfection control. The cells were transfected with T7-GFP 48 h post-infection and with BrU after additional 24 h. (b) A graph presenting cytosolic vs nuclear fluorescence intensity in vector transfected or viperin transfected individual cells. (c) Quantification of the results presented in B. Results are mean ± standard error of the cytoplasm to nuclear fluorescence intensity ratio from 31 cells in each treatment (***p < 0.0001, Student t-test). Bars = 20 µM.