FIG. 6.
Mutational analysis of the VP35 protein. (A) Expression of VP35 variants. Retroviral vectors expressing GFP, VP35, VP35(R312A), or VP35(dN200) were transfected together with HIVtrans and vesicular stomatitis virus G into 293T cells. Forty-eight hours after transfection, supernatant was used to transduce Vero cells. Expression of VP35 from transduced Vero cells was examined by Western blotting using anti-VP35 and anti-FLAG antibodies. (B) Effects of VP35 variants on viral resistance to interferon. Transduced Vero cells were either treated with 1,000 U/ml human alpha interferon (IFN+) (Sigma) for 20 h or left untreated (IFN−) and infected with a Δγ134.5 mutant at 0.05 PFU per cell. Viruses were harvested at 72 h after infection, and the virus titers on Vero cells were determined. The data represent the averages of three experiments with the standard deviations indicated by error bars. (C) Cytopathic effects. Images were taken from a representative experiment as described above for panel B. (D) Phosphorylation states of PKR and eIF-2α. Transduced Vero cells were either treated with human alpha interferon (+IFNα) (Sigma) or left untreated (−IFNα) and then infected with a Δγ134.5 mutant infection at 1.0 PFU per cell. Infected-cell lysates were prepared at 24 h after infection and subjected to immunoblot analysis using the indicated antibody. The ratio between phosphorylated eIF-2α and total eIF-2α in each lane was measured as described in the legend to Fig. 3. P-PKR, phosphorylated PKR.