Figure 4. ISGylation is responsible for the inhibition of viral RNA production in 67 mutant virus-infected cells.

(a) Viral protein synthesis in wt and 67 mutant virus-infected A549 cells expressing different levels of IFN-β-induced ISGylation. Cells were transfected with the indicated siRNAs (Ctrl: control siRNA; USP: USP18-specific siRNA; I: ISG15-specific siRNA; E1: Ube1L-specific siRNA) for 24 h, followed by 16 h of treatment with 1,000 IU ml⁻¹ human IFN-β and subsequent infection with 5 p.f.u. per cell of wt or 67 mutant virus. At 24 h after infection, cells were collected, and extracts were analysed by immunoblots probed with the indicated Abs. Quantitation of immunoblots was performed using ImageJ software (NIH). See also (Supplementary Fig. 1c,d). (b) Viral RNA synthesis in wt and 67 mutant virus-infected A549 cells expressing different levels of IFN-β-induced ISGylation. Cells were transfected with the indicated siRNAs for 24 h, followed by 16 h of treatment with 1,000 IU ml⁻¹of human IFN-β and subsequent infection with 5 p.f.u. per cell of wt or 67 mutant virus. At 24 h after infection, cells were collected, and the levels of the indicated viral mRNAs and vRNAs were determined by RT–PCR, normalized to the level of β-actin mRNA. The levels of viral mRNAs and viral vRNAs are shown as fold changes relative to the levels in the USP18 siRNA-treated, wt virus-infected sample. Results shown are mean +/− s.d. of three independent experiments, with each measurement performed in triplicate. P values were calculated using two-way analysis of variance (ANOVA) test. (c) Viral RNA synthesis in wt and 67 mutant virus-infected cells expressing endogenous levels of IFN-β-induced ISGylation. Cells were transfected with a control siRNA before IFN-β treatment followed by virus infection. Results shown are mean +/− s.d. of three independent experiments, with each measurement performed in triplicate. P values were calculated using two-way ANOVA test. ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.