Fig 2: RuV infection induces STAT1 activation and ISG expression. (A) Protein lysates from U373MG cells (a) or CCF-STTG1 cells (b) that were uninfected (M) or infected with RuV RA27/3 strain (+) at 2 and 4 days postinfection (dpi) (MOI of 1.8) were analyzed by immunoblotting for STAT1, Tyr701-phosphorylated STAT1 (p-STAT1), and RuV-encoded capsid protein. GAPDH protein expression was analyzed as a reference. Densitometric analysis of the capsid protein was performed with NIH ImageJ software. Signal intensity of the capsid protein was normalized to that of GAPDH, then shown as a relative ratio of U373MG cells infected with RuV at 2 dpi. (B) STAT1-EGFP fusion protein (green) translocated to the nucleus was detected in RuV-infected U373MG-STAT1-EGFP cells at 2 dpi by fluorescence microscopy. Typical cells positive for nuclear localization of EGFP fusion protein are shown. Nuclei were counterstained with Hoechst 33342 (blue). Images were acquired at a magnification of 40× objective lens. (C) Total RNAs extracted from U373MG cells infected with RuV RA27/3 strain (+) or heat-inactivated RuV RA27/3 strain (HI) or uninfected (M) at 2 and 4 dpi were subjected to RT-PCR for the detection of expression of MDA5, ISG56, OAS1, MX1, and viperin mRNAs. TBP mRNA was analyzed as a reference. The PCR products were analyzed by agarose gel electrophoresis. (D) Protein lysates from U373MG cells (a) or CCF-STTG1 cells (b) that were uninfected (M) or infected with RuV RA27/3 strain (+) at 2 and 4 dpi (MOI of 1.8) were analyzed by immunoblotting for the protein expression of ISGs (RIG-I, MDA5, ISG56, MX1, and viperin). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein was analyzed as a reference.

Fig 3: Silencing of MDA5 expression by siRNA reduced RuV-triggered IFN-β expression. (A) Effect of siRNA on the expression of MDA5 and RIG-I mRNAs. U373MG cells transfected with MDA5-targeting siRNA (siMDA5), RIG-I-targeting siRNA (siRIG-I), or a non-silencing control siRNA (siCtrl) at 9 nM were harvested at 48 h post-transfection. The mRNA expression levels of MDA5 and RIG-I were analyzed by qRT-PCR. Relative MDA5 or RIG-I mRNA levels in each sample were normalized to TBP and expressed as fold change relative to the corresponding control. Data are shown as the mean ± SEM (n = 3). Data were compared using a two-tailed Student t test with Welch’s correction. **, p < 0.01; ***, p < 0.001. (B) The expression of RuV-triggered IFN-β mRNA in siRNA-transfected U373MG cells. U373MG cells were infected with RuV RA27/3 strain at MOI of 1.8 at 48 h post-transfection of indicated siRNAs, harvested at 24 h postinfection, and subjected to qRT-PCR. Data are shown as the mean ± SEM (n = 3). Data were compared using a two-tailed Student t test with Welch’s correction. *, p < 0.05.