Figure 3. Overexpression of viperin controls CHIKV replication.

(A) Schematic representation of the domain organization of viperin and the various mutant constructs used. Viperin contains an ER targeting N-terminal amphipathic α-helical domain (aa 1–42) and a radical SAM catalytic domain (aa 77–209). WT viperin, SAM domain mutant of viperin (C₈₃A/C₈₇A/C₉₀A), and a truncated viperin mutant (aa 43–361) are shown. (B) HEK 293T cells were transfected with the various plasmids for 24 hours before infection with HI CHIKV or CHIKV (MOI 2.5). Cells were harvested at 12 hpi and analyzed for CHIKV infectivity by flow cytometry. Histogram plots of percent CHIKV Ag⁺ cells in various cell populations are representative of 3 independent experiments. (C) Graphical presentation of histogram plots in B. Data are expressed as mean ± SD of percent CHIKV Ag⁺ cells relative to vector-transfected cells infected with CHIKV (n = 3). *P < 0.05, 1-way ANOVA with Tukey’s post-test. Horizontal dotted line represents the mean percent CHIKV Ag⁺ cells detected in control. (D) Viral load was determined by qRT-PCR using specific primers against the negative-strand nsP1 RNA. Data are mean ± SD (n = 3). Horizontal dotted line represents the mean amount of RNA detected in control cells. **P < 0.01. (E) Viperin expression was detected with anti–c-Myc antibody. CHIKV nsP2 expression in the infected cells was detected with anti-nsP2 antibody. Detection of α-actin expression served as a loading control. Immunoblots are representative of 3 independent experiments.