K124/K128 and K139/K142 of SFV capsid are critical for the VSR activity. (A and B) S2 cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and dsEGFP (0.3 μg), together with either empty plasmid or a plasmid encoding SFV capsid deletion mutant as indicated or FB2 (1 μg for each). At 48 hpt, the total RNAs were extracted for Northern blotting. Rp49 mRNA was used as the loading control. The expression of capsid mutations in S2 cells was detected by Western blotting (B). (C and D) The amino acid sequence alignments of alphaviral capsids are as follows: SFV4 (KP699763.1), SFV6 (KT009012.1), SFV-L10 (KP271965.1), SFV-A7 (Z48163.2), CHIKV (AOT86261.1), RRV (P08491.3), and SINV (AKZ17419.1). S2 cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and dsEGFP (0.3 μg), together with either empty plasmid or the plasmid encoding the indicated single-point mutations of SFV capsid (1 μg for each). At 48 hpt, the total RNAs were extracted for Northern blotting. The expression of capsid mutations in S2 cells was detected by Western blotting (D). (E to G) Schematic illustration of double-point mutations of SFV capsid. S2 cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and dsEGFP (0.3 μg), together with either empty plasmid or the plasmid encoding the indicated double-point mutations of SFV capsid (1 μg for each). At 48 hpt, the total RNAs were extracted for Northern blotting (E) and qRT-PCR (G); the expression of capsid mutations in S2 cells was detected by Western blotting (F). (H and I) 293T cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and EGFP-specific shRNA (shEGFP) (0.3 μg), together with either empty plasmid or a plasmid encoding SFV capsid protein or mutations (NB2; 1 μg for each). (H) At 48 hpt, the total RNAs were extracted, and the level of EGFP mRNA was examined by Northern blotting. GAPDH mRNA was used as the loading control. (I) The expression of capsid mutations in 293T cells was detected by Western blotting. (J and K) 293T cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and EGFP-specific siRNA (siEGFP) (50 nM), together with either empty plasmid or a plasmid encoding capsidWT, capsidK124A/K128A, capsidK139A/K142A, or NB2 (1 μg for each). At 48 hpt, the total RNAs were extracted, and the level of EGFP mRNA was examined by Northern blotting. (K) The expression of capsid mutations in 293T cells was detected by Western blotting.