Fig. 4: The role of VEEV non-structural proteins in SIE.

a Experimental design of SIE assay in which Vero cells were transfected with mCherry control or non-structural protein (nsPx) expression plasmids (dashed lines indicate individually expressed proteins resulting from FMDV-2A ribosome skipping), followed by transduction of b VRP-eGFP (10 VRPs/cell), or infection of c SINV-eGFP (10 TCID₅₀/cell) 24 h post transfection. At 48 h post transfection, analyses were performed. The CMV-driven nsP1-4 expression plasmid (VEEV replicon) carried the VEEV 5’ and 3’UTRs (black lines), VEEV non-structural genes, and 26 S promoter that enhanced the expression of mCherry. In the nsP2, nsP3, and nsP23 expression plasmids, mCherry was fused to the non-structural genes via an FMDV 2A linker (ribosome skipping element). b, c The average percentage of relative dual-positive cells and standard deviation (two independent experiments with each of eight biological replicates) were determined by fluorescent cell quantification using fluorescence microscopy and the CellProfiler pipeline. Distinct letters indicate significant differences in relative dual-positive cells (P < 0.01, detailed information Supplementary Table 5). d Based on the nsP3 expression plasmid, five additional plasmids were developed containing the conserved macrodomain (macro), conserved alphavirus unique domain (AUD), hypervariable domain (HVD), or a combination of two domains. Dashed lines indicate individually expressed proteins resulting from FMDV-2A ribosome skipping. e Vero cells were transfected with mCherry control, nsP1–4 expression plasmid, or nsP3 domain expression plasmids, followed by transduction of VRP-eGFP (10 VRPs/cell) 24 h post transfection. The average percentage of relative dual-positive cells and standard deviation (two independent experiments with each of eight biological replicates) were determined by fluorescent cell quantification using fluorescence microscopy and the CellProfiler pipeline 48 hours post transfection. Asterisks indicate significant differences in relative dual-positive cells compared to control (**P < 0.01, ****P < 0.0001, detailed information in Supplementary Table 6).