Venezuelan equine encephalitis virus non-structural protein 3 dictates superinfection exclusion in mammalian cells

Abstract

Superinfection exclusion (SIE) prevents secondary infections of already infected cells. Arthritogenic alphaviruses induce SIE via early proteolytical cleavage of replicase precursor by non-structural protein 2 (nsP2). Here, we explore the SIE mechanism of the encephalitic Venezuelan equine encephalitis virus (VEEV). Using single-cell imaging techniques and VEEV replicons encoding green or red fluorescent proteins, we observed full SIE capacity in three hours. Transient expression of VEEV nsP3, but not nsP2, reduced alphavirus replication, suggesting a key role for VEEV nsP3 in the SIE mechanism. In particular, the VEEV nsP3 C-terminal hypervariable domain (HVD) was found to be required and sufficient for the SIE of VEEV and the more distantly related Sindbis virus. As the nsP3 HVD is known to bind multiple host proteins to form RNA replication complexes and modulate the cellular stress response, we propose that sequestering essential host protein(s) by VEEV nsP3 interferes with RNA replication of the superinfecting alphavirus.

Fig. 1. Single-cell measurements of VEEV replicon RNA amplification in Vero cells.

a Graphical illustration of the VEEV replicon (VEEVrep) RNA, and virus genomes (VEEV and SINV-eGFP) used in this study. b Schematic representation of experimental approach. VRP-mCherry (10 VRPs/cell) and/or VRP-eGFP (10 VRPs/cell) were transduced in Vero cells. Twenty-four hours post transduction, cells were fixated and Hoechst stained. Fluorescence microscopy images were analyzed using a customized CellProfiler pipeline to quantify mCherry-, eGFP-, and dual-positive cells of the total number of Hoechst-stained cells. Image generated with Adobe Illustrator using Office Word icons. c Percentage of mCherry- and eGFP-positive cells after single and co-transductions of VRPs. Bars represent the mean and standard deviation of two independent experiments with each of eight biological replicates, ****P < 0.0001(detailed information in Supplementary Table 1).

Fig. 2. VEEV-induced SIE over time.

a Schematic representation of the experimental approach. Vero cells were transduced with VRP-mCherry (10 VRPs/cell) and, at indicated time points, transduced with VRP-eGFP (10 VRPs/cell). b Twenty-four hours post primary transduction, fluorescent cells were visualized using fluorescence microscopy. c Images were analyzed using a customized CellProfiler pipeline to quantify mCherry-, eGFP-, and dual-positive cells of total fluorescent cells. Bars represent the mean and standard deviation of two independent experiments with each of eight biological replicates. Asterisks indicate significant differences in the ratio of mCherry-, eGFP-, and dual-positive cells compared to 0 h and between the following time intervals (****P < 0.0001, detailed information in Supplementary Table 3).

Fig. 3. Replication kinetics of VEEV and SINV-eGFP in the presence of VRPs.

Vero cells were single, simultaneous, or sequential transduced with VRPs (10 VRPs/cell) and infected with a VEEV or b SINV-eGFP (10 TCID₅₀/cell). The virus titers were determined by end-point dilution assays (detection limit 1 × 10³ TCID₅₀/ml). Error bars indicate the standard deviation of the mean titers (n = 2).

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).