Introduction

Many viruses have established a mechanism that prevents a secondary infection of a closely related virus in the same host cell, while the host cell is still susceptible to infection with more distantly related or unrelated viruses. This selective exclusion mechanism is referred to as superinfection exclusion (SIE). SIE was first discovered as a cross-protection mechanism in crops; plants pre-infected with a mild virus isolate were protected against more severe viral diseases1,2,3,4. Later, SIE was also demonstrated for a number of animal viruses, such as human papillomavirus5, vaccinia virus6, and hepatitis C virus7. Discovery of SIE induced by arboviruses, which are viruses transmitted by arthropod vectors that cause disease in vertebrates, raised the question whether SIE can be utilized to limit virus dispersal by mosquitoes in a similar cross-protective manner as in plants8,9,10,11,12.

Alphaviruses (genus Alphavirus, family Togaviridae) are enveloped, positive-strand RNA viruses that are typically transmitted by mosquitoes to vertebrate hosts in which they cause severe arthritogenic or encephalitic disease, depending on the viral species. The alphavirus genome contains two open reading frames (ORFs). The first ORF encodes a large polyprotein which is cleaved into four non-structural proteins (nsP) in a cleavage process that is strictly regulated by the viral protease nsP213,14. The polyprotein cleavage intermediates and mature non-structural proteins form replication complexes for negative and positive-strand RNA synthesis. The second alphavirus ORF encodes the structural polyprotein, which is translated from a subgenomic RNA and results in the expression of capsid and envelope glycoproteins15.

The SIE mechanism is not universal among virus families; some viruses block host cell entry5,7,16,17, while others inhibit genome replication of the superinfecting virus16,18,19. Previous studies demonstrated that alphavirus SIE is independent of interferon signaling, host cell transcription, cell entry, and viral structural protein translation20,21,22,23,24,25,26. Thus, alphavirus SIE appears to act at the level of RNA replication. However, the exact molecular mechanism of alphavirus SIE and the viral proteins involved remain elusive.

Several hypotheses have been proposed to explain the limited co-replication of two alphaviruses in a single cell. One of these hypotheses attributes SIE to a block in negative-strand RNA synthesis of the superinfecting virus by premature proteolytic cleavage of the non-structural polyprotein by the viral nsP2 protease of the first, incoming alphavirus. This is supported by findings that mutations in the arthritogenic chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) nsP2 increased co-replication of another alphavirus in the same cell12,27,28. However, this may not be the only molecular mechanism as other studies contradict the universal role of nsP2 in SIE8,26. Based on quantitative live-cell and single-molecule imaging, it was suggested that the constraint in superinfecting viral RNA replication is coupled to a limitation in cellular resources, defined as the total cellular carrying capacity29.

To increase our understanding of alphavirus SIE, we here investigate SIE induced by an encephalitic alphavirus, Venezuelan equine encephalitis virus TC-83 (VEEV). We identify the time frame in which VEEV establishes SIE, and the specific roles of nsP2, nsP3, and individual protein domains therein.

Results

Competition between co-transduced VEEV replicon particles

To assess the efficiency of VEEV-induced SIE, we transduced VEEV-based virus replicon particles (VRPs) that expressed mCherry (VRP-mCherry) or eGFP (VRP-eGFP) fluorescent markers in Vero cells alone or in combination (Fig. 1a, b). These VRPs encapsulate self-amplifying VEEV replicon RNA vectors based on VEEV RNA templates that lack structural genes. Instead of the structural genes, the VEEV replicon RNA vector carries a fluorescent marker gene (mCherry or eGFP), which allows visualization of VEEV replicon RNA amplification by fluorescence microscopy. Based on fluorescence microscope images, mCherry-, eGFP-, and dual-positive cells were quantified using a customized pipeline in the CellProfiler software.

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

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).

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Co-transductions of VRP-mCherry and VRP-eGFP resulted in a significant reduction of the percentage of mCherry- and eGFP-positive cells compared to single transductions of either VRP-mCherry or VRP-eGFP (Fig. 1c). In these simultaneous co-transductions 42% mCherry-positive and 59% eGFP-positive cells were detected, compared to 60% mCherry-positive and 80% eGFP-positive cells in single VRP transductions. Furthermore, only 5.5% of all cells were dual-positive, expressing both green and red fluorescent proteins (Supplementary Table 1). This percentage was less than the statistically independent probability prediction of 25% (0.42 * 0.59 × 100 = 25%). A low percentage of dual-positive cells was also observed with co-electroporation of VEEV replicon-mCherry and VEEV replicon-eGFP in Vero cells, but this was not further quantified. Altogether, this demonstrated competition between the two VEEV replicon vectors, which hindered the co-replication of both VEEV replicon vectors in the same cell.

VEEV SIE increases over time and is completed in 3 h

To capture the time frame of VEEV-induced SIE, we first transduced Vero cells with VRP-mCherry followed by sequential transductions of VRP-eGFP with different time intervals (Fig. 2a). When VRP-eGFP was transduced 1 h post VRP-mCherry transduction, no significant differences were seen compared to the simultaneous VRP co-transduction in the ratios of mCherry-, eGFP-, and dual-positive cells (Fig. 2b, c). Both intervals (0 and 1 h) resulted in a minor percentage of dual-positive cells (5.8–8.4% of total fluorescent cells), while the majority of cells expressed a single fluorescent marker. Increasing the time interval between the first and second transduction caused a significant change in the ratios of mCherry-, eGFP-, and dual-positive cells. Transducing the VRP-eGFP two hours post primary VRP-mCherry transduction resulted in reduced levels of eGFP- and dual-positive cells compared to simultaneous transductions, whereas the percentage of mCherry-positive cells increased. Increasing the VRP-eGFP transduction up to three hours further reduced the percentage of eGFP- and dual-positive cells, and increased the percentage of mCherry-positive cells. This trend stagnated after three hours; the percentage of mCherry-positive cells did not further increase after prolonging the time interval between VRP transductions. The plateau in mCherry-positive cells (65–70% mCherry-positive cells of total cells) corresponded to the percentage of mCherry-positive cells after a single VRP-mCherry transduction (Fig. 1c). Note that the reduced percentage of eGFP-positive cells was not caused by the difference in incubation time (time between transduction and fluorescence microscopy), as an incubation time of 16 h instead of 24 h did not cause a significant difference in eGFP-positive cells after a single VRP-eGFP transduction (Supplementary Table 2).

Fig. 2: VEEV-induced SIE over time.
figure 2

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).

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In conclusion, the decreased percentage of eGFP- and dual-positive cells correlated with the extended time interval between primary VRP-mCherry and secondary VRP-eGFP transduction. In three hours, the primary transduced VRP-mCherry established a maximum level of SIE in Vero cells, preventing replication of the secondary transduced VRP-eGFP. Interestingly, this timing correlated with the progression in the RNA synthesis30, suggesting a relationship between SIE and RNA synthesis.

VEEV induces homologous and heterologous SIE

In our previous research, we demonstrated that primary transduction of VRPs in Vero cells reduced the alphavirus production by 1 h and later added VEEV, Barmah Forest virus, CHIKV, and Una virus31. Also simultaneously introducing VRPs and wild-type alphaviruses resulted in a comparable level of inhibition. Here, we repeated this experiment with VEEV and SINV-eGFP (Fig. 1a). Similar to VEEV, SINV replication was inhibited by primary or simultaneous VRP introduction (Fig. 3). Although the overall effect is similar, the molecular mechanism underlying homologous (VEEV) and heterologous (SINV-eGFP) SIE might differ.

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

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).

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Transient expression of VEEV nsP3 inhibits co-expression of superinfecting alphavirus

The previous experiments demonstrated limited co-replication of VEEV replicons expressing mCherry and those expressing eGFP. We hypothesized that one or more of the non-structural proteins are involved in SIE rather than any of the structural proteins that encapsulated the replicon RNA as VRPs. To examine the role of the non-structural proteins in SIE, expression plasmids were developed that transiently expressed solely mCherry or mCherry coupled to VEEV nsP1–4 (VEEV replicon), nsP2, nsP3, or nsP23 via an FDMV 2A ribosome skipping element (Fig. 4a).

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

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).

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Vero cells were transfected with the expression plasmids, followed by VRP-eGFP transduction or SINV-eGFP infection to compare the level of co-expression of mCherry and eGFP, and thereby the construct’s ability to induce SIE (Fig. 4a). As the percentage of mCherry-positive cells varied between the expression plasmids due to differences in transfection efficiency (Supplementary Table 4), a direct comparison of dual-positive cells would not allow a fair comparison of the induced level of SIE. Therefore, the relative percentage of dual-positive cells was determined based on the number of dual-positive cells divided by the total number of mCherry-positive cells per expression plasmid (Fig. 4b, c). The transfection with the replicon construct (nsP1–4) resulted in a significantly lower level of relative dual-positive cells in comparison to the control construct that only expressed mCherry (Fig. 4b, c). Similar significant differences were observed after nsP3 and nsP23 construct transfections, whereas the nsP2 constructs showed a comparable level of relative dual-positive cells to the control construct (Fig. 4b, c). Altogether, this suggested a role for nsP3 in SIE, which was also executed in the nsP23 conformation.

The nsP3 hypervariable domain has a leading role in SIE

Next, we evaluated the influence of the individual nsP3 domains on SIE. NsP3 consists of three domains: the conserved macrodomain, the conserved alphavirus unique domain (AUD), and the hypervariable domain (HVD)32,33. We developed five expression plasmids containing individual domains or a combination of two domains (Fig. 4d). The expression plasmids were transfected into Vero cells, and VRP-eGFP were transduced 24 h post transfection as previously described (Fig. 4a). Of all expression plasmids, the level of mCherry and eGFP co-expression was only significantly lower compared to the control plasmid for the expression plasmids containing the HVD (Fig. 4e). However, the reduction in co-expression was not as strong as the nsP1–4 or nsP3 expression plasmids (Fig. 4e). This indicates a key role for the HVD in the VEEV SIE mechanism compared to the conserved macrodomain and AUD, but also demonstrated a synergetic effect of the combination of the three domains in nsP3. We conclude that the VEEV nsP3 HVD is essential and sufficient for the SIE of the homologous VEEV.

Discussion

SIE has been described for a broad spectrum of viruses, implying a conserved evolutionary advantage that might promote viral survival in general and limit the accumulation of defective recombined genomes. Despite the universal existence of this phenomenon, the mode of action can be diverse yet is often unknown. There are only two general characteristics: the mechanism of exclusion is induced post primary virus entry and only observed in the infected cell (cell-intrinsic)26. For alphaviruses, the current consensus proposes an early initiation of SIE by the non-structural proteins of the primary alphavirus, which inhibits RNA replication of the superinfecting alphavirus26,28,29. However, the molecular mechanism of this process is still elusive. To shed some light on the alphavirus SIE mechanism, we here examined the timing of VEEV-induced exclusion and the role of VEEV nsP2, nsP3, and nsP3 individual protein domains in this mechanism.

By using single-cell imaging and VRPs encoding two distinct fluorescent markers, we demonstrated immediate initiation of the VEEV exclusion mechanism in Vero cells. The degree of inhibition of the secondary VEEV replicon increased over time and reached its maximum capacity within the short time span of three hours. These results match previous results in alphavirus SIE studies on SINV and SFV, demonstrating that 15 min of primary infection was sufficient to inhibit replication of a superinfecting alphavirus in mammalian cells. Increasing the time interval between primary and superinfection further decreased the level of co-replication, which stagnated in 1–3 h25,27,34. Similarly, studies in mosquito cells demonstrated the establishment of exclusion in one hour, which endured as alphavirus infections persisted in these cells35,36.

Research on the molecular mechanism of alphavirus SIE has led to contrasting findings. Several studies have focused on the nsP2 hypothesis that proposes premature proteolytic cleavage of the second cleavage site in the polyprotein of the superinfecting virus by the primary alphavirus nsP2 protease, which prevents negative-strand RNA synthesis by the superinfecting virus. This proposed mechanism has been supported by weakened SIE phenotypes after mutations in the nsP2 gene of the primary virus. Mutations in CHIKV and SINV nsP2 that blocked the proteolytic cleavage of the viral polyprotein resulted in an increased co-expression of alphaviruses in Aedes albopictus (C6/36) cells28. Similar, two-point mutations in the SFV replicon nsP2 gene increased co-replication of alphaviruses in hamster fibroblast (BHK) cells27, whereas the same mutations in the transiently expressed CHIKV nsP2 protease resulted in the contrary in mouse embryo fibroblast (3T3) cells26. The latter study reported that CHIKV SIE is not mediated by a single viral protein, as transient expression of non-structural proteins did not protect cells from a CHIKV infection. This contrasting finding was explained by the addition of a start-codon in front of the nsP2 gene that allowed transient expression of nsP2 from the expression plasmid but also impacted protease activity according to Cherkashchenko et al.28. In our study, VEEV nsP2 was transiently expressed without this additional start codon since we applied a FMDV 2A linker that mediated ribosome skipping, but we did not observe nsP2-induced exclusion. This expression strategy yields a VEEV nsP2 with a residual proline at the N-terminus, which may affect its enzymatic activity. This means we cannot conclusively rule out an additional role for nsP2 in SIE.

We here reported a relationship between VEEV nsP3 and SIE. Transient expression of VEEV nsP3 caused a similar reduction of VRP-eGFP and SINV-eGFP, as compared to the VEEV nsP1–4 and the VEEV nsP23 constructs. Individual expression of the nsP3 domains demonstrated that the HVD played a main role in the exclusion. These results may indeed suggest that SIE is a result of a limited cellular carrying capacity during RNA replication29, but based on our methods we cannot dismiss restrictions in cellular entry or translation. The limited carrying capacity might be a consequence of insufficient cellular host proteins essential in the formation of nsP3-assembled replication complexes. In this scenario, the host proteins (e.g., FMRP or CD8137,38,39), are sequestered by binding sites in the nsP3 HVD. When the host protein(s) are sequestered by the nsP3 of the first alphavirus, this may limit the superinfecting alphavirus in replication complexes formation to synthesize RNA. VEEV variants with mutated HVDs that abrogate interactions with host factors replicate different efficiencies in BHK-21 cells40. It would be interesting to investigate if such mutants are still susceptible to SIE. In addition to a direct role of VEEV nsP3 in SIE, for heterologous SIE of VEEV VRPs on SINV-eGFP, another contributing factor may be the faster RNA replication of the (shorter) VEEV replicon, which is also reflected by significantly higher virion production of VEEV vs. SINV (Fig. 3).

The contrary findings on alphavirus SIE make it hard to draw firm conclusions on the molecular mechanism(s), which might be more complex than a universal direct effect of a single non-structural protein. Variations in the alphavirus SIE mechanism would explain the divergent observations of SIE dependent on the experimental design, e.g. differences in viruses, cells, and delivery methods. Cherkashchenko et al.28 showed that the observed nsP2-induced exclusion did not completely depend on the protease-activity, but was also caused by a suppressed replicase activity. Additional modes of viral inhibition have also been reported after accomplishing RNA replication exclusion, e.g. inhibition of virus binding and arrest in virus-endosome fusion25. Furthermore, variation in the level of exclusion has been demonstrated for various host cell types, despite the lack of involvement of de novo cellular transcription36. We focused on SIE in Vero cells, but SIE in more relevant cell types for VEEV infection (e.g., dendritic cells and macrophages) might differ and requires further investigation. The mode of exclusion might also vary between alphaviruses, similar to the difference in alphavirus cellular transcriptional shutoff by either nsP2 or capsid for arthritogenic and encephalitic alphaviruses, respectively41,42. The existence of different molecular mechanisms of SIE has also been described for distinct members of the subfamily Alphaherpesvirinae and the family Closteroviridae4,43,44. Thus, one defined molecular SIE mechanism within a group of related viruses does not directly exclude the existence of another mechanism in that group, and this also appears to apply to alphavirus.

Unraveling the alphavirus-induced SIE mechanism contributes to our understanding of mutual alphavirus interactions. The mechanism may explain the maintenance of viral genome integrity by prevention of co-infections. Furthermore, this fundamental knowledge can be applied to develop antiviral strategies or optimize alphavirus-based therapeutics. As persistently infected mosquito cells continuously exclude superinfecting alphaviruses24,35,36, this mechanism has been studied to create resistance in mosquitoes against superinfecting alphaviruses by primary infections of insect-specific alphaviruses or defective viral genomes8,10. The other way round, prevention of SIE might allow co-replication of co-transduced therapeutic VRPs, which require the expression of multiple VRPs in the same cell27. We anticipate that additional studies on the alphavirus SIE mechanism will be relevant to assess interactions between alphaviruses and to develop more efficient biotechnological applications.

Methods

Cells and viruses

BHK-21 cells (clone 13, ECACC 85011433) were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 5% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Sigma-Aldrich). Vero cells were cultured at 37 °C with 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. One day prior to the experiment, Vero cells were seeded in wells of a 24-well plate in a culture medium with HEPES (Gibco).

Venezuelan equine encephalitis virus TC-83 (VEEV, NCBI Genbank L01443.1) and recombinant Sindbis virus expressing eGFP (SINV)45 stocks were grown on Vero cells. Stock titers were determined by end-point dilution assays on Vero cells.

Production of VEEV replicon particles

VEEV-based virus replicon particles (VRPs) were produced to transduce the VEEV replicon RNA in cells. In short, the pUC57-T7-VEEV replicon and pUC57-T7-helper plasmids encoding respectively, VEEV non-structural proteins and structural proteins were isolated (NucleoBond Xtra Midi EF purification kit, Macherey-Nagel), linearized (NotI restriction enzyme, New England BioLabs), in vitro-transcribed (T7 RNA polymerase, New England BioLabs) and capped (Cap structure analog, New England BioLabs). The synthesized RNA constructs were introduced into 8×106 BHK-21 cells by electroporation (two pulses of 850 V/25 μF, 0.4 cm cuvettes, Gene Pulser Xcell, Biorad). After electroporation, cells were resuspended in 10 ml DMEM supplemented with 10% FBS and 1% penicillin–streptomycin, and incubated at 37 °C with 5% CO2. After 24-h incubation, the supernatant containing the VRPs was harvested, centrifuged (15 min, 500g), and stored at −80 °C. VRP concentrations were determined by serial dilutions on Vero cells and quantified based on the expression of the fluorescent marker while considering the dilution factors.

Construction of expression plasmids

Several CMV-driven expression plasmids carrying mCherry and one or several VEEV non-structural genes derived from the VEEV TC-83 genome were constructed. The VEEV nsP1-4 (replicon) expression plasmid, pUC57-CMV-VEEV replicon, carried the VEEV 5’ and 3’UTRs, the VEEV non-structural genes, and subgenomic 26 S promoter sequence followed by a multiple cloning site in which mCherry gene was inserted to allow expression from the subgenomic mRNA. The nsP2, nsP3, and nsP23 expression plasmids were based on the pDest40-mCherry expression plasmid in which the VEEV non-structural gene(s) were connected to mCherry gene by a foot-and-mouth disease virus (FMDV) 2A linker46,47. The 2A linker mediates ribosome skipping, resulting in the production of mCherry and the nsP construct under study from the same mRNA molecule.

VEEV replicon particle transductions and virus infections

VRPs expressing mCherry (VRP-mCherry, 10 VRPs/cell) and VRPs expressing eGFP (VRP-eGFP, 10 VRPs/cell) were simultaneously transduced in Vero cells. In sequential transductions, VRP-mCherry (10 VRPs/cell) was first transduced followed by VRP-eGFP (10 VRPs/cell) after various time intervals. In simultaneous transductions/infections, VRP-mCherry and VEEV or SINV were added to Vero cells at the same time. In the sequential transductions/infections, VRP-mCherry was administered first followed by virus infection at indicated time points. As controls, VRP transductions and virus infections were performed independently of each other. In all cases, the cells with the VRPs and/or viruses were first incubated for 1 h at 37 °C. Thereafter, the mixture was replaced for a secondary VRP transduction, virus infection, or fresh culture medium with HEPES. In case of virus infections, supernatant samples were collected at several time points post infection to determine the virus titer by endpoint dilution assay on Vero cells. Furthermore, cells were prepared for fluorescence microscopy imaging 24 h post primary transduction/infection.

Expression plasmid transfections, VRP transductions, and virus infections

Vero cells were transfected with 250 ng of expression plasmid DNA using Lipofectamine2000 transfection reagent (Invitrogen) in Opti-MEM reduced serum medium (Gibco) according to the manufacturer’s instructions. Two hours post transfection, the transfection mixture was replaced by a fresh culture medium with HEPES, and cells were incubated for 24 h at 37 °C. Thereafter, cells were transduced with VRP-eGFP (10 VRPs/cell) or infected with VEEV or SINV-eGFP (10 TCID50 units/cell) for 2 hours, followed by medium replacement. After an additional incubation period of 24 h at 37 °C, cells were prepared for fluorescence microscopy imaging, and supernatant samples were collected to determine virus titers by end-point dilution assays on Vero cells.

Fluorescence microscopy imaging and single-cell image analysis

To prepare cells for fluorescence microscopy imaging, cells were washed with PBS, fixated in 4% paraformaldehyde in PBS for 10 min, permeabilized in 0.1% sodium dodecyl sulfate in PBS for 10 min, and stained with 1:100 Hoechst staining (ThermoFisher Scientific) in PBS for 2 min. Between all steps, cells were washed and eventually incubated in PBS. Hoechst-stained cell monolayers were imaged using the Zeiss Axio Observer Z1 inverted fluorescence microscope. These images were computationally analyzed by using the open-source image analysis tool CellProfiler 4.2.1. To allow high-throughput automated quantification of fluorescent cells, a customized pipeline was developed in which cell objects in the DAPI, rhodamine, and GFP channels were first separately classified, followed by an overlay of the cell objects and quantification. The pipeline and detailed protocols will be shared upon request.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9 software. Data were analyzed using ordinary one-way ANOVA followed by Turkey’s multiple comparisons test. Results were considered statistically different for P-values < 0.01.