SIE has been described for a broad spectrum of viruses, implying a conserved evolutionary advantage that might promote viral survival in general and limit 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 alphavirus 26, 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 minutes 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 one to three hours 25, 27, 33. Similarly, studies in mosquito cells demonstrated establishment of exclusion in one hour, which endured as alphavirus infections persist in these cells 34, 35.
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 superinfecting non-structural polyprotein 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) cells 28. Similar, two-point mutations in the SFV replicon nsP2 gene increased co-replication of alphaviruses in hamster fibroblast (BHK) cells 27, whereas the same mutations in the transiently expressed CHIKV nsP2 protease resulted in the contrary in mouse embryo fibroblast (3T3) cells 26. 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. (2022). 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.
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 29. This 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 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.
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. Variation 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. (2022) 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 fusion (Singh et al. 1997). Furthermore, variation in the level of exclusion has been demonstrated for various host cell types, despite the lack of involvement of de novo cellular transcription (Condreay and Brown 1986). 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, respectively 36, 37. The existence of different molecular mechanisms of SIE has also been described for distinct members of the subfamily Alphaherpesvirinae and the family Closteroviridae 4, 38, 39. 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 for 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 alphaviruses 24, 34, 35, this mechanism has been studied to create resistance in mosquitoes against superinfecting alphaviruses by primary infections of insect-specific alphaviruses or defective viral genomes 8, 10. The other way round, prevention of SIE might allow co-replication of co-transduced therapeutic VRPs, which require expression of multiple VRPs in the same cell 27. 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.