New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes

Abstract

The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.

Author Summary

Many viruses encode proteins containing intrinsically disordered domains, whose functions are as yet unknown. Here we show that such a domain (HVD) in the alphavirus nsP3 protein orchestrates assembly of viral replication complexes through interaction with RNA-binding cellular factors. Surprisingly, geographically isolated viruses have evolved to utilize different cellular proteins: the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV) binds all members of the FXR family, while nsP3 HVDs of Sindbis and chikungunya viruses interact with G3BP proteins. Despite being in different families, G3BPs and FXRs have similar domain organization, and assemble into higher order complexes, such as stress granules. Alphaviruses exploit their abilities for complex self-assembly and RNA binding to build RNA-containing pre-replication complexes. Using CRISPR/Cas9 mediated knockouts, we show that deletion of all homologs strongly affects virus replication, while knockout of a single FXR or G3BP homolog has no or mild effect. Our data suggest that an alphavirus HVD serves as a hub to recruit host factors for replication complex assembly and may determine virus adaptation to distinct cellular environments. Notably, the improved understanding of HVD interactions allows alphavirus replication to be switched from an FXR- to G3BP-dependent mode and opens new possibilities for development of antiviral therapeutics.