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.
Highlights
The Alphavirus genus of the Togaviridae family contains a wide variety of human and animal pathogens
Many viruses encode proteins containing intrinsically disordered domains, whose functions are as yet unknown. 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
Geographically isolated viruses have evolved to utilize different cellular proteins: the nsP3 hypervariable domain (HVD) of Venezuelan equine encephalitis virus (VEEV) binds all members of the Fragile X syndrome (FXR) family, while nsP3 HVDs of Sindbis and chikungunya viruses interact with G3BP proteins
Summary
The Alphavirus genus of the Togaviridae family contains a wide variety of human and animal pathogens. The alphavirus genome is a single-stranded RNA of positive polarity. It is approximately 11.5 kb in length, mimics the structure of cellular mRNAs and serves as a template for translation of four nonstructural proteins, nsP1-4. These proteins are initially synthesized as polyprotein precursors P123 and P1234 and processed into their individual components: nsP1, nsP2, nsP3 and nsP4. This differential processing regulates the synthesis of the negative-strand genome replication intermediate, viral genome and subgenomic RNA (G RNA and SG RNA) at different steps of virus replication. The SG RNA is translated into the viral structural proteins: capsid, E2 and E1, which package the viral genome into infectious virions [1]
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