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.

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Alphaviruses are a group of significant human and animal pathogens. The nearly 30 members of this genus are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts (54). Alphaviruses cause different diseases but have similar replication strategies and life cycles. In insect vectors, they cause persistent lifelong infections and viral replication does not critically affect the viability of the hosts (51). These viruses also often establish persistent infection in cultured mosquito cells. In contrast, vertebrate hosts usually develop acute infection that often results in disease prior to virus clearance by the immune system (27, 31). The infection of susceptible vertebrate cells typically leads to rapid cytopathic effect (CPE) (54) and cell death.Sindbis virus (SIN) is the prototype member of the Alphavirus genus. It can replicate productively in a wide variety of cell lines of insect and vertebrate origin and causes age-dependent encephalomyelitis in mice (28). As do all other alphaviruses, SIN enters the cells via receptor-mediated endocytosis. pHdependent fusion of viral and endosomal membranes leads to release of nucleocapsids into the cytoplasm (12) followed by nucleocapsid disassembly and genome replication (64).The SIN genome is a single RNA molecule of positive polarity, 11.7 kb in length. It contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tract and is translated by host-cell machinery similar to cellular mRNAs (52). The 5Ј two-thirds of the genome encodes the nonstructural proteins (nsPs), which comprise the viral components of the RNA replicase/transcriptase required for replication of the viral genome and transcription of subgenomic RNA. The 4.1-kb subgenomic RNA corresponds to the 3Ј third of the genome and is translated into structural proteins that form virus particles. Replication of SIN is very rapid and leads to high-level accumulation of virusspecific RNAs and structural proteins. Finally, a large number of viral particles released by budding from the cell surface disseminate the infection. Viral replication profoundly affects cell metabolism, with downregulation of host cell protein synthesis playing the central role (31). Cells lose integrity and die within 24 to 48 h postinfection (p.i.). In many cell types, death is accompanied by apoptotic changes (37). Replication of viral RNA and/or accumulation of SIN nsPs is sufficient to cause translational shutoff and cell death. However, expression of viral structural proteins significantly accelerates the development of CPE (18).In recent studies, we selected SIN self-replicating RNAs (replicons) that were capable of persisting in some vertebrate cell lines for an unlimited number of passages without causing CPE (1, 16). These noncytopathic replicons replicated less efficiently than the parent and contained point mutations in the gene encoding one of the nsPs, nsP2. One adaptive mutation, P 726 3L, was at the same position as that found in the SIN-1 variant that also exhibits reduced cytopathogenicity (11,63).

Replication of alphaviruses in vertebrate cells strongly affects cell physiology and ultimately leads to development of a cytopathic effect (CPE) and cell death. Sindbis virus (SIN) replication causes major changes in cellular macromolecular synthesis, in which the strong downregulation of transcription of cellular mRNAs and rRNAs plays a critical role. SIN nonstructural protein nsP2 was previously proposed as one of the main regulators of virus-host cell interactions, because point mutations in the carboxy-terminal part of nsP2 could make SIN and other alphaviruses and replicons less cytopathic and capable of persisting in some vertebrate cell lines. These mutants were incapable of inhibiting transcription and downregulating a viral stress-induced cell response. In the present work, we demonstrate that (i) SIN nsP2 is critically involved in CPE development, not only during the replication of SIN-specific RNAs, but also when this protein is expressed alone from different expression cassettes; (ii) the cytotoxic effect of SIN nsP2 appears to be at least partially determined by its ability to cause transcriptional shutoff; (iii) these functions of SIN nsP2 are determined by the integrity of the carboxy-terminal peptide of this protein located outside its helicase and protease domains, rather than by its protease activity; and (iv) the cytotoxic activity of SIN nsP2 depends on the presence of this protein in a free form, and alterations in P123 processing abolish the ability of nsP2 to cause CPE.The alphavirus genus of the Togaviridae family contains a number of human and animal pathogens. Nearly 30 members of the genus are widely distributed on all continents. They are transmitted by mosquitoes to vertebrates that serve as amplifying hosts (17,20,38). In infected vertebrates, some of the alphaviruses cause an acute disease, often characterized by high-titer viremia or host death (16,18). Accordingly, these viruses exhibit a highly cytopathic phenotype in cell cultures of mammalian and avian origin (38).Sindbis virus (SIN) is a prototype member of the genus and one of the least pathogenic alphaviruses. SIN is widely used in experimental research, since it can infect a wide variety of commonly used vertebrate cell lines, where it replicates to high titers approaching 10 10 PFU/ml. Moreover, SIN replication leads to the rapid development of a cytopathic effect (CPE) and cell death within 24 to 48 h postinfection (10). The SIN genome is a single-stranded RNA of almost 11.5 kb which has a positive polarity (37) and contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tail. After release from the nucleocapsids (42, 43), the genome is translated into the viral nonstructural proteins nsP1 to nsP4, which are encoded by the 5Ј two-thirds of the genome. Together with host factors these proteins form the replicative enzyme complex (RC) required for viral genome replication and transcription of the subgenomic RNA (38). The latter RNA is encoded by the 3Ј one-third of the genome and translated into the structural proteins t...

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