Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

Abstract

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome-and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.The genus Alphavirus in the family Togaviridae contains a number of widely distributed human and animal pathogens. Some of the alphaviruses, including Venezuelan (VEEV), eastern, and western equine encephalitis viruses, constitute a serious public health threat in the United States (53,63,65,66) and cause severe encephalitis in humans and animals that can result in death or neurological sequelae (10,21,27,41). Other family members cause a mild infection, a self-limited febrile illness characterized by fever, skin rashes, and arthritis (21). In spite of differences in their abilities to cause disease, alphaviruses demonstrate strong homology in their encoded proteins and appear to have similar mechanisms of RNA replication (59). Under natural conditions, alphaviruses circulate between mosquito vectors, in which they cause a persistent, life-long infection, and vertebrate hosts, in which the infection is always acute and characterized by a short-term, high-titer viremia that is required for infection of new mosquitoes during blood meals (64). Thus, alphaviruses are capable of replicating in both vertebrate and invertebrate cells and, accordingly, utilize very different intracellular environments for the efficient synthesis of virus-specific RNAs and the production of viral particles.The alphavirus genome is a single-...

The Amino-Terminal Domain of Alphavirus Capsid Protein Is Dispensable for Viral Particle Assembly but Regulates RNA Encapsidation through Cooperative Functions of Its Subdomains

“…However, in the last few years, our understanding of the alphavirus RNA replication mechanism has undergone some modifications. It was demonstrated that for some of the alphaviruses, such as SINV and VEEV, replication complexes are formed at the plasma membrane, and a major fraction of them remain there, even at later stages of the infection and continue to function as sites of viral RNA synthesis (49)(50)(51). Thus, RNA replication and its packaging into virions are likely to be more closely compartmentalized than we previously thought.…”

Ability of minus strands and modified plus strands to act as templates in Semliki Forest virus RNA replication

“…Thus, the recruitment of the minus strand for replication inside cells appears to happen with a very low efficiency or not at all. Based on several lines of evidence, it is generally thought that the minus strands are present in infected cells in dsRNA form in complex with the plus strands (Gorchakov et al, 2008;KulasegaranShylini et al, 2009;Mai et al, 2009;Spuul et al, 2010). The inability of the minus strand to serve as a template in any RNA synthesis might therefore also mean that for plusstrand synthesis the replicase would require a (partially) dsRNA template.…”

Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci

“…nsP3 is the most enigmatic of all nsPs, with unclear yet essential roles in alphavirus RNA replication (11). nsP3 is highly phosphorylated (12,23), is part of the viral replication complex (RC) (5), and has been shown to interact with Ras-GAP SH3 domain-binding proteins (G3BP1 and G3BP2) in two independent coimmunoprecipitation studies (3,7). Infection with another Togaviridae family member; rubella virus, has been shown to alter the distribution of host cell G3BP, suggesting that G3BP plays an important role in the outcome of viral infection (16).…”