No abstract

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...

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-...

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.Alphaviruses are a widely distributed group of significant human and animal pathogens. Some of them, including Venezuelan, eastern, and western equine encephalitis viruses, cause serious febrile illness and encephalitis (26). Others cause diseases with mild symptoms that usually include rash, fever, and arthritis (19). Alphavirus structural and nonstructural proteins (nsPs) demonstrate an obvious homology, suggesting that replication of their genomes, interactions with host cell biology, and formation of viral particles have much in common (52).The alphavirus genome is a single-stranded RNA of positive polarity and almost 12 kb in length that mimics the structure of cellular mRNAs. It contains both a 5Ј methylguanylate cap and a 3Ј polyadenylate tail (27, 51). These features allow the translation of viral proteins by host cell machinery directly from the genome RNA. The 5Ј two-thirds of the genome is translated into nonstructural proteins that comprise the viral components of the replicative enzyme complex (RC) that is required for replication of the viral genome and transcription of the subgenomic RNA. The subgenomic RNA corresponds to the 3Ј third of the genome. It is synthesized from the subgenomic promoter and translated into viral structural proteins, which are dispensable for RNA replication. The RNAs...

The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, ϳ35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C VEE 33-68 includes two domains with distinct functions: the ␣-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.