Changes of the Secondary Structure of the 5′ End of the Sindbis Virus Genome Inhibit Virus Growth in Mosquito Cells and Lead to Accumulation of Adaptive Mutations

Abstract

Both the 5 end of the Sindbis virus (SIN) genome and its complement in the 3 end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5 untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5 sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5 UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5 UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.The Alphavirus genus of the Togaviridae family contains nearly 30 known members, including a number of human and animal pathogens (reviewed in reference 12). The majority of alphaviruses are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts. In insect vectors, alphaviruses cause persistent life-long infections and do not greatly affect the viability of their hosts (3). Accordingly, they establish persistent infections in cultured mosquito cells (2). In vertebrates, alphaviruses usually cause acute infections that often result in disease (14), and the infection of susceptible cultured vertebrate cells leads to a progressive cytopathic effect (CPE) and cell death (8,20).Sindbis virus (SIN) is a prototype member of the Alphavirus genus, and it has always been a highly valuable source of information about the mechanisms of alphavirus replication and virus-host cell interactions (reviewed in reference 44). This virus can productively replicate in a wide variety of cell lines of insect and vertebrate origins. Like other alphaviruses, SIN enters cells via receptor-mediated endocytosis (4). The fusion of viral and endosomal membranes leads to the release of nucleocapsids into the cytoplasm (10), and after their ribosome-mediated disassembly, viral genomes become capable of translation and replication (42,49).The SIN genome is a single-stranded RNA of positive polarity that is almost 12 kb long (43). It is ca...

Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

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

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