The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG (8-10) VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. The Alphavirus genus of the Togaviridae contains almost 30 species of viruses. These viruses are globally distributed and cause a variety of disease, ranging from mild febrile illness to encephalitis and death. Alphaviruses are arthropod borne and enzootically maintained in a cycle between mosquitoes and birds or small mammals. In vertebrate hosts, infection is acute, producing high viral yields prior to clearance or death. In cultured vertebrate, cells with large amounts of viral RNA are rapidly generated prior to apoptotic cell death. Factors likely contributing to the efficiency of RNA synthesis include the sequestration of machinery in cytoplasmic membrane-associated centers (8,12,13,19) and specific regulation of the viral RNA-dependent RNA polymerase (RdRp) activity. The morphogenesis of viral RNA synthetic complexes and regulation of RNA synthetic activity are integrally linked to programmed proteolytic processing of the viral components of the complex over the course of virus infection.Sindbis virus (SIN) is the type species of the Alphavirus genus and serves as a model for the study of viral RNA synthesis. The SIN genome is 11.7 kb of message-sense RNA that is capped and polyadenylated (49, 51), allowing for efficient translation. Following viral entry and release of the genome to the cytoplasm, the large 5Ј open reading frame of the genomic RNA is translated into the nonstructural polyproteins P123 or P1234, required for viral RNA synthesis. P1234 results from a readthrough event at the 3Ј end of the nsP3-coding region (22,31,50). Approximately 10% of translating ribosomes read through the stop codon to give rise to P1234. Cleavage of the nonstructural polyproteins is catalyzed by the nsP2-associated proteinase activity (3, 16), ultimately resulting in four mature proteins, nsP1, nsP2, nsP3, and nsP4. Cleavage of the polyproteins is sequential and temporally regulated, creating viral RNA synthetic complexes with distinct functions over the course of the single-cell replication cycle (21,23,25,46). Initial cleavage of P1234 releases mature nsP4 and occurs rapidly (2, 45). nsP4 contains the RdRp catalytic core but requires the other nsP proteins for activity. P123 and nsP4 form t...
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