Mutations in the Sindbis virus capsid gene can partially suppress mutations in the cytoplasmic domain of the virus E2 glycoprotein spike.
Assembly and budding of alphaviruses are postulated to occur by protein-protein interactions between sites on the cytoplasmic domain of the transmembranal envelope E2 glycoprotein and on the surface of the nucleocapsid protein subunits. Genetic data to support this model have been obtained by isolating revertants of two slow-growth mutants of Sindbis virus and analyzing the sequences of the genes encoding their structural proteins. The slow-growth phenotypes of the mutants were previously shown to result from site-directed mutations of 2 amino acids in the sequence corresponding to the 33 amino acids at the carboxyl terminus of E2, which are localized to the cytoplasmic face of the plasma membrane. Putative revertants of these two mutants with faster growth rates were isolated by sequential passaging of virus grown on insect cells or chicken embryo fibroblasts. Sequence analysis of plaque-purified viruses that grew significantly better than the original mutant revealed that the original E2 mutation was present and that there were additional amino acid changes in the virus capsid. Two of the latter were introduced separately into the wild-type virus cDNA and into the genomes of the original mutants. The new strains of virus that contained both capsid and E2 mutations produced many more extracellular particles than those with the E2 mutations alone, indicating substantial suppression of the original E2 mutation. Both capsid mutations appear to be localized near a hydrophobic pocket of the capsid, which is postulated to be the site for docking of hydrophobic amino acids of the E2 cytoplasmic domain. This genetic study provides strong support for the current models of alphavirus assembly.
Ryan
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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA. milton@borcim.wustl.edu
Ivanova
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Schlesinger
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D000818:Animals; D001665:Binding Sites; D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D009033:Culicidae; D003593:Cytoplasm / Q000378:metabolism; D016296:Mutagenesis; D012845:Sindbis Virus / Q000235:genetics* / Q000254:growth & development; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000378:metabolism
D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C064950:glycoprotein E2, Sindbis virus