Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease.
We have cloned and sequenced the cDNA made to the region of RNA encoding the structural proteins of three complementation group C mutants of Sindbis virus, ts2, ts5, and ts13, and of their revertants. These mutants possess defects in the posttranslational processing of their structural proteins at the nonpermissive temperature. Comparison of the deduced amino acid sequences of the mutants with those of the revertants and with the parental HR strain of virus showed all three mutants to have single amino acid substitutions in the highly conserved COOH-terminal half of the capsid protein that give rise to temperature sensitivity. ts2 and ts5 were found to have the same lesion and thus represent independent isolations of the same mutant, whereas ts13 possessed a different change. Reversion to temperature insensitivity in all three mutants occurred by reversion of the mutated nucleotide to the parental nucleotide, restoring the original amino acid. It has been previously postulated that the capsid protein possesses an autoproteolytic activity that cleaves the capsid protein from the nascent polyprotein during translation. Comparison of the amino acid sequence of the capsid protein with that of serine proteases leads us to hypothesize that histidine-141, aspartate-147, and serine-215 of the Sindbis capsid protein form the catalytic triad of a serine protease. This hypothesis is supported by the finding that all three temperature-sensitive lesions mapped occur near these residues: ts2 and ts5 change proline-218 to serine and in ts13 lysine-138 has been replaced by isoleucine.
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002642:Chick Embryo; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D010450:Endopeptidases / Q000235:genetics*; D005347:Fibroblasts; D007700:Kinetics; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases; D012845:Sindbis Virus / Q000201:enzymology / Q000235:genetics*; D013045:Species Specificity; D013696:Temperature
D004247:DNA; D010450:Endopeptidases; D012697:Serine Endopeptidases