Sequence analysis of the E2 gene of a hyperglycosylated, host restricted mutant of sindbis virus and estimation of mutation rate from frequency of revertants

Abstract

SV ap 15 21 , a strain of Sindbis virus (SV) derived form our standard laboratory strain of SV (SV std) after repeated passage on Aedes alboictus cells, grows normally in mosquito cells but is host restricted (hr) in vertebrate cells. It is also temperature sensitive (ts) and produces pinpoint plaques on vertebrate cells (sp). E2 glycoprotein of SV std differs from that of the more widely used SV HR (from which SV std was derived)_by an additional (i.e., third) N-linked glycan. The E2 SV ap 15 21 , in turn, differs from the of SV std by the addition of a fourth glycn. We have determined the nucleotide sequence of the E2 genes of SV ap 15 21 and of SV std, as well as that of our isolate of SV HR. The nucleotide sequence of the SV std E2 gene predicted the occurrence of an additional N-linked glycn attachment site, not present in the SV HR E2, at Asn 232 (Asp in SV HR. The sequence of the SV ap 15 21 E2 gene demonstrated three mutations relative to the SV std gene, including one that predicted the occurrence of another potential N-linked glycan attachment site at Asn 275. Sequence analysis of 15 revertants of SV ap 15 21 which are longer host-restricted revealed that all had lost the glycosylation site at Asn 275, confirming the connection between the hyperglycosylation and the host dependent block in assembly. Most of these revertants had also lost the temperature sensitivity and small plaque traits (i.e., were ts + and lp). Each revertant of this class was characterized by one of three different mutations in two separate codons (Asn 275 and Thr 277), resulting in the loss of the glycosylation iste at Asn 275. A fourth mutation, resulting in an Asn 275→ Tyr substitution, was associated with a hr + ts phenotype in three isolates. Finally an additional mutation in a different part of the E2 gene was found in two hr + ts sp isolates that had also lost the glycosylation site at Asn 275 through a mutation resulting in a Thr 277→ Ile substitution. Knowledge of the nucleotide sequences associated with the ts defect in SVap 15 21 and with its reversion permit an estimation of the mutation rate of this virus. This calculation indicates mutatin rate of less than 10 −6 errors per base incorporation.