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

SVap15/21, a strain of Sindbis virus (SV) derived from our standard laboratory strain of SV (SVstd) after repeated passage on Aedes albopictus 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 SVstd differs from that of the more widely used SVHR (from which SVstd was derived) by an additional (i.e., third) N-linked glycan. The E2 of SVap15/21, in turn, differs from that of SVstd by the addition of a fourth glycan. We have determined the nucleotide sequence of the E2 genes of SVap15/21 and of SVstd, as well as that of our isolate of SVHR. The nucleotide sequence of the SVstd E2 gene predicted the occurrence of an additional N-linked glycan attachment site, not present in the SVHR E2, at Asn232 (Asp in SVHR). The sequence of the SVap15/21 E2 gene demonstrated three mutations relative to the SVstd gene, including one that predicted the occurrence of another potential N-linked glycan attachment site at Asn275. Sequence analysis of 15 revertants of SVap15/21 which are no longer host-restricted revealed that all had lost the glycosylation site at Asn275, 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 (Asn275 and Thr277), resulting in the loss of the glycosylation site at Asn275. A fourth mutation, resulting in an Asn275----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 Asn275 through a mutation resulting in a Thr277----Ile substitution. Knowledge of the nucleotide sequences associated with the ts defect in SVap15/21 and with its reversion permit an estimation of the mutation rate of this virus. This calculation indicates a mutation rate of less than 10(-6) errors per base incorporation.