Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype

Abstract

SVLM21 is a mutant of Sindbis virus which in contrast to the standard virus, SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. Previously, by making use of the infectious Toto plasmids, we had constructed recombinant viruses containing various SVLM21 sequences, and were thereby able to map the mutations associated with the SVLM21 phenotype to the gene for the nonstructural protein nsP1. Two mutations were found in the nsP1 gene of SVLM21. These led to predicted amino acid changes at residue 87 from Arg to Leu, and at residue 88 from Ser to Cys. In the work presented here, we assess the relative contributions of these two mutations to the SVLM21 phenotype using site-directed mutagenesis to obtain virus encoding only the change to Leu at residue 87 of nsP1 (SVMS319), or only the change to Cys at residue 88 (SVMS321). In addition we show that SVLM10, which was isolated during the selection procedure for SVLM21, encodes only the change at residue 88. In addition to its ability to grow in methionine-deprived mosquito cells, SVLM21 differs from SVSTD in two other respects: (1) it shows an increased sensitivity to neplanocin A (NPA) and (2) it generates increased levels of methyltransferase in infected cells. Whether we looked at resistance to low methionine, sensitivity to NPA, or levels of methyltransferase generated, SVMS319, SVMS321, and SVLM10 all expressed only a partial SVLM21 phenotype. Furthermore we were not able in these experiments to distinguish between these three viruses. We conclude therefore that both amino acid changes, i.e., at residues 87 and 88, are required to produce the full SVLM21 phenotype, and that both changes contribute equally.