Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.