Close to four hundred arthropod-borne viruses (arboviruses), many of which cause serious human disease, have been identified. These viruses fall into five different families. Sindbis virus, which is a mosquito- transmitted arbovirus, and the prototype virus of the family Togaviridae, genus alphavirus, is the subject of this proposal. Much effort is being directed at understanding how the four nonstructural (ns) proteins encoded by Sindbis virus function in the synthesis of the viral RNAs. Making use of two mutants of Sindbis virus isolated in my laboratory (SVLM21, which is able to grow in methionine-deprived mosquito cells and SVMPA, which is resistant to mycophenolic acid) we have obtained strong evidence which associates the RNA guanylyltransferase and methyltransferase activities (the activities which cap and methylate the 5' end of the viral mRNAs) with the ns protein, nsP1. Although all cytoplasmic RNA viruses whose mRNAs have a 5' cap, must in some way encode the activities needed to modify the 5' terminus of their mRNAs, in no case has the catalytic site involved in methylation or the binding site for S-adenosylmethonine, the methyl donor, been identified. The thrust of this proposal is to localize and identify the catalytic sites on nsP1 associated with the methylation (and the guanylylation) of viral mRNAs. To that end we will express deleted forms of nsP1 and test them for methyltransferase activity. We will use site-directed mutagenesis to identify amino acid residues required for MTr activity, and by means of photoaffinity labeling we shall identify the binding site for AdoMet. In addition, we shall develop assays for the other activities involved in capping and methylation of viral mRNAs (i.e. RNA triphosphatase and guanylyltransferase) and will determine if expressed nsP1 has these activities. Because the SVLM21 and SVMPA mutations map to the nsP1 coding sequence and because we have already expressed enzymatically active nsP1 in E. coli we are well positioned to carry out these experiments. Definition of the catalytic site on nsP1 which is involved in the methylation of viral mRNAs will facilitate the rational design of antiviral compounds which might be effective in inhibiting not only alphaviruses, but also other viruses in the Sindbis virus superfamily.
