CRITICAL RESIDUES OF SEMLIKI-FOREST-VIRUS RNA CAPPING ENZYME INVOLVEDIN METHYLTRANSFERASE AND GUANYLYLTRANSFERASE-LIKE ACTIVITIES

The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransf erase (MT) and guanylyltransferase-like (GT) activities, which are inv olved in the capping of viral mRNAs., MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covale nt complex m(7)GMP-nsP1. These reactions are virus specific and thus p otential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the l arge alphavirus-like superfamily. Mutations of D64 D90, R93, C135, C14 2, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1, All MT-negative mutants lost also the GT activity, confirming t hat methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT re action without destroying MT activity, Conservation of residues essent ial for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Re sidues D64 and D90 were necessary for AdoMet binding, as measured by U V cross-linking, Secondary structure predictions of nsP1 and other pro teins of the superfamily place these residues in positions correspondi ng to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related.