Putative RNA capping activities encoded by brome mosaic virus: Methylationand covalent binding of guanylate by replicase protein 1a

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded p roteins, la and 2a. The amino-terminal half of la is a distant homolog of a lphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs, In this study, we examined the enzymatic activities of BMV la e xpressed in yeast, where the protein is fully functional in RNA replication . la methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-ade nosylmethionine (AdoMet) as the methyl donor, Product analysis by nuclear m agnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7, Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, a nd was accompanied by transfer of H-3-methyl from AdoMet to the covalent 1a -guanylate complex. The covalent complex could be immunoprecipitated by la antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further meth yltransferase reactions. Mutation of conserved amino acids in the N-termina l half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels, Covalent 1a-guanylate comple x formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts, These results show that BMV 1a has activit ies similar to those of alphavirus nsP1, demonstrating conservation of thes e putative capping functions across a wide span of sequence divergence with in the alphavirus-like superfamily, Conservation of this unusual combinatio n of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.