Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which co ntains RNA capping and helicase-like domains, and 2a, which is related to p olymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutation s at the predicted capping and helicase active sites of 1a and analyzed the ir effects on BMV RNA3 replication in yeast, The helicase mutants showed no function in any assays used: they were strongly defective in template recr uitment for RNA replication, as measured by la-induced stabilization of RNA 3, and they synthesized no detectable negative-strand or subgenomic RNA. Ca pping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative- strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping de fects, we deleted chromosomal gene XRN1, encoding the major exonuclease tha t degrades uncapped mRNAs, XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of larg e amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants shelved no complementation, Instead, at high levels of expre ssion, a helicase mutant dominantly interfered with the function of the wil d-type protein. These results are discussed in relation to the interconnect ed functions required for different steps of positive-strand RNA virus repl ication.