Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis.

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.