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

Abstract

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 la protein. To examine the role of la, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the la 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 la 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 la than of 2a, the other BMV-encoded protein involved in replication.

Functions of alphavirus nonstructural proteins in RNA replication

“…The role of helicase in the replication cycle of positive-strand RNA viruses remains mysterious, although it is often thought to either separate double-stranded RNAs or remove secondary structures from template RNAs. A ts mutant within the helicase domain of BMV la encodes a protein defective in all forms of RNA synthesis, consistent with this line of thinking (126). On the other hand, the phenotype of SIN RNA-negative ts mutants located in the helicase is not very clear-cut but has been interpreted to support a role in the conversion from negative-strand to positive-strand synthesis (119).…”

Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses

“…A reovirus-like pathway for (+)RNA replication (FIG. 4b,c) would also explain the common early shutoff of (-)RNA synthesis in BMV and other (+)RNA viruses, while (+)RNA synthesis continues unabated 112,117,118 , and the dependence of (-)RNA synthesis on continuing protein synthesis 118 . In such a model, (-)RNAs might be synthesized primarily during or immediately after replication-complex assembly from newly synthesized proteins, forming dsRNAs that would preferentially or exclusively be templates for (+)RNA synthesis in the resulting stable replication complexes.…”

“…106) and BMV 1a (REF. 112), µ2 also seems to be a co-factor for (+)RNA synthesis 111 . For dsRNA viruses, packaging of (+)RNAs is associated with, or followed by, (-)RNA synthesis, yielding dsRNAs that are protected in cores or corelike precursors 90,91 .…”

“…Replicationcomplex assembly and negative-strand synthesis seem to cease on exhaustion of a limiting host factor, possibly the target membrane for replication-complex formation 15,118 . Pre-formed replication complexes, however, remain active in (+)RNA synthesis 112,118 .…”

Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

“…RNA1 and RNA2 encode viral RNA synthesis factors 1a and 2a, which are required for genome replication and sgRNA synthesis. 2a is the viral RNA polymerase, while 1a is a multifunctional RNA replication factor that directs assembly of the membrane-associated RNA replication complex, recruits viral RNA templates and 2a polymerase, and contributes RNA capping and potentially helicase functions to RNA synthesis (4,18,30,34). RNA3 encodes movement protein 3a and coat protein (CP), which are dispensable for RNA replication but required for systemic spread in plants.…”