Use of bromovirus RNA2 hybrids to map cis- and trans-acting functions in a conserved RNA replication gene

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with tripartite genomes. RNA replication by either virus requires genomic RNAs 1 and 2, which encode protein 1a and the polymeraselike, 94-kilodalton 2a protein, respectively. Proteins 1a and 2a share extensive sequence similarity with proteins encoded by a wide range of other positive-strand RNA viruses of animals and plants. Heterologous combinations of BMV and CCMV RNAs 1 and 2 do not support viral RNA replication, and although BMV RNA2 is amplified in CCMV-infected cells, CCMV RNA2 is not amplified by BMV. Construction of hybrids by precise exchange of segments between BMV and CCMV RNA2 has now allowed preliminary mapping of such virus-specific replication functions in RNA2 and the 2a protein. The ability to support replication in trans with BMV RNA1 segregated with a 5' BMV RNA2 fragment encoding the first 358 2a gene amino acids, while a 5' fragment extending over 281 BMV 2a codons transferred only cis-acting competence for RNA2 amplification in cells coinfected with wild-type BMV. Successful trans-acting function with CCMV RNA1 segregated with a CCMV RNA2 3' fragment that included the last 206 2a gene codons. Thus, the less conserved N- and C-terminal 2a segments appear to be involved in required interaction(s) of this polymeraselike protein with the 1a protein or RNA1 or both. Moreover, when individual hybrid RNA2 molecules that function with either BMV or CCMV RNA1 were tested, BMV- and CCMV-specific differences in recognition and amplification of RNA3 templates appeared to segregate with RNA1.