Use of Bromovirus RNA3 Hybrids to Study Template Specificity in Viral RNA Amplification

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.