Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus

Abstract

Complete cDNA copies of genomic RNA1, RNA2, and RNA3 of cowpea chlorotic mottle virus (CCMV) were cloned 1 base downstream from a T7 RNA polymerase promoter. The mixture of capped in vitro transcripts from all three clones produced normal CCMV infections in barley protoplasts and cowpea plants. By using transcripts from these clones and from a similar set of biologically active clones of the related brome mosaic virus (BMV), all possible single component exchanges between the BMV and CCMV tripartite genomes were tested. Viral RNA replication was not observed with any heterologous combination of RNA1 and RNA2, which encode trans-acting viral RNA replication factors. However, substitution of the heterologous RNA3 into either genome produced viable hybrid viruses, both of which replicated in barley protoplasts and produced lesions on Chenopodium hybridum, a local lesion host for both parent viruses. In hybrid infections, BMV and CCMV coat proteins each readily packaged RNAs from the heterologous virus, but BMV RNAs were replicated to a higher level than CCMV RNAs, even when trans-acting RNA replication factors were provided by CCMV genes. Neither hybrid systemically infected the natural host of either parent virus, suggesting that host specificity determinants in BMV and CCMV are encoded by RNA3 and at least one other genomic RNA.