Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment

Abstract

The genome of broad bean mottle bromovirus (BBMV) contains three positive-sense ssRNA segments, each capped with m7GpppA. Full-length transcribable cDNA clones for four strains of BBMV were constructed by employing reverse transcriptase-PCR (RT-PCR) and a high fidelity Vent DNA polymerase. The transcribed BBMV RNAs contained a 5' non-viral G residue and, although delayed, produced symptoms similar to those observed in plants infected with authentic virion RNAs. The transcripts replicated inefficiently in protoplasts. In contrast, transcript-derived progeny BBMV RNAs had the repaired termini, were as infectious as the authentic BBMV RNAs and replicated to high levels in protoplasts. In vitro translation of synthetic RNAs confirmed the previously proposed gene expression strategy for BBMV. Sequencing of virion RNAs from the Bawden strain revealed two forms of BBMV RNA3 components, the longer form containing 21 5' extra nucleotides derived by the duplication of two short 5' leader regions. The relative concentration of the two RNA 3 forms was found to be host-dependent, with the longer form prevailing in broad bean and Nictiana clevelandii infections and the shorter form in bean infections.