The coat protein binding site on the RNA of southern bean mosaic virus is implicated in assembly of the virus

The assembly of icosahedral plant viruses such as the cowpea strain of southern bean mosaic virus (SBMV-C) is a poorly understood process. Models of assembly propose that this process is initiated by the co-condensation of coat protein subunits with specific viral RNA sequences to form a RNA-protein complex (RNPC). A 23 nucleotide sequence on the RNA of SBMV-C that binds to SBMV-C coat protein has been identified and is proposed to fold into a hairpin. Biochemical studies of the SBMV-C RNPC were conducted to study the protein-protein and protein-RNA interactions in the RNPC. Crosslinking of coat proteins in the RNPC yielded an apparent coat protein dimer. This supports a proposal that the coat protein subunits in the RNPC interact as dimers. Trypsin cleavage of coat protein in the RNPC and of unassembled coat protein yielded similar sized polypeptides suggesting that trypsin has access to the same cleavage sites on both sources of coat protein. In addition, a role for the coat protein binding site (CPBS) in assembly of SBMV-C was addressed. Mutations that disrupt the CPBS were introduced into an infectious SBMV-C clone by site directed mutagenesis. Cowpeas were infected with transcripts from this clone. Results show that the mutant bases revert to wild type on the RNA from virus extracted from systemic leaves. The importance of the CPBS in SBMV-C life cycle is therefore demonstrated and a role for the CBFS in assembly is suggested.