Studies on hybrid comoviruses reveal the importance of three-dimensional structure for processing of the viral coat proteins and show that the specificity of cleavage is greater in trans than in cis

A series of cowpea mosaic virus (CPMV)-based hybrid comoviral RNA-2 molecul es have been constructed. In these, the region encoding both the large (L) and small (5) viral coat proteins was replaced by the equivalent region fro m bean pod mottle virus (BPMV). The hybrid RNA-Z molecules were able to rep licate in cowpea protoplasts in the presence of CPMV RNA-1. Though processi ng of the hybrid polyproteins by the CPMV-specific 24K proteinase at the si te between the 58/48K and L proteins could readily be achieved, no processi ng at the site between the L and S coat proteins could be obtained even whe n the sequence of amino acids between the two coat proteins was made CPMV-l ike. As a result, none of the hybrids was able to form functional virus par ticles, and they could not infect cowpea plants. Comparison with the proces sing of the L-S site in cis in reticulocyte lysates demonstrated that the r equirements for processing are more stringent in trans than in cis. The res ults suggest that the L-S cleavage site is defined by more than just a line ar sequence of amino acids and probably involves interactions between the L -S loop and the beta barrels of the viral coat proteins. (C) 1999 Academic Press.