Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles -- Jagadish et al. 72 (7): 1543 -- Journal of General Virology

This study demonstrates that the coat protein of Johnsongrass mosaic virus (JGMV), a potyvirus, can be successfully expressed in Escherichia coli and Saccharomyces cerevisiae and spontaneously assembles into virus-like particles (PVLPs). The researchers used expression vectors to produce full-length coat protein in both microbial hosts. The resulting particles resembled native JGMV in their flexuous morphology and width but were heterogeneous in length, featuring a stacked-ring structure similar to coat protein assemblies without viral RNA. Analysis using electron microscopy and immunoblotting confirmed particle assembly. Importantly, two coat protein mutants created by site-directed mutagenesis—one changing tryptophan-130 and tyrosine to glycine-proline, and another changing arginine-194 and glutamine-195 to aspartate-leucine—failed to form PVLPs despite being expressed. These results establish a cell-free microbial system for studying potyvirus assembly mechanisms and identifying critical protein regions required for polymerization.

Key findings

• Full-length JGMV coat protein expressed in E. coli and yeast spontaneously assembled into virus-like particles without viral RNA
• Assembled particles were heterogeneous in length with stacked-ring structure, resembling native virus morphology in width and flexibility
• Site-directed mutagenesis of highly conserved amino acids (Trp-130/Tyr and Arg-194/Gln-195) abolished particle assembly despite protein expression
• Lysyl endopeptidase treatment revealed that expressed coat protein adopted a native-like structure with protected core domains
• This microbial expression system enables future studies of assembly mechanisms, genome encapsidation, and identification of critical assembly domains