ASSEMBLY OF PHYSALIS MOTTLE VIRUS CAPSID PROTEIN IN ESCHERICHIA-COLI AND THE ROLE OF AMINO AND CARBOXY TERMINI IN THE FORMATION OF THE ICOSAHEDRAL PARTICLES

The coat protein gene of physalis mottle tymovirus (PhMV) was over exp ressed in Escherichia coil using pET-3d vector. The recombinant protei n was found to self assemble into capsids in vivo. The purified recomb inant capsids had an apparent s value of 56.5 S and a diameter of 29(/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lackin g the first 11 and 26 amino acid residues and two carboxy-terminal del etions lacking the last five and ten amino acid residues were construc ted and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vi vo, as evident from electron microscopy, ultracentrifugation and agaro se gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids wer e as stable as the empty capsids formed in vivo and encapsidated a sma ll amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids w hile it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were m ost stable. These results demonstrate that the N-terminal 26 amino aci d residues are not essential for T=3 capsid assembly in PhMV. In contr ast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not ass emble into capsids, suggesting that these residues are crucial for fol ding and assembly of the particles. (C) 1997 Academic Press Limited.