Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles

Abstract

When the full-length coat protein (CP) of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed in Escherichia coli or yeast, it assembled to form potyvirus-like particles. The particles were heterogeneous in length with a stacked-ring appearance and resembled JGMV particles in their flexuous morphology and width. This cell-free assembly system should permit analysis of the mechanisms of particle assembly and genome encapsidation. Two mutant forms of CP produced by site-directed mutagenesis failed to assemble into virus-like particles.

Site-Directed Mutagenesis of a Potyvirus Coat Protein and its Assembly in Escherichia Coli

“…In the mutant pTTQ19:CP m94D (Fig. 1), only the residue R TM was changed to D in contrast to the previously described double mutant pTTQ19:CP R-Q* (Jagadish et al, 1991) where both R TM and Q~9~ were mutated. This was done to eliminate any effects, in the new mutant pTTQ19:CP m94D, that might have been caused by the introduction of the amino acid residue L in place of Q195 adjacent to R TM.…”

“…We have used site-directed mutagenesis followed by expression of a modified Johnsongrass mosaic virus coat protein gene in Escherichia coli and electron microscopic analysis, to initiate identification of some of the residues within the core region crucial to self-polymerization. We have previously shown that complete coat protein synthesized in the yeast Saccharomyces cerevisiae, or with an additional 16 amino acids at its N terminus in E. coli, readily assembled to form potyvirus-like particles (Jagadish et al, 1991). These particles were similar in their morphology to those assembled in vitro in the absence of viral RNA (McDonald & Bancroft, 1977).…”

“…1). This was achieved by using ssDNA of the phagemid pT3T718U:CP ~*Q* (Jagadish et al, 1991) and the Dut-Ung site-directed mutagenesis method (Kunkel et al, 1987) fragment was cloned into pTTQ19. In the mutant pTTQ19:CP m94D (Fig.…”

“…1). To obtain the above mutational changes, PCR procedures were employed using SCMBI-1 phagemid DNA (Jagadish et al, 1991), or DNA from one of the existing constructs, as a template and appropriate primers designed to include BamHI-NsiI restriction enzyme recognition sequences and the desired mutations. The PCR products were purified, restricted with BamHI and NsiI and were used to replace the corresponding fragments in the construct pTTQ19:CP or the mutant construct pTTQ19:CP RQ-D~/D2asR to obtain the desired combination of amino acid changes as shown in Fig.…”

“…The general procedures for the use of recombinant DNA technology were as described (Sambrook et al, 1989). The mutant constructs were transformed into E. coli strain DH1 and expressed by IPTG induction as described before (Jagadish et al, 1991).…”