Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo.

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.