Virion associated proteins of human enteric caliciviruses

Williams, Judith Carol (1998) Virion associated proteins of human enteric caliciviruses. University of Southampton, Doctoral Thesis.

Abstract

A 3' terminal ORF (ORF3) in SRSVs and animal caliciviruses is predicted to encode a small basic protein of unknown function. It has been speculated that this protein may be a minor structural component of the virus or that it is involved in capsid assembly.

A small protein (VPg) covalently linked to the RNA genome has been identified in animal caliciviruses. Therefore by analogy it is reasonable to assume that SRSVs also contain a genome-linked protein and that such a protein would be present as a minor component of the mature virion. A region of the SRSV genome predicted to encode the VPg protein was cloned and expressed in E. coli. The expressed recombinant protein was purified by metal chelate affinity chromatography and used to raise hyperimmune antisera in mice. It is currently not possible to prepare sufficient quantities of purified RNA directly from stool for confirmation of the presence of a VPg-like protein. However, antisera to the VPg protein were used to demonstrate the presence of VPg directly in stool samples. Furthermore this antiserum cross-reacted with VPg antigen from both genetic groups of SRSVs suggesting that VPg is potentially a useful diagnostic antigen which would be independent of strain variation.

The N-terminus of the LV 3B-like (VPg) protein and the 3C-protease were determined by Edman degradation. The 3C-like protease cleaved the N-terminus of the 3B-like protein at EG (aa 875-876) and EA (as 1008-1009) at the C-terminus. The sequence between these cleavage points was cloned and expressed in E. coli. Hyperimmune antisera were raised to the purified translation product. These antisera when analysed for cross-reactivity against the purified translation product from the equivalent region from SV gave a strong band. Immunoanalysis revealed that the 3B-like protein was detectable by human convalescent sera.

The LV full length clone was repaired and placed under control of the T7 promoter. TNT experiments with this clone identified a similar polyprotein cleavage pattern to SV. Three major bands of 113kDa, 41kDa and 39kDa were observed. Further optimisation of the in vitro TNT system generated products of 36kDa, 29kDa and 25kDa due to further proteolytic cleavage of the 3CD-like protein. RIPAs with antisera raised to the 3B-like protein supported these observations.

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