Expression of the hepatitis E virus ORF1

Abstract

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

“…We failed in our attempts to demonstrate these enzymatic activities for fragments of 470 and 527 residues, suggesting a rather large translational product may be required for enzymatic activity. Therefore, the capping enzyme domain may extend to what was initially predicted to be a protease domain (17), but which appears not to act as a protease (34).…”

“…In another study, prolonged in vivo expression yielded N-terminal 78-kDa and C-terminal 107-kDa fragments (34). Transfection of in vitro-synthesized RNA, consisting of the complete HEV genome, to HepG2 cells resulted in synthesis of ORF1, ORF2, and ORF3 products as well as release of small amounts of infectious virus into the culture medium (29).…”

“…None of the predicted functions has been experimentally verified. Indeed, it was recently shown that one of the putative protease active site residues is not conserved in different isolates and that mutation of the predicted active site cysteine did not abrogate P186 processing (34). Thus, the predicted protease appears not to exist or have enzymatic activity.…”

Hepatitis E: Molecular Virology and Pathogenesis

“…The Cys483 residue is highly conserved among all HEV isolates, whereas His590 is not. 28 ORF1 polyprotein processing has been observed in transfection studies of infectious HEV cDNA clone. 9 The exact cleavage sites of the ORF1 polyprotein are still not known.…”

“…Two potential processed products of 107 kDa and 78 kDa were observed in vivo following extended incubations (24-36 h). 28 However, site-specific mutagenesis of cysteine 483, failed to abolish this cleavage. Contradicting this study, no processing of the ORF1 polyprotein was observed into discrete units, either in vitro or in HepG2 cells 29 and Huh7 cells.…”