Expression of the hepatitis E virus ORF1

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 NH$_2$-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys(483), 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, His(590), 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.