Expression of the hepatitis E virus ORF1

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose gen ome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which shar e homology with the products of the corresponding ORF of members of the alp havirus-like superfamily of plus-strand RNA viruses. Among animal virus mem bers 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. T o determine if the NSP of HEV is similarly processed, ORF1 was introduced i nto a plasmid vector which allowed for expression both in vitro using a cou pled transcription/translation system and in vivo using a vaccinia virus-dr iven transient expression system. A recombinant vaccinia virus expressing O RF1 was also constructed. Both in vitro and in vivo expression under standa rd conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have be en shown to activate other viral proteases, failed to change this expressio n pattern. However, in vivo following extended incubations (24-36 hours), t wo potential processing products of 107 kDa and 78 kDa were observed, N- an d C-terminus-specific immunoprecipitation and deletion mutagenesis were use d to determine that the order of these products within the NSP is NH2-78 kD a-107 kDa-COOH. However, site-specific mutagenesis of Cys(483), predicted b y 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 catalyti c 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 thi s protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary mo nkey hepatocytes were not successful and therefore this proteolytic cleavag e could not be authenticated. Overall, the results of this study indicate t hat either the HEV NSP is not processed or that it is cleaved at one site b y a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.