Hepatitis B virus open reading frame X

Abstract

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154 amino acid polypeptide. I constructed two recombinant plasmids, each with the capacity to direct the synthesis of a fusion polypeptide containing 145 of the 154 amino acids predicted by HBV ORF X. pHBV-X700 encodes a polypeptide, designated {dollar}\beta{dollar}-gal{dollar}\sb{lcub}1007{rcub}{dollar}:X{dollar}\sb{lcub}145{rcub}{dollar}, which consists of the N-terminal 1007 amino acids of {dollar}\beta{dollar}-galactosidase followed by the amino acids encoded by ORF X. This fusion protein was over-expressed in bacteria, partially purified, and used to generate polyclonal antisera in rabbits.;The second plasma PHBV-X500 encodes a polypeptide {dollar}\beta{dollar}-gal{dollar}\sb8{dollar}:X{dollar}\sb{lcub}145{rcub}{dollar} composed of only eight amino acids of the N-terminus of {dollar}\beta{dollar}-galactosidase followed by the same viral sequence. The second, smaller fusion protein was expressed and characterized by in vitro transcription and translation. This fusion protein was used in immunoprecipitations to demonstrate that the rabbit antiserum generated to the first, large fusion protein contained antibodies which recognized determinants encoded by ORF X.;Immunoprecipitations using the smaller ORF X fusion protein as a radioactively-labelled antigen were performed also to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in some samples of sera characterized as positive for hepatitis B surface antigen (HBsAg) but were not found in sixteen normal human sera. As such, these findings represented evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggested that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe{dollar}\sb3{dollar}Ag (anti-HBe{dollar}\sb3{dollar}).;The rabbit antisera identified as containing anti-X antibodies were used in an attempt to detect and identify the X protein in virus particles, preparations of infected liver, and in cell lines transfected with HBV DNA. I was not able to detect the presence of an HBV ORF X-encoded protein in these materials. These results suggested that the X protein is not present in mature virions or may be present in very low copy number.