Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-enco ding region, indicating a potential overlapping open reading frame (ORF). F our peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to tes t sera for specific antibodies from inn chronic HCV patients, 44 healthy co ntrols, and 60 patients with non-HCV liver disease, At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) wa s significantly associated with chronic HCV infection, when compared to non -HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodie s to A4 were not detected in any serum sample. Our western blot assays conf irmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-enc oding region. Because this novel HCV protein stimulates specific immune res ponses, it has potential value in diagnostic tests and as a component of va ccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.