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

Abstract

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-encoding region, indicating a potential overlapping open reading frame (ORF). Four 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 test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, 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) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p , 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

“…This finding emerged from a bioinformatics analysis of eight diverse HCV RNA sequences that identified specific nucleotide positions at which mutations that would be expected to occur due to the natural selection process but were suppressed by constraints additional to the requirement for a conserved core protein (17). Furthermore, Walewski et al (16,17) provided indirect evidence for expression of the ARF/ core11 ORF by designing peptides representing core11 aminoacid sequences and using them as antigens for the detection of specific antibodies in serum samples from HCV-infected patients.…”

“…The p16 protein is known as ARFP (alternative reading frame protein) (17), F (for frameshift) (19) or core11 (to indicate the position of the new open reading frame) protein (18,20).…”

“…Since its discovery, several studies have shown that the core11/ARFP/F protein is specifically recognized by antibodies in serum (17)(18)(19)(33)(34)(35)(36)(37)(38)(39)(40) and by the memory T cells (34,36) of HCV-infected patients, providing evidence that the core11 ORF is produced during natural HCV infection in humans. However, initial studies gave variable frequencies for the detection of core11-humoral immune responses in HCV-infected individuals.…”

“…However, initial studies gave variable frequencies for the detection of core11-humoral immune responses in HCV-infected individuals. Walewski et al (17) reported an association between chronic HCV infection and antibody reactivity to core11, by showing that 13 of 100 (13%) serum samples from HCV patients with chronic infection reacted with two synthetic HCV-1a core11 peptides consisting of aa 4-17 and 37-56, in immunoblot assays. We subsequently showed, with lysates of E. coli cells producing a recombinant GST-core11 protein from an HCV-1 isolate lacking the first 10 core amino acids [designated D(1-10)core11/F], the detection on western blots of specific antibodies in 77% of the HCV-positive sera from chronically infected patients tested (26 of 34 sera) (18).…”

“…A survey of the HCV sequences compiled in the database indicated that the core11 ORF was present in all seven HCV genotypes and all the infectious viral clones described to date (17). The existence of an alternative ORF overlapping the core protein gene is inconsistent with the conventional Flaviviridae genetic map, but the HCV core11 ORF is not the only exception.…”