The hepatitis C virus (HCV), now recognized as the principal causative agent of non-A, non-B hepatitis, was first discovered in 1989. However, the biology of the virus remains poorly understood.

Because of the Division of Liver Diseases’ growing clinical involvement with hepatitis C, Dr. Andrea Branch, an outstanding authority on plant viroids and on the delta agent (hepatitis D virus, HDV), has developed a program of basic studies of the HCV virus. Recognizing that viral genomes often have overlapping genes, Dr. Branch and her associates, in particular Dr. Jose Walewski and Decherd Stump have developed software that facilitates their carrying out comparative sequence analyses on published HCV isolates, seeking overlapping, dual-use regions.

Their strategy for identifying such regions is based on the degeneracy of the genetic code. Thus, the code for a number of amino acids is fully specified by the first two bases of a codon. Since the third base in this situation is of no consequence for defining the encoded amino acid, it would be anticipated that, over time, random mutations would result in the given amino acid being encoded by any of four sequences, with the same bases in positions 1 and 2, and A, T, G, or C in position 3. This variability in position 3 would have no effect on the amino acid sequence of the encoded polypeptide. However, if there were an overlapping reading frame, an insignificant change in base position 3 in one reading frame could produce a significant alteration in the amino acid composition of a polypeptide encoded in an overlapping, alternative reading frame.

Based on these theoretical considerations, diverse HCV sequences were obtained from GenBank and aligned. The proximal portion of the main open reading frame (ORF) was found to contain a region of highly constrained sequence, in which the 3rd base in numerous codons is far more restricted than would be anticipated by chance. When read in the alternative 1+ reading frame, nearly 90 percent of the retrieved sequences contained at least 124 codons without a stop codon, representing a candidate second or alternate ORF. By contrast, within the same region of HCV RNA, the 2+ reading frame was riddled with stop codons.

To determine if this alternate ORF (A-ORF) was biologically significant, two peptides, representing different regions of the encoded A-ORF polypeptide were synthesized and used to develop assays to detect antibodies against the A-ORF protein. Analysis of sera from patients with chronic HCV has demonstrated that an appreciable proportion of them contain antibodies to the A-ORF protein, indicating that the protein must be synthesized during chronic HCV infection.

Efforts are now ongoing to identify the protein itself in liver samples from infected patients. The identified protein may be of value in improved HCV diagnostics. Moreover, the new protein, as well as the uniquely conserved region of the HCV genome that encodes it, are potential targets for a variety of new anti-HCV therapeutic strategies. It is likely that therapies developed on the basis of these observations will eventually undergo clinical evaluation within our program at Mount Sinai.