Use of the Vaccinia Virus/T7 Expression System for Studying HCV Protein Processing

HCV and related viruses are now classified as a separate genus in the family Flaviviridae (1 ), which includes two other genera, Flavivirus (2 ), and Pestivirus (3 ). The positive-strand HCV genome RNA is approx 9.4 kb in length and contains a highly conserved 5′ noncoding region followed by a long open reading frame encoding a polyprotein of 3010–3033 amino acids (4 ,5 ) Recently, it was determined that the 3′ end contains another highly conserved noncoding region approx 100 bp in length (6 –8 ). Because a cell-culture system supporting efficient HCV replication is lacking, efforts to define potential HCV-encoded polypeptides have utilized expression of HCV cDNA in cell-free translation systems and in cell cultures. The HCV polyprotein appears to be cleaved at multiple sites to produce at least 10 structural and nonstructural (NS) proteins (9 ). The order and nomenclature of these cleavage products for the HCV-H strain are NH₂ -C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, where C, El, and E2 are putative structural proteins and the remaining NS proteins are believed to be replicase components (9 –12 ). Host signal peptidase in the endoplasmatic reticulum lumen appears to catalyze cleavages in the structural-NS2 region (C/El, E1/E2, E2/p7, and p7/NS2 sites) (9 ,13 ), whereas an HCV-encoded serine proteinase located in the N-termmal one-third of the NS3 protein is responsible for four cleavages in the NS region (3/4A, 4A/4B, 4B/5A, and 5A/5B sites) (11 ,14 –16 ).