Anti-HCV Nonstructural Protein 5 Monoclonal antibody, Clone A389

The Japanese variant of the hepatitis C virus (HCV-N) genome, consisting of 9440 nucleotides in length, was cloned from a small amount (2 ml) of plasma from a single Japanese carrier by using RT-PCR and modified RT-PCR. The HCV-N genome has a long open reading frame that encodes a 3014 amino acid polyprotein with 340 and 57 bases of 5' and 3' non-coding sequences, respectively. HCV-N has a 4-amino-acid insertion in the NS5 region as compared to other HCV isolates, but this insertion is found to be very rare upon direct sequencing of that region. Comparative sequence analysis of all the complete and partial HCV sequences that were reported indicates that HCV can be subdivided into at least 4 groups. The HCV-N isolate has a high homology with HCV-J and HCV-BK (> 90%) and so belongs to group II, but shows less similarity to HCV-1 (> 78%, group I) and least to HC-J6 (> 67%, group III). Among these HCV isolates, the 5' non-coding region was the most conserved (> 93%) since it plays an important role in replication. The RT-PCR assay to detect HCV-RNA, using the primers deduced from this region, was very sensitive and specific. The putative core protein could become an important target for immunoassay because of a high degree of amino acid sequence similarity in that region. A high degree of diversity and a low similarity between each HCV isolate in the putative envelope protein play an important role in the chronicity of HCV infection and development of immunopreventive agents, such as immunoglobulin and vaccine for that infection.

Hepatitis C virus (HCV) possesses a positive-sense RNA genome which encodes a large polyprotein of 3,010 amino acids. Previous data and sequence analysis have indicated that this polyprotein is processed by cellular proteases and possibly by a virally encoded serine protease localized in the N-terminal domain of nonstructural protein NS3. To characterize the molecular aspects of HCV protein biogenesis and to clearly identify the protein products derived from the HCV genome, we have examined HCV polyprotein expression by using the vaccinia virus T7 transient expression system in transfected cells and by cell-free translation studies. HCV proteins were identified by immunoprecipitation with region-specific antisera. Here we show that the amino-terminal region of the HCV polyprotein is processed in vitro by cellular proteases releasing three structural proteins: p21 (core), gp37 (El), and gp61 (E2). Processing of the nonstructural region of HCV was evident in transfected cells. Two proteins of 24 and 68 kDa were immunoprecipitated with anti-NS2 and NS3 antisera, respectively. Antiserum against NS4 recognized three proteins of 6, 26, and 31 kDa, while antisera specific for NS5 immunoprecipitated two polypeptides of 56 and 65 kDa, indicating that each of these two genes encodes at least two different proteins. When the NS3 protease domain was inactivated by replacing the proposed catalytic Ser-1165 with Ala, processing at several sites was abolished. When Ser-1164 was mutated to Ala, no effect on the processing was observed. Cleavage activities at three of the four sites affected by NS3 were shown to occur in trans, while processing at the carboxy terminus of NS3 could not be mediated in trans. These results provide a detailed description of the protein products obtained from the processing of the HCV polyprotein. Furthermore, the data obtained implicate NS3 as a serine protease and demonstrate that a catalytically active NS3 is necessary for cleavage of the nonstructural region of HCV.