Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics
| Autor: | Arash Grakoui, Jian Xu, Charles M. Rice, Béla M. Prágai, Chao Lin |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
viruses
Molecular Sequence Data Restriction Mapping Immunology Hepacivirus Viral Nonstructural Proteins Biology Kidney Cleavage (embryo) Polymerase Chain Reaction Microbiology Cell Line Serine chemistry.chemical_compound Proteinase 3 Cricetinae Virology Chlorocebus aethiops Tumor Cells Cultured Animals Humans Amino Acid Sequence Binding site NS5B DNA Primers chemistry.chemical_classification NS3 Binding Sites Base Sequence Sequence Homology Amino Acid virus diseases biochemical phenomena metabolism and nutrition Molecular biology digestive system diseases Amino acid Kinetics chemistry Biochemistry Insect Science Serine Proteinase Inhibitors Protein Processing Post-Translational Research Article |
| Zdroj: | Journal of Virology. 68:8147-8157 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.68.12.8147-8157.1994 |
| Popis: | The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases. |
| Databáze: | OpenAIRE |
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