Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation.
Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site. These results suggest that a stable NS3-NS4A interaction is important for cleavage at the NS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D005822:Genetic Vectors; D016174:Hepacivirus / Q000378:metabolism*; D007668:Kidney; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D014162:Transfection; D014616:Vaccinia virus; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000906:Antibodies; D000939:Epitopes; D011498:Protein Precursors; D021382:Protein Sorting Signals; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus