An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.
Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.
Failla
C
C
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Rome, Italy.
Tomei
L
L
De Francesco
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D006367:HeLa Cells; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017931:DNA Primers; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080106:NS4A protein, flavivirus