In vitro activity of hepatitis C virus protease NS3 purified from recombinant Baculovirus-infected Sf9 cells.
A recombinant Baculovirus expression system was used for the production of a 20-kDa protein encompassing the hepatitis C virus NS3 protease domain. The protein was purified to apparent homogeneity after detergent extraction of cell homogenates. It was shown to be a monomer in solution and to cleave the in vitro translated precursor proteins NS4A-NS4B and NS5A-NS5B, but not the NS4B-NS5A or the NS3-NS4A precursors. The enzyme also cleaved a 20-mer peptide corresponding to the NS4A-NS4B junction with kcat/Km = 174 m(-1) s(-1). Peptides harboring NS4A sequences comprising amino acids 21-54 (Pep4A21-54) and 21-34 (Pep4A21-34) were found to induce an up to 2.8-fold acceleration of cleavage. Kinetic analysis revealed that this acceleration was due to an increase in kcat whereas no significant effect on Km could be detected. Pep4A21-54 was also an absolute requirement for cleavage of in vitro translated NS4B-NS5A by the purified protease. From these data we conclude that: (i) the purified protease domain shows substrate specificity and cleavage requirements similar to those previously reported on the basis of transfection experiments, (ii) activation of the purified protease by the NS4A co-factor can be mimicked by synthetic peptide analogs, and (iii) a central hydrophobic region of NS4A with a minimum core of 14 amino acids is responsible for the interaction with NS3.
Steinkühler
C
C
Istituto di Richerche di Biologia Molecolare P. Angeletti Pomezia, Rome, Italy.
Tomei
L
L
De Francesco
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases