Modulation of Hepatitis C Virus NS3 Protease and Helicase Activities through the Interaction with NS4A

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• 作者：Paola Gallinari ; Chantal Paolini ; Debra Brennan ; Chiara Nardi ; Christian Steinkü ; hler ; and Raffaele De Francesco
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：April 27, 1999
• 年：1999
• 卷：38
• 期：17
• 页码：5620 - 5632
• 全文大小：164K
• 年卷期：v.38,no.17(April 27, 1999)
• ISSN：1520-4995

文摘

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in theN-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminalportion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A,a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We haveexpressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4Aoccurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complexwith a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergentand glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency(k_cat/K_m) of NS3-NS4A-associated protease under optimized conditions was 55 000 s^-1 M^-1, very similarto that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand,both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouplethe ATPase/ssRNA binding and RNA unwinding activities associated with NS3.