Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

被引:61
作者
Gallinari, P [1 ]
Paolini, C [1 ]
Brennan, D [1 ]
Nardi, C [1 ]
Steinkühler, C [1 ]
De Francesco, R [1 ]
机构
[1] Ist Ric Biol Mol P Angeletti, I-00040 Rome, Italy
关键词
D O I
10.1021/bi982892+
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. 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 have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and 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 similar to 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 uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3,
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页码:5620 / 5632
页数:13
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