Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A.
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 (kcat/Km) 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.
Gallinari
P
P
Istituto di Ricerche di Biologia Molecolare "P. Angeletti", Rome, Italy.
Paolini
C
C
Brennan
D
D
Nardi
C
C
Steinkühler
C
C
De Francesco
R
R
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; 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
D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D046911:Macromolecular Substances; D012367:RNA, Viral; 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; C093542:NS4 protein, hepatitis C virus