Hepatitis C virus encodes a large polyprotein precursor that is proteo
lytically processed into at least 10 distinct products, in the order N
H2C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. A serine proteinase enc
oded in the N-terminal 181 residues of the NS3 nonstructural protein i
s responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5
B) in the nonstructural region. NS4A, a 54-residue nonstructural prote
in which forms a stable complex with the NS3 proteinase, is required a
s a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances pro
cessing at the 4A/4B end 5A/5B sites. Recently reported crystal struct
ures demonstrated that NS4A forms an integral part of the NS3 serine p
roteinase. In this report, we present evidence that NS4A forms a nonio
nic detergent-stable complex with the NS4B5A polyprotein substrate, wh
ich may explain the requirement of NS4A for the 4B/5A cleavage. Isoleu
cine-29 of NS4A, which has been previously shown to be essential for i
ts proteinase cofactor activity and formation of the NS3 complex was f
ound to be important for the interaction between NS4A and the NS4B5A s
ubstrate. In addition, two more hydrophobic residues in the NS4A centr
al region (valine-23 and isoleucine-25) were also shown to be essentia
l for the cofactor activity and for the interaction with either the NS
3 proteinase or the NS4B5A polyprotein substrate. Finally, the possibl
e mechanisms by which these viral proteins interact with each other ar
e discussed.