The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins.

Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.