COMPLEX-FORMATION BETWEEN THE NS3 SERINE-TYPE PROTEINASE OF THE HEPATITIS-C VIRUS AND NS4A AND ITS IMPORTANCE FOR POLYPROTEIN MATURATION

Processing of the hepatitis C virus polyprotein is mediated by host ce ll signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third o f NS3 is responsible for cleavage at the four sites carboxy terminal o f NS3. The activity of this proteinase is modulated by NS4A, a 54-amin o-acid polyprotein cleavage product essential for processing at the NS 3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency bet ween NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to expre ss hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase an d NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the mi nimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Simil arly, deletion of amino-terminal NS3 sequences impairs complex formati on as well as cleavage at the NS4B/5A site but not at the NS4A-indepen dent NS5A/5B site. These results suggest that a stable NS3-NS4A intera ction is important for cleavage at the NS4A-dependent sites and that a mino-terminal NS3 sequences and the central NS4A domain are directly i nvolved in complex formation.