Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyprotein are effected by the virus-encoded serine protease contained within NS3. Using transient expression in HeLa cells of cDNA fragments that code for regions of the HCV polyprotein, we studied whether viral functions other than NS3 are required for proteolytic processing at these sites. We found that, in addition to NS3, a C-terminal 33-amino-acid sequence of the NS4A protein is required for cleavage at the NS3-NS4A and NS4B-NS5A sites and that it accelerates the rate of cleavage at the NS5A-NS5B junction. In addition, we show that NS4A can activate the NS3 protease when supplied in trans. Our data suggest that HCV NS4A may be the functional analog of flavivirus NS2B and pestivirus p10 proteins.