Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the 
leading cause of liver disease in the U.S. and is responsible for most liver 
transplants. Current treatments for this infectious disease are inadequate; 
therefore, new therapies must be developed. Several labs have obtained evidence 
for a protein complex that involves many of the nonstructural (NS) proteins 
encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact 
structurally and functionally. In this study, we investigated the interaction 
between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments 
and surface plasmon resonance data indicate a direct interaction between NS3 and 
NS5B that is primarily mediated through the protease domain of NS3. This 
interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates 
product formation in RNA unwinding experiments under conditions of excess 
nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of 
nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under 
pre-steady-state conditions, in which NS3 and substrate concentrations are 
similar, product formation increased in the presence of NS5B. The increase was 
consistent with 1:1 complex formed between the two proteins. A fluorescently 
labeled form of NS3 was used to investigate this interaction through 
fluorescence polarization binding assays. Results from this assay support 
interactions that include a 1:1 complex formed between NS3 and NS5B. The 
modulation of NS3 by NS5B suggests that these proteins may function together 
during replication of the HCV genome.