Dynamics of HCV RNA-dependent RNA Polymerase NS5B in Complex with RNA

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNAdependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuation are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3’ terminus at the enzyme active site. The hepatitis C virus (HCV) is recognized as a major human pathogen with approximately 170 million infected people worldwide (1,2). Chronic HCV infection is associated with severe liver disease, including cirrhosis and hepatocellular carcinoma (HCC) (3). HCV is a positive strand virus that belongs to the Flaviviridae family (4). It contains a single stranded (ss) 9.6 kb RNA genome, which encodes a polyprotein that is processed into several structural and non-structural proteins (3). The nonstructural protein 5B (NS5B), which is a prime target in current drug discovery efforts, shows RNA-dependent RNA polymerase (RdRp) activity