An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non-nucleoside inhibitors.

A major target for antiviral therapy against hepatitis C virus (HCV) is the HCV polymerase nonstructural protein 5B (NS5B). Huge efforts have been devoted to the development of nucleoside and non-nucleoside inhibitors (NNIs) of NS5B. An offshoot of these efforts has been the structural characterization of the interaction of NS5B with NNIs by X-ray crystallography. These works have shown that the conformation of recombinant NS5B is very similar across strains, constructs and complexes, making evaluation of the long-range conformational effects of NNIs nontrivial. Using procedures appropriate to the evaluation of such minor but potentially important differences, we objectively assessed the conformational diversity in the 78 available genotype 1b NS5B structures in the Protein Data Bank. We find that there are 20 significantly different NS5B conformations available, but all are geometrically close to a closed, RNA synthesis initiation-competent one. Within this fairly restricted range, differences can be mapped to movements of NS5B domains and subregions. Most of this information is actually defined by small but significant changes in complexes with NNIs. We thus establish rigorously the moving parts of the NS5B molecular machine and the previously unrecognized hinge points that come into play upon NNI binding. We propose that NNIs binding at three of the four distinct sites specifically inhibit the initiation step by the same mechanism: they prevent NS5B's "thumb" from quite reaching the proper initiation-competent position. Furthermore, we suggest that a small number of critical hinges in the NS5B structure may emerge as sites of resistance mutations during future antiviral treatment.