Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

Abstract

bThe replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory ␤-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory ␤-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

“…A ␤-hairpin protrudes in the active site and represents an obstacle for RNA translocation (16,17). The structure of an engineered HCV NS5B enzyme that lacks this ␤-hairpin shows that a 6mer RNA primer-template can be accommodated under these conditions (18). The data also reveals significant movements of the thumb relative to the fingers, which creates sufficient room for the double stranded (ds)RNA region that is located at the dsRNA exit (Fig.…”

“…d, HCV NS5B (genotype 2a JFH-1 strain) crystal structure containing a dsRNA substrate (PDB code 4E7A). A regulatory ␤-hairpin loop located in the fingers subdomain is removed from the NS5B construct to allow for binding of a short dsRNA substrate (18).…”

“…1. a, highlight of residue Arg-394 interacting with the phosphate backbone of the primer strand (PDB code 4E7A) (18). b, Lys-98 and Lys-100 are located in close proximity of the RNA template at the entrance site (PDB code 1NB7) (15).…”

Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase

“…1) whose organization can be likened to a closed right hand (3)(4)(5)(6). Two unique features of NS5B are an extension of the fingers domain that reaches over the active site to contact the thumb domain to enclose the active site and a ␤-loop that projects from the thumb domain into the active site to block the binding of duplex RNA (Fig.…”

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

“…The fact that BMS-791325 is unable to block elongation suggests that the interaction between the fingers and thumb that occurs during the formation of active replication complexes is fundamentally different during elongation. It is possible that the fingers domain is less flexible during elongation and blocks the binding of BMS-791325 or that the shape of the thumb binding pocket is different and cannot bind inhibitor (17,38). Experiments with non-nucleoside inhibitors that bind to different polymerase structural domains are ongoing to further investigate the impact of the finger-thumb interaction on HCV NS5B replication.…”