Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides

Abstract

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Å away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-Å resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage 6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the 6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.About 3% of the world population is chronically infected with hepatitis C virus (HCV), an infection that leads to liver cancer in many patients. There is neither vaccine nor effective therapy available against this pathogen, and, therefore, there is an urgent need for efficient means of combating and, ultimately, curing this viral disease. The RNA-dependent RNA polymerase (RdRp) activity of HCV protein NS5B is an absolute requirement for replication of the virus. This enzyme exhibits important differences with cellular polymerases and is therefore a good target for developing specific anti-HCV therapies. The crystal structure of unliganded NS5B has been determined by three groups independently (2,8,27); this work shows that NS5B contains the classic structural domains, denoted fingers, palm, and thumb, of other single-chain polynucleotide polymerases (21). The strictly conserved aspartic acids that chelate the catalytic Mg 2ϩ ions are found in the palm, the fold of which is conserved among all members of the polymerase I and ␣ families (43). In contrast to most other single-chain polymerases of known structure, the finger and thumb domains are connected in the HCV enzyme and are therefore not free to change conformation independently of each other. This is also the case for other RdRps, as confirmed by the recently reported structure of the double-stranded RNA bacteriophage 6 polymerase (12). The overall similarity between the phage 6 ...

Predictive and comparative analysis of Ebolavirus proteins

“…The alignment between Ebolavirus RdRP and Bunyavirus RdRP includes both the catalytic domain and a helical bundle connected to its C-terminus. These two domains are conserved among known structures of RdRPs from RNA viruses [84][85][86][87][88] ( Fig. 4A-C).…”

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

“…This ␤-hairpin generates a narrow gate that prevents the 3Ј terminus of the template from slipping through the active site and that ensures initiation of RNA synthesis from the 3Ј-end of the template. Another interesting feature of NS5B is a low affinity GTP-binding site located at the interface of the thumb and finger subdomain and residing on the surface of the molecule (51). Binding of GTP to this site may induce a conformational change resulting in efficient initiation of RNA synthesis (52).…”

Unravelling hepatitis C virus replication from genome to function

“…46). NS5B also has a low-affinity GTP-binding site, distal from the active site, which is thought to be an allosteric regulator of the finger-thumb interaction 47 . NS5B is tethered to membranes by a C-terminal peptide anchor 48 and interacts with itself to form higher-order RdRP complexes that may have functional relevance to the membrane-bound replicase, described below 49 .…”