Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Abstract

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp 220 , Asp 318 , and Asp 319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzymebound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.Recent estimates indicate that more than 170 million people worldwide are infected with the hepatitis C virus (HCV) 1 (1). It is estimated that about 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2-5). There is thus an urgent need for the development of antiviral drugs aimed at inhibiting this pathogen.The HCV nonstructural 5B protein (NS5B) has been shown to be an RNA-dependent RNA polymerase (6 -11). The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (3). This lack of specificity for HCV RNA supports the notion that additional viral or cellular factors are required for specific recognition of the viral replication signal. The HCV RNA polymerase activity has been shown to be supported by both magnesium and manganese ions (7, 13, 21-25). However, the re...

Characterization of the Elongation Complex of Dengue Virus RNA Polymerase

“…Several earlier studies reported different roles for divalent metal ions in the polymerase function (24, 25). We examined the effect of MgCl 2 on the formation of the elongation complex (Fig.…”

Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA

“…Based on amino acid similarity within eight distinct RdRp motifs, protein A belongs to the positive-strand RNA RdRp supergroup I (34, 35). The sixth RdRp motif contains a GDD motif that is conserved among RdRps of all positive-strand RNA viruses and is required for metal ion coordination in the polymerase active site (4,9,34,35). Mutation of the GDD motif inhibits polymerase activity in many viruses, such as hepatitis C virus, poliovirus, and FHV (9, 31).…”

Dual Allosteric Inhibitors Jointly Modulate Protein Structure and Dynamics in the Hepatitis C Virus Polymerase

“…17 The three-dimensional structure of 1QUV was determined without metal ions coordinated within the active site. However, two magnesium ions were added to the structure in all simulations because they have been shown biochemically to be needed for efficient enzyme function 18,19 and have been observed to fundamentally alter the structure and dynamics of the enzyme in previous molecular simulations by our group. 15 Two allosteric inhibitors, 3MS and VGI, were employed in this work.…”