Characterization of the Metal Ion Binding Properties of the Hepatitis C Virus RNA Polymerase

Abstract

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg 2؉ or Mn 2؉ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K d values for the binding of Mg 2؉ and Mn 2؉ ions to the free enzyme were 3.1 and 0.3 mM, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ioninduced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state. Currently, hepatitis C virus (HCV)1 is the leading etiological agent of non-A non-B hepatitis, with more than 170 million people worldwide being infected with HCV (1). 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). HCV is a positive, single-stranded RNA virus of the Flaviviridae family. The genome is ϳ10,000 nucleotides long and encodes a single polyprotein of about 3,010 amino acids (6). The polyprotein is processed by both host cell and viral proteases into three major structural proteins and several nonstructural proteins necessary for viral replication (6).One key enzyme encoded by HCV is the nonstructural 5B protein (NS5B), which has been shown to be an RNA-dependent RNA polymerase (7-12). The HCV NS5B protein contains characteristic motifs, such as the GDD motif, shared by RNAdependent RNA polymerases (13). The NS5B protein is thus believed to be responsible for the genome replication of HCV. Indeed, polymerase activity has been demonstrated with recombinant NS5B expressed in both insect cells and Escherichia coli (8, 14 -26). The activity has been extensively studied because it is one of the major targets for the development of antiviral drugs. The NS5B protein can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (27). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8). This lack of spec...

Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

“…8A) (30,38,39), the displacement of Mg 2ϩ (or Mn 2ϩ ) by iron can explain the inhibition of the RdRp activity of NS5B. A direct iron-mediated inhibition of NS5B activity is indeed illustrated by the data in Fig.…”

“…6 and 7). This powerful technique was previously employed to monitor the metal binding properties of NS5B⌬21 and to determine the apparent dissociation constant (K d ) for Mg 2ϩ (3.1 mM) and Mn 2ϩ (0.3 mM) (30). We show that the apparent K d for Fe 2ϩ is 6 M, and the apparent K d for Fe 3ϩ is 60 M (Table I).…”

“…Expression and Purification of NS5B⌬21-NS5B⌬21, a truncated form of HCV NS5B protein lacking the 21 C-terminal amino acids, was expressed and purified as described previously (30).…”

“…This experiment showed that Fe 2ϩ competes with Mg 2ϩ for binding to the protein. Because Mg 2ϩ (or Mn 2ϩ , or a combination of both) are absolutely required for NS5B to function as an RdRp (30,38,39), we reasoned that the binding of iron may impair its enzymatic activity. This was directly assessed by a primer-independent RNA synthesis assay (Fig.…”

“…The NS5B⌬21 protein was highly purified (Fig. 6A) and analyzed by endogenous tryptophan fluorescence emission spectroscopy (30) for the binding of ferrous (Fe 2ϩ ) iron. Fluorescence spectra were quenched in the presence of increasing micromolar concentrations of Fe 2ϩ without affecting the emission maximum and the spectra bandwidth (Fig.…”