Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence

Tomei, Licia; Vitale, Rosa; Incitti, Ilario; Serafini, Sergio; Altamura, Sergio; Vitelli, Alessandra; Francesco, Raffaele De

doi:10.1099/0022-1317-81-3-759

Cited by 80 publications

(87 citation statements)

References 25 publications

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“…Expression, Purification, and Intrinsic Fluorescence Properties of HCV NS5B Protein-The HCV NS5B protein contains motifs shared by RNA polymerases, and its activity has been shown to be dependent on the presence of either magnesium or manganese ions (8,14,(22)(23)(24)(25)(26). To further characterize the metal binding activity of the enzyme, the NS5B protein was expressed in E. coli as described under "Experimental Procedures."…”

Section: Resultsmentioning

confidence: 99%

“…To further characterize the metal binding activity of the enzyme, the NS5B protein was expressed in E. coli as described under "Experimental Procedures." A truncated form of NS5B (NS5B⌬21) lacking the previously identified 21-amino acid hydrophobic domain (14,22,24,25) was expressed and purified. SDS-PAGE analysis showed that the 65-kDa NS5B⌬21 protein was the predominant polypeptide in the purified fraction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8). 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.Previous reports showed that both magnesium and manganese ions can support the polymerase activity (8,14,(22)(23)(24)(25)(26), although manganese ions appear to be more effective in promoting optimal catalytic action (14). No information is currently available on the precise affinity of the enzyme for metal ions nor on their possible roles in catalysis, but manganese has recently been found in the crystal structure of NS5B (28).…”

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confidence: 99%

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

Bougie

Charpentier

Bisaillon

2003

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Bougie

Charpentier

Bisaillon

2003

Journal of Biological Chemistry

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“…The biological activity of the compounds against NS5B polymerase were evaluated in a reaction buffer containing 20 mM TriseHCl (pH 7.0), 100 mM NaCl, 100 mM sodium glutamate, 0.1 mM DTT, 0.01% BSA, 0.01% Tween-20, 5% glycerol, 20 U/mL of RNase Out, 0.25 mM of polyrA/U12, 25 mM UTP, 2 mCi [alpha-32 P]UTP, 300 ng of NS5BCD21and 1.0 mM MnCl2 with or without inhibitors (100 mM) in a total volume of 25 mL for 1 h at 30 C as previously described [26,27]. Reactions were terminated by the addition of ice-cold 5% (v/v) trichloroacetic acid (TCA) containing 0.5 mM pyrophosphate.…”

Section: Ns5b Inhibition Assaymentioning

confidence: 99%

Synthesis of novel diflunisal hydrazide–hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors

Şenkardeş

Kaushik‐Basu

Durmaz

et al. 2016

European Journal of Medicinal Chemistry

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a b s t r a c tHepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. We have previously identified the hydrazideehydrazone scaffold as a promising hepatitis C virus (HCV) and hepatocelluler inhibitor. Herein we describe the design a number of 2 0 ,4 0 -difluoro-4-hydroxy-N'-(arylmethylidene) biphenyl-3-carbohydrazide (3a-t) as anti-HCV and anticancer agents. Results from evaluation of anti-HCV activity indicated that most of the synthesized hydrazone derivatives inhibited viral replication in the Huh7/Rep-Feo1b and Huh 7.5-FGR-JCI-Rluc2A reporter systems. Antiproliferative activities of increasing concentrations of 2 0 ,4 0 -difluoro-4-hydroxy-N'-(2-pyridyl methylidene)biphenyl-3-carbohydrazide 3b and diflunisal (2.5e40 mM) were assessed in liver cancer cell lines (Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with sulforhodamine B assay for 72 h. Compound 3b with 2-pyridinyl group in the hydrazone part exhibited promising cytotoxic activity against all cell lines with IC 50 values of 10, 10.34 16.21 4.74, 9.29 and 8.33 mM for Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475 cells, respectively, and produced dramatic cell cycle arrest at SubG1/G0 phase as an indicator of apoptotic cell death induction.

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“…Another TNTase-free HCV NS5B with deletion of the 21 C-terminal hydrophobic amino acids copied HCV X-RNA inefficiently by internal initiation (Kashiwagi et al, 2002). Tomei et al reported that the full-length NS5B has a slightly enhanced processivity compared to the truncated form of NS5B (Tomei et al, 2000). Since all the HCV NS5B proteins used for structure determination were missing these hydrophobic residues (Bressanelli et al, 1999;Lesburg et al, 1999;Butcher et al, 2001), knowledge of the topological characteristics of the C-terminal hydrophobic domain of NS5B is still needed to understand the role of this region in enzymatic function of NS5B.…”

Section: Introductionmentioning

confidence: 99%

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

Choi

Kim

Oh³

2003

Exp Mol Med

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A bstractThe hepatitis C virus (HCV) RNA-dependent RNA polym erase, NS5B protein, is the key viral enzym e responsible for replication of the HCV viral RNA genom e. Although several full-length and truncated form s of the HCV NS5B proteins have been expressed previously in insect cells, contam ination of host term inal transferase (TNTase) has ham pered analysis of the RNA synthesis initiation m echanism using natural HCV RNA tem plates. W e have expressed the HCV NS5B protein in insect cells using a recom binant baculovirus and purified it to near hom ogeneity w ithout contam inated TNTase. The highly purified recom binant HCV NS5B w as capable of copying 9.6-kb full-length HCV RNA tem plate, and m ini-HCV RNA carrying both 5'-and 3'-untranslated regions (UTRs) of the HCV genom e. In the absence of a prim er, and other cellular and viral factors, the NS5B could elongate over HCV RNA tem plates, but the synthesized products were prim arily in the double stranded form , indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA tem plates representing the 3'-end region of HCV m inus-strand RNA and the X-RNA at the 3'-end of HCV RNA genom e w as also initiated de novo. N o form ation of dim er-size self-prim ed RNA products resulting from extension of the 3'-end hydroxyl group w as observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polym erase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genom e.

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Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence

Cited by 80 publications

References 25 publications

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

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

Synthesis of novel diflunisal hydrazide–hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

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