Direct fluorometric measurement of hepatitis C virus helicase activity

Boguszewska-Chachulska, Anna M.; Krawczyk, Mariusz; Stankiewicz, Anna; Gozdek, Agnieszka; Haenni, Anne‐Lise; Strokovskaya, L. I.

doi:10.1016/j.febslet.2004.04.072

Cited by 51 publications

(44 citation statements)

References 34 publications

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“…Cells were grown at 37°C until the cultures reached an optical density at 600 nm of ϳ1.0; they were induced with 1 mM IPTG and harvested after 4 h at 37°C. The recombinant protein was purified according to the protocol established for proteins purified from insect cell cultures (4). The concentration of the HCV helicase domain 1 was determined at 280 nm using the molar extinction coefficient of 7,680 M Ϫ1 cm Ϫ1 , which was calculated on the basis of protein composition by the ProtParam program.…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of the peptide was determined at 280 nm using the molar extinc- Expression and purification of recombinant proteins. The expression and purification of the full-length HCV helicase and its domain 2 were performed as previously described (4). Domain 1 of the HCV helicase was cloned into the bifunctional YpET-30a vector, a recombinant of pET-30a (Clontech) and pFL38, kindly provided by M. Zagulski (Institute of Biochemistry and Biophysics [IBB]), by using homologous recombination in the Saccharomyces cerevisiae BY strain.…”

Section: Methodsmentioning

confidence: 99%

“…The ATPase assay was performed as described previously (3,4). The peptide was tested up to a concentration of 100 M. The reactions were carried out in 25 l of reaction buffer with 100 nM helicase for 60 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Gozdek

Zhukov

Polkowska

et al. 2008

Antimicrob Agents Chemother

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Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ( 1 H and 15 N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family (11). HCV infection, affecting 3% of the world population, leads to chronic hepatitis in up to 85% of the cases, in 10 to 20% of the cases it develops into cirrhosis that requires constant treatment and provokes permanent infirmity, while 1 to 5% of chronically infected patients are diagnosed with hepatocellular carcinoma (9). No efficient treatment exists; even the new dual therapy with pegylated interferon alpha 2a or 2b and ribavirin is effective only in up to 60% of the cases, depending on the genotype of the virus and the duration of the treatment (21). To date, no vaccine against HCV has been developed in spite of numerous attempts and advanced trials, principally because of the high variability of the RNA genome and association of HCV particles with host lipoproteins and immunoglobulins (1, 15). Thus, nonstructural proteins involved in viral replication are being examined as targets of antiviral therapy. One of them is NS3 (serine protease/RNA helicase), whose helicase activity is indispensable for replication of the viral RNA (25). The helicase part of NS3 folds into three domains of comparable size (domains 1, 2, and 3) that form a triangular molecule. Five structures of the NS3 helicase have been resolved by X-ray crystallography. The latest resolved structure shows two helicases bound to a single DNA molecule and reveals an apparent interface between two protein molecules (33). The existence of oligomeric structures o...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Gozdek

Zhukov

Polkowska

et al. 2008

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

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“…Because the assay does not require electrophoretic separation of the strands unwound by the helicase, it can be readily adapted for high-throughput screening or for measurement of real-time kinetics [14][15][16]. We previously applied the assay to the monitoring of NS3 helicase activity, using a dsRNA substrate [17].…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

Furuta

Salam

Tani

et al. 2014

Methods in Molecular Biology

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Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

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“…A previously reported fluorescence resonance energy transfer (FRET) assay (18,20) was performed to detect the DNA strands separation in real time. In this assay, the fluorescent strand of the duplex DNA was modified with an Alexa Fluor 488 fluorescent dye, whereas the quencher strand was modified with a spectrally paired quencher dye, black hole quencher.…”

Section: Pt Modifications Occur At G Ps Aac/g Ps Ttc Sites In R Anatmentioning

confidence: 99%

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif

Zheng

Jiang

Cao

et al. 2016

Journal of Biological Chemistry

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Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PTmodifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.

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Direct fluorometric measurement of hepatitis C virus helicase activity

Cited by 51 publications

References 34 publications

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif

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