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Farouk F, Ibrahim IM, Sherif S, Abdelhamed HG, Sharaky M, Al-Karmalawy AA. Investigating the effect of polymerase inhibitors on cellular proliferation: Computational studies, cytotoxicity, CDK1 inhibitory potential, and LC-MS/MS cancer cell entrapment assays. Chem Biol Drug Des 2024; 103:e14500. [PMID: 38467555 DOI: 10.1111/cbdd.14500] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2023] [Revised: 02/04/2024] [Accepted: 02/26/2024] [Indexed: 03/13/2024]
Abstract
Directly acting antivirals (DAAs) are a breakthrough in the treatment of HCV. There are controversial reports on their tendency to induce hepatocellular carcinoma (HCC) in HCV patients. Numerous reports have concluded that the HCC is attributed to patient-related factors while others are inclined to attribute this as a DAA side-effect. This study aims to investigate the effect of polymerase inhibitor DAAs, especially daclatasivir (DLT) on cellular proliferation as compared to ribavirin (RBV). The interaction of DAAs with variable cell-cycle proteins was studied in silico. The binding affinities to multiple cellular targets were investigated and the molecular dynamics were assessed. The in vitro effect of the selected candidate DLT on cancer cell proliferation was determined and the CDK1 inhibitory potential in was evaluated. Finally, the cellular entrapment of the selected candidates was assessed by an in-house developed and validated LC-MS/MS method. The results indicated that polymerase inhibitor antiviral agents, especially DLT, may exert an anti-proliferative potential against variable cancer cell lines. The results showed that the effect may be achieved via potential interaction with the multiple cellular targets, including the CDK1, resulting in halting of the cellular proliferation. DLT exhibited a remarkable cell permeability in the liver cancer cell line which permits adequate interaction with the cellular targets. In conclusion, the results reveal that the polymerase inhibitor (DLT) may have an anti-proliferative potential against liver cancer cells. These results may pose DLT as a therapeutic choice for patients suffering from HCV and are liable to HCC development.
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Affiliation(s)
- Faten Farouk
- Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt
| | - Ibrahim M Ibrahim
- Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt
| | - Salma Sherif
- Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt
| | | | - Marwa Sharaky
- Pharmacology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt
- Biochemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt
| | - Ahmed A Al-Karmalawy
- Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, Egypt
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Del Rosario García-Lozano M, Dragoni F, Gallego P, Mazzotta S, López-Gómez A, Boccuto A, Martínez-Cortés C, Rodríguez-Martínez A, Pérez-Sánchez H, Manuel Vega-Pérez J, Antonio Del Campo J, Vicenti I, Vega-Holm M, Iglesias-Guerra F. Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses. Bioorg Chem 2023; 133:106408. [PMID: 36801791 DOI: 10.1016/j.bioorg.2023.106408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 01/23/2023] [Accepted: 02/01/2023] [Indexed: 02/05/2023]
Abstract
Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.
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Affiliation(s)
- María Del Rosario García-Lozano
- Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain; SeLiver Group at the Institute of Biomedicine of Seville (IBIS), Virgen del Rocío University Hospital CSIC University of Seville, Seville, Spain
| | - Filippo Dragoni
- Department of Medical Biotechnologies, Siena University Hospital, Policlinico Le Scotte, Viale Bracci 16, 53100 Siena, Italy
| | - Paloma Gallego
- Unit for Clinical Management of Digestive Diseases and CIBERehd, Valme University Hospital, 41014 Seville, Spain
| | - Sarah Mazzotta
- Department of Chemistry, University of Milan, 20133 Milan, Italy
| | - Alejandro López-Gómez
- Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain
| | - Adele Boccuto
- Department of Medical Biotechnologies, Siena University Hospital, Policlinico Le Scotte, Viale Bracci 16, 53100 Siena, Italy; VisMederi Research srl, Siena, Italy
| | - Carlos Martínez-Cortés
- Structural Bioinformatics and High Performance Computing (BIO-HPC) Research Group, UCAM Universidad Católica de Murcia, 30107 Murcia, Spain
| | - Alejandro Rodríguez-Martínez
- Department of Physical Chemistry and Institute of Biotechnology, University of Granada, Campus Fuentenueva sn, 18071 Granada, Spain
| | - Horacio Pérez-Sánchez
- Structural Bioinformatics and High Performance Computing (BIO-HPC) Research Group, UCAM Universidad Católica de Murcia, 30107 Murcia, Spain
| | - José Manuel Vega-Pérez
- Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain
| | | | - Ilaria Vicenti
- Department of Medical Biotechnologies, Siena University Hospital, Policlinico Le Scotte, Viale Bracci 16, 53100 Siena, Italy.
| | - Margarita Vega-Holm
- Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain.
| | - Fernando Iglesias-Guerra
- Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain
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3
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Saraswat S, Chaudhary M, Sehgal D. Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays. Front Cell Infect Microbiol 2020; 9:478. [PMID: 32039053 PMCID: PMC6989534 DOI: 10.3389/fcimb.2019.00478] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Accepted: 12/27/2019] [Indexed: 12/11/2022] Open
Abstract
Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432–592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of “Papain-like cysteine protease,” as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.
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Affiliation(s)
- Shweta Saraswat
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Meenakshi Chaudhary
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Deepak Sehgal
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
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Ventura GT, da Costa ECB, Capaccia AM, Mohana-Borges R. pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. PLoS One 2014; 9:e115941. [PMID: 25551442 PMCID: PMC4281115 DOI: 10.1371/journal.pone.0115941] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 11/29/2014] [Indexed: 11/29/2022] Open
Abstract
The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.
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Affiliation(s)
- Gustavo Tavares Ventura
- Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Emmerson Corrêa Brasil da Costa
- Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Anne Miranda Capaccia
- Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Ronaldo Mohana-Borges
- Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
- * E-mail:
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5
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Nonstructural protein 5A (NS5A) and human replication protein A increase the processivity of hepatitis C virus NS5B polymerase activity in vitro. J Virol 2014; 89:165-80. [PMID: 25320291 DOI: 10.1128/jvi.01677-14] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
UNLABELLED The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.
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Hazuda DJ, Burroughs M, Howe AYM, Wahl J, Venkatraman S. Development of boceprevir: a first-in-class direct antiviral treatment for chronic hepatitis C infection. Ann N Y Acad Sci 2013; 1291:69-76. [PMID: 23859802 DOI: 10.1111/nyas.12218] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The identification of hepatitis C virus (HCV) as the causative agent of non-A and non-B hepatitis, over 20 years ago, fueled an intensive effort to develop direct-acting antivirals targeting the viral polymerase and protease, two key proteins critical for HCV replication. However, it took more than two decades for these efforts to be realized with boceprevir, one of the two HCV protease inhibitors approved for treatment of HCV infection in 2011. The development of boceprevir is a major advancement in the ability to treat HCV infection and a significant step toward the long-term goal of eradicating chronic HCV infection. Both as a first-in-class agent and an entirely new modality for treating HCV infection, many challenges were encountered during the discovery and development of this compound. The lessons learned in overcoming these obstacles offer insights and pave the way for the newly emerging field of HCV antiviral therapeutics. This paper will describe the discovery and development of a first-in-class direct antiviral treatment for chronic hepatitis C infection, boceprevir, marketed around the world as Victrelis™.
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Affiliation(s)
- Daria J Hazuda
- Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
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7
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Howe AYM, Venkatraman S. The Discovery and Development of Boceprevir: A Novel, First-generation Inhibitor of the Hepatitis C Virus NS3/4A Serine Protease. J Clin Transl Hepatol 2013; 1:22-32. [PMID: 26357603 PMCID: PMC4548358 DOI: 10.14218/jcth.2013.002xx] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2013] [Revised: 02/22/2013] [Accepted: 02/25/2013] [Indexed: 12/11/2022] Open
Abstract
An estimated 2-3% of the world's population is infected with hepatitis C virus (HCV), making it a major global health problem. Consequently, over the past 15 years, there has been a concerted effort to understand the pathophysiology of HCV infection and the molecular virology of replication, and to utilize this knowledge for the development of more effective treatments. The virally encoded non-structural serine protease (NS3) is required to process the HCV polyprotein and release the individual proteins that form the viral RNA replication machinery. Given its critical role in the replication of HCV, the NS3 protease has been recognized as a potential drug target for the development of selective HCV therapies. In this review, we describe the key scientific discoveries that led to the approval of boceprevir, a first-generation, selective, small molecule inhibitor of the NS3 protease. We highlight the early studies that reported the crystal structure of the NS3 protease, its role in the processing of the HCV polyprotein, and the structural requirements critical for substrate cleavage. We also consider the novel attributes of the NS3 protease-binding pocket that challenged development of small molecule inhibitors, and the studies that ultimately yielded milligram quantities of this enzyme in a soluble, tractable form suitable for inhibitor screening programs. Finally, we describe the discovery of boceprevir, from the early chemistry studies, through the development of high-throughput assays, to the phase III clinical development program that ultimately provided the basis for approval of this drug. This latest phase in the development of boceprevir represents the culmination of a major global effort to understand the pathophysiology of HCV and develop small molecule inhibitors for the NS3 protease.
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8
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Xue W, Wang M, Jin X, Liu H, Yao X. Understanding the structural and energetic basis of inhibitor and substrate bound to the full-length NS3/4A: insights from molecular dynamics simulation, binding free energy calculation and network analysis. MOLECULAR BIOSYSTEMS 2012; 8:2753-65. [DOI: 10.1039/c2mb25157d] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
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9
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Martin MM, Condotta SA, Fenn J, Olmstead AD, Jean F. In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile. Biol Chem 2011; 392:927-35. [PMID: 21749281 DOI: 10.1515/bc.2011.076] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.
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Affiliation(s)
- Morgan M Martin
- Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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10
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Smirnova OA, Ivanov AV, Ivanova ON, Valuev-Elliston VT, Kochetkov SN. Cell defense systems against oxidative stress and endoplasmic reticulum stress: Mechanisms of regulation and the effect of hepatitis C virus. Mol Biol 2011. [DOI: 10.1134/s0026893311010122] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
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11
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Kerrigan JJ, Xie Q, Ames RS, Lu Q. Production of protein complexes via co-expression. Protein Expr Purif 2010; 75:1-14. [PMID: 20692346 DOI: 10.1016/j.pep.2010.07.015] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2010] [Revised: 07/22/2010] [Accepted: 07/31/2010] [Indexed: 12/21/2022]
Abstract
Multi-protein complexes are involved in essentially all cellular processes. A protein's function is defined by a combination of its own properties, its interacting partners, and the stoichiometry of each. Depending on binding partners, a transcription factor can function as an activator in one instance and a repressor in another. The study of protein function or malfunction is best performed in the relevant context. While many protein complexes can be reconstituted from individual component proteins after being produced individually, many others require co-expression of their native partners in the host cells for proper folding, stability, and activity. Protein co-expression has led to the production of a variety of biological active complexes in sufficient quantities for biochemical, biophysical, structural studies, and high throughput screens. This article summarizes examples of such cases and discusses critical considerations in selecting co-expression partners, and strategies to achieve successful production of protein complexes.
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Affiliation(s)
- John J Kerrigan
- Biological Reagents & Assay Development, Platform Technology & Science, GlaxoSmithKline R&D, 1250 South Collegeville Road, Collegeville, PA 19426, USA
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12
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Dahl G, Arenas OG, Danielson UH. Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of Protease Inhibitors. Biochemistry 2009; 48:11592-602. [DOI: 10.1021/bi9016928] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Göran Dahl
- Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
| | - Omar Gutiérrez Arenas
- Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
| | - U. Helena Danielson
- Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
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13
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Rajagopalan R, Misialek S, Stevens SK, Myszka DG, Brandhuber BJ, Ballard JA, Andrews SW, Seiwert SD, Kossen K. Inhibition and Binding Kinetics of the Hepatitis C Virus NS3 Protease Inhibitor ITMN-191 Reveals Tight Binding and Slow Dissociative Behavior. Biochemistry 2009; 48:2559-68. [DOI: 10.1021/bi900038p] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- Ravi Rajagopalan
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Shawn Misialek
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Sarah K. Stevens
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - David G. Myszka
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Barbara J. Brandhuber
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Joshua A. Ballard
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Steven W. Andrews
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Scott D. Seiwert
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
| | - Karl Kossen
- InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, Biosensor Tools, 1588 East Connecticut Drive, Salt Lake City, Utah 84132, and Array Biopharma, 3200 Walnut Street, Boulder, Colorado 80301
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14
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The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase. J Virol 2009; 83:3268-75. [PMID: 19153239 DOI: 10.1128/jvi.01849-08] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.
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15
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Bermúdez-Aguirre AD, Padilla-Noriega L, Zenteno E, Reyes-Leyva J. Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A. TOHOKU J EXP MED 2009; 218:165-75. [DOI: 10.1620/tjem.218.165] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Affiliation(s)
- Alejandro Daniel Bermúdez-Aguirre
- Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México
- Laboratorio de Virología, Centro de Investigaciones Biomédicas de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5
| | - Luis Padilla-Noriega
- Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México
| | - Edgar Zenteno
- Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México
- Facultad de Medicina Humana, Universidad Ricardo Palma
| | - Julio Reyes-Leyva
- Laboratorio de Virología, Centro de Investigaciones Biomédicas de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5
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16
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Beran RKF, Pyle AM. Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem 2008; 283:29929-37. [PMID: 18723512 PMCID: PMC2573085 DOI: 10.1074/jbc.m804065200] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.
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Affiliation(s)
- Rudolf K F Beran
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA
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17
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Dahl G, Sandström A, Åkerblom E, Danielson UH. Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3. FEBS J 2007; 274:5979-86. [DOI: 10.1111/j.1742-4658.2007.06120.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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18
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Dahl G, Sandström A, Åkerblom E, Danielson UH. Resistance Profiling of Hepatitis C Virus Protease Inhibitors using Full-Length NS3. Antivir Ther 2007. [DOI: 10.1177/135965350701200504] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Background The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3. Experimental The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain. Results The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme. Conclusions These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.
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Affiliation(s)
- Göran Dahl
- Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
| | - Anja Sandström
- Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
| | - Eva Åkerblom
- Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
| | - U Helena Danielson
- Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
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19
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Wen G, Chen C, Luo X, Wang Y, Zhang C, Pan Z. Identification and characterization of the NTPase activity of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) expressed in bacteria. Arch Virol 2007; 152:1565-73. [PMID: 17447110 DOI: 10.1007/s00705-007-0969-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2007] [Accepted: 03/09/2007] [Indexed: 11/26/2022]
Abstract
The nonstructural protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK(232)T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA(354) for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K(m) and k(cat)) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.
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Affiliation(s)
- G Wen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
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20
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Zhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY, Kao C, Luo G. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J Virol 2005; 79:8687-97. [PMID: 15994762 PMCID: PMC1168731 DOI: 10.1128/jvi.79.14.8687-8697.2005] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2005] [Accepted: 03/22/2005] [Indexed: 01/28/2023] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.
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Affiliation(s)
- Chen Zhang
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA
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21
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Abstract
Hepatitis C virus (HCV) has infected millions of people worldwide and has emerged as a global health crisis. The currently available therapy is interferon (IFN) either alone or in combination with ribavirin. However, the disappointing efficacy of IFN has led to the considerable need for improved treatments and a number of new therapies are under evaluation in clinical trials. These include pegylated IFNs, which have altered physiochemical characteristics allowing once-weekly dosing. Combination of pegylated IFN with ribavirin should further improve sustained response rates. However, not all patients are successfully treated with IFNs, particularly those infected with genotype 1 of the virus, and it is likely that potent, specific drugs will be required. The majority of new approaches currently trying to combat this viral disease are aimed at inhibition of viral targets. Most effort has been directed towards inhibition of the NS3 serine protease, and potent inhibitors have now been described. However, a clinical candidate is yet to emerge against this difficult target. Considerable work by leading researchers has provided crystal structures of the key replicative enzymes, NS3 protease, NS3 helicase, NS5B polymerase and full-length NS3 protease-helicase, and there is much hope that such structural information will bear fruit. More recently, inhibition of host targets, particularly inosine monophosphate dehydrogenase (IMPDH), has become of interest and there are on-going clinical trials with such inhibitors. Research aimed at novel treatments for HCV disease is gathering pace and very recent developments in cell-based assay systems can only hasten the discovery of improved therapies.
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Affiliation(s)
- B W Dymock
- Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Herts, AL7 3AY, UK.
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22
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Tackett AJ, Chen Y, Cameron CE, Raney KD. Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro. J Biol Chem 2005; 280:10797-806. [PMID: 15634684 DOI: 10.1074/jbc.m407971200] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
NS3 (nonstructural protein 3) from the hepatitis C virus is a 3' --> 5' helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in K(D) = 2.7 +/- 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the K(D) for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (K(D) = 1.3 +/- 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (K(D) = 11.3 +/- 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.
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Affiliation(s)
- Alan J Tackett
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, Arkansas 72205, USA
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23
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Kyono K, Miyashiro M, Taguchi I. Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. J Biochem 2004; 135:245-52. [PMID: 15047727 DOI: 10.1093/jb/mvh029] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) ANAL: BIOCHEM: 257, 120-126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 protease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn(2+), and that the optimal pH was 6.0-6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.
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Affiliation(s)
- Kiyoshi Kyono
- Medicinal Chemistry Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505.
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24
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Lam AMI, Rypma RS, Frick DN. Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding. Nucleic Acids Res 2004; 32:4060-70. [PMID: 15289579 PMCID: PMC506820 DOI: 10.1093/nar/gkh743] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The molecular basis of the low-pH activation of the helicase encoded by the hepatitis C virus (HCV) was examined using either a full-length NS3 protein/NS4A cofactor complex or truncated NS3 proteins lacking the protease domain, which were isolated from three different viral genotypes. All proteins unwound RNA and DNA best at pH 6.5, which demonstrate that conserved NS3 helicase domain amino acids are responsible for low-pH enzyme activation. DNA unwinding was less sensitive to pH changes than RNA unwinding. Both the turnover rate of ATP hydrolysis and the K(m) of ATP were similar between pH 6 and 10, but the concentration of nucleic acid needed to stimulate ATP hydrolysis decreased almost 50-fold when the pH was lowered from 7.5 to 6.5. In direct-binding experiments, HCV helicase bound DNA weakly at high pH only in the presence of the non-hydrolyzable ATP analog, ADP(BeF3). These data suggest that a low-pH environment might be required for efficient HCV RNA translation or replication, and support a model in which an acidic residue rotates toward the RNA backbone upon ATP binding repelling nucleic acid from the binding cleft.
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Affiliation(s)
- Angela M I Lam
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA
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25
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Kuang WF, Lin YC, Jean F, Huang YW, Tai CL, Chen DS, Chen PJ, Hwang LH. Hepatitis C virus NS3 RNA helicase activity is modulated by the two domains of NS3 and NS4A. Biochem Biophys Res Commun 2004; 317:211-7. [PMID: 15047170 DOI: 10.1016/j.bbrc.2004.03.032] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2004] [Indexed: 11/21/2022]
Abstract
To determine whether the two domains of hepatitis C virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins--the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3-NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3-NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein, R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3-NS4A. Together, these results suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities.
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Affiliation(s)
- Wan-Fen Kuang
- Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC
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26
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Frick DN, Rypma RS, Lam AMI, Gu B. The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Biol Chem 2004; 279:1269-80. [PMID: 14585830 PMCID: PMC3571687 DOI: 10.1074/jbc.m310630200] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3'-5' helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.
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Affiliation(s)
- David N Frick
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
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27
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Abstract
Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.
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Affiliation(s)
- François Penin
- Institut de Biologie et Chimie des Protéines, Lyon, France.
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28
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Hou LH, Du GX, Guan RB, Tong YG, Wang HT. In vitro assay for HCV serine proteinase expressed in insect cells. World J Gastroenterol 2003; 9:1629-32. [PMID: 12854181 PMCID: PMC4615522 DOI: 10.3748/wjg.v9.i7.1629] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.
METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.
RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 × 107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients' sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.
CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.
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Affiliation(s)
- Li-Hua Hou
- Department of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Beijing 100071, China.
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29
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Perni RB, Kwong AD. Inhibitors of hepatitis C virus NS3.4A protease: an overdue line of therapy. PROGRESS IN MEDICINAL CHEMISTRY 2003; 39:215-55. [PMID: 12536674 DOI: 10.1016/s0079-6468(08)70072-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Robert B Perni
- Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, USA
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30
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Du GX, Hou LH, Guan RB, Tong YG, Wang HT. Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease. World J Gastroenterol 2002; 8:1088-93. [PMID: 12439931 PMCID: PMC4656386 DOI: 10.3748/wjg.v8.i6.1088] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/18/2002] [Revised: 08/01/2002] [Accepted: 08/09/2002] [Indexed: 02/06/2023] Open
Abstract
AIM To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease. RESULTS The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not. CONCLUSION A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.
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Affiliation(s)
- Gui-Xin Du
- Department of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Fengtai, Beijing 100071, China.
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31
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Sico C, White S, Tsao E, Varma A. Enhanced kinetic extraction of parvovirus B19 structural proteins. Biotechnol Bioeng 2002; 80:250-6. [PMID: 12226856 DOI: 10.1002/bit.10509] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Recombinant structural proteins (VP1 and VP2) of the human parvovirus B19 have been expressed simultaneously using the baculovirus expression system to form virus-like particles (VLPs) that have potential use as vaccines. In this study, we report optimization of extraction conditions to recover these VLPs from cell paste. Under hypotonic conditions with neutral pH these VLPs were poorly extracted (up to 3% extraction). Addition of reducing agents, detergents, salts, and sonication did not improve the extractability. While screening for conditions to improve the extractability of the VLPs, we discovered that a combination of higher pH and elevated processing temperature significantly increased the extraction. Whereas increasing pH alone increased extractability from 3% to 6% (pH increased from 8.0 to 9.5), the effect of elevated temperature was much more substantial. At 50 degrees C, we observed the extraction to be more than fivefold higher than that at room temperature (up to 25% extracted at pH 9.0). The kinetics of extraction at elevated temperatures showed a rapid initial rate of extraction (on the order of minutes) followed by a plateau. In addition, we compared the extraction of VP1 expressed alone. VP1 expressed alone is incapable of forming VLPs. We observed that non-VLP VP1 was easily extractable (up to 60% extracted) under conditions in which the VP1 + VP2 VLPs were not extractable. From these studies we conclude that parvovirus B19 structural proteins expressed to form VLPs have a hindered extractability as compared with non-VLP protein. This hindrance to extraction can be significantly reduced by processing at elevated temperatures and an increased pH, possibly due to the enhanced rates of solubilization and diffusion.
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Affiliation(s)
- Colleen Sico
- MedImmune, Inc., 35 West Watkins Mill Road, Gaithersburg, Maryland 20878, USA
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32
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Poliakov A, Hubatsch I, Shuman CF, Stenberg G, Danielson UH. Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus. Protein Expr Purif 2002; 25:363-71. [PMID: 12182815 DOI: 10.1016/s1046-5928(02)00042-6] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.
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Affiliation(s)
- Anton Poliakov
- Department of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
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Pang PS, Jankowsky E, Planet PJ, Pyle AM. The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 2002; 21:1168-76. [PMID: 11867545 PMCID: PMC125889 DOI: 10.1093/emboj/21.5.1168] [Citation(s) in RCA: 180] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2001] [Revised: 12/03/2001] [Accepted: 01/09/2002] [Indexed: 01/22/2023] Open
Abstract
The RNA helicase/protease NS3 plays a central role in the RNA replication of hepatitis C virus (HCV), a cytoplasmic RNA virus that represents a major worldwide health problem. NS3 is, therefore, an important drug target in the effort to combat HCV. Most work has focused on the protease, rather than the helicase, activities of the enzyme. In order to further characterize NS3 helicase activity, we evaluated individual stages of duplex unwinding by NS3 alone and in complex with cofactor NS4A. Despite a putative replicative role in RNA unwinding, we found that NS3 alone is a surprisingly poor helicase on RNA, but that RNA activity is promoted by cofactor NS4A. In contrast, NS3 alone is a highly processive helicase on DNA. Phylogenetic analysis suggests that this robust DNA helicase activity is not vestigial and may have specifically evolved in HCV. Given that HCV has no replicative DNA intermediate, these findings suggest that NS3 may have the capacity to affect host DNA.
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Affiliation(s)
- Phillip S. Pang
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, Medical Scientist Training Program, Columbia College of Physicians and Surgeons, New York, NY, Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, Integrated Program in Cellular, Molecular and Biophysical Sciences and Howard Hughes Medical Institute, Columbia University, New York, NY, USA Corresponding author e-mail:
| | - Eckhard Jankowsky
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, Medical Scientist Training Program, Columbia College of Physicians and Surgeons, New York, NY, Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, Integrated Program in Cellular, Molecular and Biophysical Sciences and Howard Hughes Medical Institute, Columbia University, New York, NY, USA Corresponding author e-mail:
| | - Paul J. Planet
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, Medical Scientist Training Program, Columbia College of Physicians and Surgeons, New York, NY, Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, Integrated Program in Cellular, Molecular and Biophysical Sciences and Howard Hughes Medical Institute, Columbia University, New York, NY, USA Corresponding author e-mail:
| | - Anna Marie Pyle
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, Medical Scientist Training Program, Columbia College of Physicians and Surgeons, New York, NY, Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, Integrated Program in Cellular, Molecular and Biophysical Sciences and Howard Hughes Medical Institute, Columbia University, New York, NY, USA Corresponding author e-mail:
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Abstract
The Hepatitis C virus is a positive-stranded RNA virus which is the causal agent for a chronic liver infection afflicting more than 170,000,000 people world-wide. The HCV genome is approximately 9.6 kb in length and the proteome encoded is a polyprotein of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into structural and non-structural proteins. The functions of most of these proteins have been established by analogy to other viruses and by sequence homology to known proteins, as well as subsequent biochemical analysis. Two of the non-structural proteins, NS4b and NS5a, are still of unknown function. The development of antivirals for this infectious agent has been hampered by the lack of robust and economical cell culture and animal infection systems. Recent progress in the molecular virology of HCV has come about due to the definition of molecular clones, which are infectious in the chimpanzee, the development of a subgenomic replicon system in Huh7 cells, and the description of a transgenic mouse model for HCV infection. Recent progress in the structural biology of the virus has led to the determination of high resolution three-dimensional structures of a number of the key virally encoded enzymes, including the NS3 protease, NS3 helicase, and NS5b RNA-dependent RNA polymerase. In some cases these structures have been determined in complex with substrates, co-factors (NS4a), and inhibitors. Finally, a variety of techniques have been used to define host factors, which may be required for HCV replication, although this work is just beginning.
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Affiliation(s)
- S Rosenberg
- Department of Chemistry, University of California, Berkeley, CA 94720, USA.
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Aoubala M, Holt J, Clegg RA, Rowlands DJ, Harris M. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. J Gen Virol 2001; 82:1637-1646. [PMID: 11413375 DOI: 10.1099/0022-1317-82-7-1637] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.
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Affiliation(s)
- Mustapha Aoubala
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | - John Holt
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | | | - David J Rowlands
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | - Mark Harris
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
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36
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Pessi A. A personal account of the role of peptide research in drug discovery: the case of hepatitis C. J Pept Sci 2001; 7:2-14. [PMID: 11245202 DOI: 10.1002/psc.310] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Although peptides themselves are not usually the end products of a drug discovery effort, peptide research often plays a key role in many aspects of this process. This will be illustrated by reviewing the experience of peptide research carried out at IRBM in the course of our study of hepatitis C virus (HCV). The target of our work is the NS3/4A protease, which is essential for maturation of the viral polyprotein. After a thorough examination of its substrate specificity we fine-tuned several substrate-derived peptides for enzymology studies, high-throughput screening and as fluorescent probes for secondary binding assays. In the course of these studies we made the key observation: that the protease is inhibited by its own cleavage products. Single analog and combinatorial optimization then derived potent peptide inhibitors. The crucial role of the NS4A cofactor was also addressed. NS4A is a small transmembrane protein, whose central domain is the minimal region sufficient for enzyme activation. Structural studies were performed with a peptide corresponding to the minimal activation domain, with a series of product inhibitors and with both. We found that NS3/4A is an induced fit enzyme, requiring both the cofactor and the substrate to acquire its bioactive conformation; this explained some puzzling results of 'serine-trap' type inhibitors. A more complete study on NS3 activation, however, requires the availability of the full-length NS4A protein. This was prepared by native chemical ligation, after sequence engineering to enhance its solubility; structural studies are in progress. Current work is focused on the P' region of the substrate, which, at variance with the P region, is not used for ground state binding to the enzyme and might give rise to inhibitors showing novel interactions with the enzyme.
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Affiliation(s)
- A Pessi
- Department of Biotechnology, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Rome, Italy.
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Back SH, Kim JE, Rho J, Hahm B, Lee TG, Kim EE, Cho JM, Jang SK. Expression and purification of an active, full-length hepatitis C viral NS4A. Protein Expr Purif 2000; 20:196-206. [PMID: 11049744 DOI: 10.1006/prep.2000.1301] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.
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Affiliation(s)
- S H Back
- Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, Kyungbuk, 790-784, Korea
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Fowler A, Price-Jones M, Hughes K, Anson J, Lingham R, Schulman M. Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified. JOURNAL OF BIOMOLECULAR SCREENING 2000; 5:153-8. [PMID: 10894758 DOI: 10.1177/108705710000500307] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.
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Affiliation(s)
- A Fowler
- Department of SPA Technology, Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, England.
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40
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Fattori D, Urbani A, Brunetti M, Ingenito R, Pessi A, Prendergast K, Narjes F, Matassa VG, De Francesco R, Steinkühler C. Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer. J Biol Chem 2000; 275:15106-13. [PMID: 10809747 DOI: 10.1074/jbc.275.20.15106] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.
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Affiliation(s)
- D Fattori
- Istituto di Ricerche di Biologia Molecolare "P. Angeletti" Via Pontina Km 30,600, Pomezia, 00040 Rome, Italy
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41
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Butkiewicz N, Yao N, Zhong W, Wright-Minogue J, Ingravallo P, Zhang R, Durkin J, Standring DN, Baroudy BM, Sangar DV, Lemon SM, Lau JY, Hong Z. Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3. J Virol 2000; 74:4291-301. [PMID: 10756044 PMCID: PMC111946 DOI: 10.1128/jvi.74.9.4291-4301.2000] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.
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Affiliation(s)
- N Butkiewicz
- Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA
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Yusof R, Clum S, Wetzel M, Murthy HM, Padmanabhan R. Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J Biol Chem 2000; 275:9963-9. [PMID: 10744671 DOI: 10.1074/jbc.275.14.9963] [Citation(s) in RCA: 230] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.
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Affiliation(s)
- R Yusof
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
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43
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Wright-Minogue J, Yao N, Zhang R, Butkiewicz NJ, Baroudy BM, Lau JY, Hong Z. Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors. J Hepatol 2000; 32:497-504. [PMID: 10735621 DOI: 10.1016/s0168-8278(00)80402-x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV METHODS: Active NS3 protease domains of genotype 1-3 (representing five subtypes: la, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B. RESULTS Our data revealed that NS3 proteases cross-interacted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner. CONCLUSION Our study suggests that the NS4A cofactor function is well conserved among HCV It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.
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Affiliation(s)
- J Wright-Minogue
- Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA
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44
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Abstract
The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Worldwide, more than 170 million people are chronically infected with Hepatitis C virus and are at risk of developing cirrhosis and/or liver cancer. In addition, infections with arthropod-borne flaviviruses (such as dengue fever, Japanese encephalitis, tick-borne encephalitis, St. Louis encephalitis, Murray Valley encephalitis, West Nile, and yellow fever viruses) are emerging throughout the world. The pestiviruses have a serious impact on livestock. Unfortunately, no specific antiviral therapy is available for the treatment or the prevention of infections with members of the Flaviviridae. Ongoing research has identified possible targets for inhibition, including binding of the virus to the cell, uptake of the virus into the cell, the internal ribosome entry site of hepaciviruses and pestiviruses, the capping mechanism of flaviviruses, the viral proteases, the viral RNA-dependent RNA polymerase, and the viral helicase. In light of recent developments, the prevalence of infections caused by these viruses, the disease spectrum, and the impact of infections, different strategies that could be pursued to specifically inhibit viral targets and animal models that are available to study the pathogenesis and antiviral strategies are reviewed.
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45
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Butkiewicz NJ, Yao N, Wright-Minogue J, Zhang R, Ramanathan L, Lau JY, Hong Z, Dasmahapatra B. Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides. Biochem Biophys Res Commun 2000; 267:278-82. [PMID: 10623610 DOI: 10.1006/bbrc.1999.1898] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.
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Affiliation(s)
- N J Butkiewicz
- Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey, 07033, USA
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46
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Leyssen P, De Clercq E, Neyts J. Perspectives for the treatment of infections with Flaviviridae. Clin Microbiol Rev 2000; 13:67-82, table of contents. [PMID: 10627492 PMCID: PMC88934 DOI: 10.1128/cmr.13.1.67] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Worldwide, more than 170 million people are chronically infected with Hepatitis C virus and are at risk of developing cirrhosis and/or liver cancer. In addition, infections with arthropod-borne flaviviruses (such as dengue fever, Japanese encephalitis, tick-borne encephalitis, St. Louis encephalitis, Murray Valley encephalitis, West Nile, and yellow fever viruses) are emerging throughout the world. The pestiviruses have a serious impact on livestock. Unfortunately, no specific antiviral therapy is available for the treatment or the prevention of infections with members of the Flaviviridae. Ongoing research has identified possible targets for inhibition, including binding of the virus to the cell, uptake of the virus into the cell, the internal ribosome entry site of hepaciviruses and pestiviruses, the capping mechanism of flaviviruses, the viral proteases, the viral RNA-dependent RNA polymerase, and the viral helicase. In light of recent developments, the prevalence of infections caused by these viruses, the disease spectrum, and the impact of infections, different strategies that could be pursued to specifically inhibit viral targets and animal models that are available to study the pathogenesis and antiviral strategies are reviewed.
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Affiliation(s)
- P Leyssen
- Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
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47
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Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure 1999; 7:1353-63. [PMID: 10574797 DOI: 10.1016/s0969-2126(00)80025-8] [Citation(s) in RCA: 319] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
BACKGROUND Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.
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Affiliation(s)
- N Yao
- Structural Chemistry Department, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539, USA
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48
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Abstract
The non-structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3-encoded zinc-dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream. The released, mature NS5A protein is a 56 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). The NS5A gene has a quasispecies distribution, meaning that various NS5A sequences co-exist, in various proportions, in infected individuals. HCV NS5A appears to be located in cytoplasmic membranes surrounding the nucleus. Its precise functions are not known. HCV non-structural proteins, including NS5A, form a large multiprotein replication complex, which probably directs the replication of the HCV genome. HCV NS5A lacking the 146 N-terminal amino acids is a potent transcriptional activator in vitro. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon (IFN) sensitivity-determining region' has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1b, but strongly conflicting evidence has been published. In fact, there would seem to be no such region in the NS5A protein, even though NS5A plays an important and complex role in HCV resistance to IFN. Structure-function studies are required to identify precisely how NS5A and IFN interact.
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Affiliation(s)
- J M Pawlotsky
- Department of Bacteriology and Virology and INSERM U99, Hôpital Henri Mondor, Université Paris XII, Créteil, France
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49
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Zhang R, Beyer BM, Durkin J, Ingram R, Njoroge FG, Windsor WT, Malcolm BA. A continuous spectrophotometric assay for the hepatitis C virus serine protease. Anal Biochem 1999; 270:268-75. [PMID: 10334844 DOI: 10.1006/abio.1999.4109] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.
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Affiliation(s)
- R Zhang
- Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA
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50
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Howe AY, Chase R, Taremi SS, Risano C, Beyer B, Malcolm B, Lau JY. A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity. Protein Sci 1999; 8:1332-41. [PMID: 10386883 PMCID: PMC2144360 DOI: 10.1110/ps.8.6.1332] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.
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Affiliation(s)
- A Y Howe
- Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
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