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Laguna-Meraz S, Jose-Abrego A, Roman S, Leal-Mercado L, Panduro A. Risk Factors Associated with Hepatitis C Subtypes and the Evolutionary History of Subtype 1a in Mexico. Viruses 2024; 16:1259. [PMID: 39205233 PMCID: PMC11359553 DOI: 10.3390/v16081259] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 07/30/2024] [Accepted: 08/04/2024] [Indexed: 09/04/2024] Open
Abstract
The Hepatitis C Virus (HCV), with its diverse genotypes and subtypes, has significantly impacted the health of millions of people worldwide. Analyzing the risk factors is essential to understanding the spread of the disease and developing appropriate prevention strategies. This study aimed to identify risk factors associated with HCV subtype transmission and calculate the emergence time of subtype 1a in Mexico. A cross-sectional study was conducted from January 2014 to December 2018, involving 260 HCV-infected adults. HCV infection was confirmed via Enzyme-Linked Immunosorbent Assay, and viral load was measured by real-time PCR. Genotyping/subtyping tools were the Line Probe Assay and Sanger sequencing of the non-structural region 5B (NS5B). The most frequent HCV subtype was 1a (58.5%), followed by subtypes 1b (19.2%), 3a (13.1%), 2b (5.4%), 2a/2c (2.7%), 2a (0.8%), and 4a (0.4%). Intravenous drug use and tattoos were significant risk factors for subtypes 1a and 3a, while hemodialysis and blood transfusion were linked with subtype 1b. For the evolutionary analysis, 73 high-quality DNA sequences of the HCV subtype 1a NS5B region were used, employing a Bayesian coalescent analysis approach. This analysis suggested that subtype 1a was introduced to Mexico in 1976, followed by a diversification event in the mid-1980s. An exponential increase in cases was observed from 1998 to 2006, stabilizing by 2014. In conclusion, this study found that HCV subtypes follow distinct transmission routes, emphasizing the need for targeted prevention strategies. Additionally, the findings provide valuable insights into the origin of HCV subtype 1a. By analyzing the history, risk factors, and dynamics of the HCV epidemic, we have identified these measures: limiting the harm of intravenous drug trafficking, enhancing medical training and infrastructure, and ensuring universal access to antiviral treatments. The successful implementation of these strategies could lead to an HCV-free future in Mexico.
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Affiliation(s)
- Saul Laguna-Meraz
- Department of Genomic Medicine in Hepatology, Civil Hospital of Guadalajara, “Fray Antonio Alcalde”, Guadalajara 44280, Jalisco, Mexico; (S.L.-M.); (A.J.-A.); (S.R.); (L.L.-M.)
- Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
| | - Alexis Jose-Abrego
- Department of Genomic Medicine in Hepatology, Civil Hospital of Guadalajara, “Fray Antonio Alcalde”, Guadalajara 44280, Jalisco, Mexico; (S.L.-M.); (A.J.-A.); (S.R.); (L.L.-M.)
- Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
| | - Sonia Roman
- Department of Genomic Medicine in Hepatology, Civil Hospital of Guadalajara, “Fray Antonio Alcalde”, Guadalajara 44280, Jalisco, Mexico; (S.L.-M.); (A.J.-A.); (S.R.); (L.L.-M.)
- Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
| | - Leonardo Leal-Mercado
- Department of Genomic Medicine in Hepatology, Civil Hospital of Guadalajara, “Fray Antonio Alcalde”, Guadalajara 44280, Jalisco, Mexico; (S.L.-M.); (A.J.-A.); (S.R.); (L.L.-M.)
- Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
- Doctoral Program in Molecular Biology in Medicine, Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
| | - Arturo Panduro
- Department of Genomic Medicine in Hepatology, Civil Hospital of Guadalajara, “Fray Antonio Alcalde”, Guadalajara 44280, Jalisco, Mexico; (S.L.-M.); (A.J.-A.); (S.R.); (L.L.-M.)
- Health Sciences Center, University of Guadalajara, Guadalajara 44340, Jalisco, Mexico
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Maunye TK, Gededzha MP, Blackard JT, Rakgole JN, Selabe SG. Hepatitis C Virus Genotype 5 Variability in Treatment-Naïve Patients in South Africa. Intervirology 2023; 66:77-87. [PMID: 37231989 PMCID: PMC10353306 DOI: 10.1159/000528178] [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: 05/02/2022] [Accepted: 11/14/2022] [Indexed: 05/27/2023] Open
Abstract
INTRODUCTION Hepatitis C virus (HCV) genotype 5 was originally identified in South Africa, where it represents 35-60% of all HCV infections. There are limited data on resistance-associated variants (RAVs) in South Africa. Thus, we investigated variability within the NS3/NS4A, NS5A, and NS5B genes of treatment-naïve individuals with HCV genotype 5 infection at the Dr. George Mukhari Academic Hospital (DGMAH) in Pretoria, South Africa. METHODS Nested PCR was performed to amplify the NS3/4A, NS5A, and NS5B genes. RAVs were evaluated using the Geno2pheno tool. RESULTS In the NS3/4A gene, F56S and T122A were detected in one sample each. The D168E mutation was detected in 7 samples. Within the NS5A gene, the T62M mutation was detected in 2 individuals. In the NS5B gene, 8 of 12 individuals (67%) had the A421V mutation, while all 12 individuals (100%) had the S486A mutation. DISCUSSION RAVs were detected frequently among treatment-naïve individuals with HCV genotype 5 infection in South Africa. Thus, resistance testing may be prudent when initiating treatment of patients with genotype 5 infection. Additional population-based studies are needed to understand the prevalence of these RAVs during HCV genotype 5 infection.
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Affiliation(s)
- Tshegofatso K Maunye
- HIV and Hepatitis Research Unit, Department of Virology, Sefako Makgatho Health Sciences University, Pretoria, South Africa
- National Health Laboratory Service, Pretoria, South Africa
| | - Maemu P Gededzha
- Department of Immunology, Faculty of Health Sciences, University of Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa
| | - Jason T Blackard
- HIV and Hepatitis Research Unit, Department of Virology, Sefako Makgatho Health Sciences University, Pretoria, South Africa,
- Division of Digestive Diseases, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA,
| | - Johnny N Rakgole
- HIV and Hepatitis Research Unit, Department of Virology, Sefako Makgatho Health Sciences University, Pretoria, South Africa
| | - Selokela G Selabe
- HIV and Hepatitis Research Unit, Department of Virology, Sefako Makgatho Health Sciences University, Pretoria, South Africa
- National Health Laboratory Service, Pretoria, South Africa
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Khalid H, Shahid S, Tariq S, Ijaz B, Ashfaq UA, Ahmad M. Discovery of Novel HCV NS5B polymerase inhibitor, 2-(3,4-dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)-N-(2-fluorobenzyl)acetamide via molecular docking and experimental approach. Clin Exp Pharmacol Physiol 2021; 48:1653-1661. [PMID: 34386985 DOI: 10.1111/1440-1681.13571] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Revised: 07/13/2021] [Accepted: 08/08/2021] [Indexed: 11/27/2022]
Abstract
Hepatitis C Virus (HCV) is a viral infection posing a severe global threat that left untreated progresses to end-stage liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Moreover, no prophylactic approach exists so far enabling its prevention. The NS5B polymerase holds special significance as the target of intervention against HCV infection. The current study kindles benzothiazine derivatives against HCV NS5B polymerase through in silico and experimental approaches. Following docking, the compound 2-(3,4-dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)-N-(2-fluorobenzyl)acetamide was revealed to form effective binding interaction in the proposed site of HCV NS5B with a score of -10 kcal/mol and subsequently was deciphered through molecular dynamics (MD) simulation study which indicated interaction of residues TYR_382, VAL_381 and HIS_467 through hydrophobic interaction and two residues such as GLU_202 and LYS_209 contributed in the formation of water bridges. The subsequent in silico pharmacological analysis revealed its safe drug profile. The cytotoxicity activity of compound 6c indicated to be non-toxic in HepG2 cells at concentration ranges from 0.001-1.0 µmol/L with >80% cell viability and diminished expression of the HCV NS5B to 98% at the dose of 1.0 µmol/L and 90% at 0.5µmol/L. Thus the hit compound 6c might be a potent NS5B polymerase inhibitor required to be validated further through in vivo and preclinical studies.
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Affiliation(s)
- Hina Khalid
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad, Pakistan
| | - Sana Shahid
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad, Pakistan
| | - Somayya Tariq
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Bushra Ijaz
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Usman Ali Ashfaq
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad, Pakistan
| | - Matloob Ahmad
- Department of Chemistry, Government College University, Faisalabad, Pakistan
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Abstract
The therapeutic targeting of the nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) of the Hepatitis C Virus (HCV) with nucleotide analogs led to a deep understanding of this enzymes structure, function and substrate specificity. Unlike previously studied DNA polymerases including the reverse transcriptase of Human Immunodeficiency Virus, development of biochemical assays for HCV RdRp proved challenging due to low solubility of the full-length protein and inefficient acceptance of exogenous primer/templates. Despite the poor apparent specific activity, HCV RdRp was found to support rapid and processive transcription once elongation is initiated in vitro consistent with its high level of viral replication in the livers of patients. Understanding of the substrate specificity of HCV RdRp led to the discovery of the active triphosphate of sofosbuvir as a nonobligate chain-terminator of viral RNA transcripts. The ternary crystal structure of HCV RdRp, primer/template, and incoming nucleotide showed the interaction between the nucleotide analog and the 2'-hydroxyl binding pocket and how an unfit mutation of serine 282 to threonine results in resistance by interacting with the uracil base and modified 2'-position of the analog. Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19.
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Affiliation(s)
- Joy Y Feng
- Gilead Sciences, Inc., Foster City, CA, United States.
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Liatsos GD. Controversies’ clarification regarding ribavirin efficacy in measles and coronaviruses: Comprehensive therapeutic approach strictly tailored to COVID-19 disease stages. World J Clin Cases 2021; 9:5135-5178. [PMID: 34307564 PMCID: PMC8283580 DOI: 10.12998/wjcc.v9.i19.5135] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 01/01/2021] [Accepted: 05/20/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Ribavirin is a broad-spectrum nucleoside antiviral drug with multimodal mechanisms of action, which supports its longevity and quality as a clinical resource. It has been widely administered for measles and coronavirus infections. Despite the large amount of data concerning the use of ribavirin alone or in combination for measles, severe acute respiratory syndrome, Middle East respiratory syndrome, and coronavirus disease 2019 (COVID-19) outbreaks, the conclusions of these studies have been contradictory. Underlying reasons for these discrepancies include possible study design inaccuracies and failures and misinterpretations of data, and these potential confounds should be addressed.
AIM To determine the confounding factors of ribavirin treatment studies and propose a therapeutic scheme for COVID-19.
METHODS PubMed database was searched over a period of five decades utilizing the terms “ribavirin” alone or combined with other compounds in measles, severe acute respiratory syndrome, Middle East respiratory syndrome, and COVID-19 infections. The literature search was performed and described according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles were considered eligible when they reported on ribavirin dose regimens and/or specified outcomes concerning its efficacy and/or possible adverse-effects. In vitro and animal studies were also retrieved. A chapter on ribavirin’s pharmacology was included as well.
RESULTS In addition to the difficulties and pressures of an emerging pandemic, there is the burden of designing and conducting well-organized, double-blind, randomized controlled trials. Many studies have succumbed to specific pitfalls, one of which was identified in naturally ribavirin-resistant Vero cell lines in in vitro studies. Other pitfalls include study design inconsistent with the well-established clinical course of disease; inappropriate pharmacology of applied treatments; and the misinterpretation of study results with misconceived generalizations. A comprehensive treatment for COVID-19 is proposed, documented by thorough, long-term investigation of ribavirin regimens in coronavirus infections.
CONCLUSION A comprehensive treatment strictly tailored to distinct disease stages was proposed based upon studies on ribavirin and coronavirus infections.
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Affiliation(s)
- George D Liatsos
- Department of Internal Medicine, "Hippokration" General Hospital, Athens 11527, Attiki, Greece
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Diversity of the hepatitis C virus NS5B gene during HIV co-infection. PLoS One 2020; 15:e0237162. [PMID: 32750098 PMCID: PMC7402467 DOI: 10.1371/journal.pone.0237162] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 07/20/2020] [Indexed: 01/06/2023] Open
Abstract
Viral diversity is an important feature of hepatitis C virus (HCV) infection and an important predictor of disease progression and treatment response. HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes, although limited data are available regarding the impact of HIV on non-structural genes of HCV, particularly in the absence of direct-acting therapies. The genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene were examined in 3 groups of women with HCV genotype 1a infection, including those with HCV mono-infection, antiretroviral (ART)-naïve women with HIV/HCV co-infection and CD4 cell count <350 cells/mm3, and ART-naïve women with HIV/HCV co-infection and CD4 cell count ≥350 cells/mm3. None had ever been treated for HCV infection. There was evidence of significant diversity across the entire NS5B gene in all women. There were several nucleotides and amino acids with distinct distributions across the three study groups, although no obvious clustering of NS5B sequences was observed based on HIV co-infection or CD4 cell count. Polymorphisms at amino acid positions associated with resistance to dasabuvir and sofosbuvir were limited, although the Q309R variant associated with ribavirin resistance was present in 12 individuals with HCV mono-infection, 8 HIV/HCV co-infected individuals with CD4 <350 cells/mm3, and 12 HIV/HCV co-infected individuals with CD4 ≥350 cells/mm3. Previously reported fitness altering mutations were rare. CD8+ T cell responses against the human leukocyte antigen (HLA) B57-restricted epitopes NS5B2629-2637 and NS5B2936-2944 are critical for HCV control and were completely conserved in 44 (51.8%) and 70 (82.4%) study participants. These data demonstrate extensive variation across the NS5B gene. Genotypic variation may have a profound impact on HCV replication and pathogenesis and deserves careful evaluation.
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The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses. J Virol 2020; 94:JVI.00204-20. [PMID: 32075935 PMCID: PMC7163137 DOI: 10.1128/jvi.00204-20] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Accepted: 02/10/2020] [Indexed: 12/12/2022] Open
Abstract
Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3Dpol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens.IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens.
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Romero-López C, Berzal-Herranz A. The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle. Int J Mol Sci 2020; 21:1479. [PMID: 32098260 PMCID: PMC7073135 DOI: 10.3390/ijms21041479] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 02/05/2023] Open
Abstract
RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.
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Affiliation(s)
- Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
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Nyström K, Waldenström J, Tang KW, Lagging M. Ribavirin: pharmacology, multiple modes of action and possible future perspectives. Future Virol 2019. [DOI: 10.2217/fvl-2018-0166] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Ribavirin is a unique guanosine analog with broad-spectrum activity against many RNA and DNA viruses. In addition to its mutational properties, ribavirin exerts extensive perturbation of cellular and viral gene expression. Furthermore, recent advances indicate that the impact of ribavirin on divergent cellular and viral pathways may be concentration dependent. This review aims at providing an overview of the pharmacology and multiple modes of action of ribavirin as well as pointing to possible novel future uses.
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Affiliation(s)
- Kristina Nyström
- Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Västra Götaland Region, Sweden
| | - Jesper Waldenström
- Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Västra Götaland Region, Sweden
| | - Ka-Wei Tang
- Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Västra Götaland Region, Sweden
| | - Martin Lagging
- Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Västra Götaland Region, Sweden
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Lohmann V. Hepatitis C virus cell culture models: an encomium on basic research paving the road to therapy development. Med Microbiol Immunol 2019; 208:3-24. [PMID: 30298360 DOI: 10.1007/s00430-018-0566-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Accepted: 10/01/2018] [Indexed: 12/17/2022]
Abstract
Chronic hepatitis C virus (HCV) infections affect 71 million people worldwide, often resulting in severe liver damage. Since 2014 highly efficient therapies based on directly acting antivirals (DAAs) are available, offering cure rates of almost 100%, if the infection is diagnosed in time. It took more than a decade to discover HCV in 1989 and another decade to establish a cell culture model. This review provides a personal view on the importance of HCV cell culture models, particularly the replicon system, in the process of therapy development, from drug screening to understanding of mode of action and resistance, with a special emphasis on the contributions of Ralf Bartenschlager's group. It summarizes the tremendous efforts of scientists in academia and industry required to achieve efficient DAAs, focusing on the main targets, protease, polymerase and NS5A. It furthermore underpins the importance of strong basic research laying the ground for translational medicine.
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Affiliation(s)
- Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), University of Heidelberg, INF 344, 1st Floor, 69120, Heidelberg, Germany.
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Mandal A, Ganta KK, Chaubey B. Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication. HEPATITIS RESEARCH AND TREATMENT 2016; 2016:9671031. [PMID: 27446609 PMCID: PMC4942654 DOI: 10.1155/2016/9671031] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/28/2016] [Revised: 05/23/2016] [Accepted: 05/30/2016] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus is major cause of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Presently available direct-acting antiviral drugs have improved success rate; however, high cost limits their utilization, especially in developing countries like India. In the present study, we evaluated anti-HCV potential of several siRNAs targeted against the HCV RNA-dependent RNA polymerase NS5B and cellular factors, La autoantigen, PSMA7, and human VAMP-associated protein to intercept different steps of viral life cycle. The target genes were downregulated individually as well as in combinations and their impact on viral replication was evaluated. Individual downregulation of La autoantigen, PSMA7, hVAP-A, and NS5B resulted in inhibition of HCV replication by about 67.2%, 50.7%, 39%, and 52%, respectively. However, antiviral effect was more pronounced when multiple genes were downregulated simultaneously. Combinations of siRNAs against La autoantigen with NS5B or hVAP-A resulted in greater inhibition in HCV replication. Our findings indicate that siRNA is a potential therapeutic tool for inhibiting HCV replication and simultaneously targeting multiple viral steps with the combination of siRNAs is more effective than silencing a single target.
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Affiliation(s)
- Anirban Mandal
- Centre for Advance Studies, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India
| | - Krishna Kumar Ganta
- Centre for Advance Studies, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India
| | - Binay Chaubey
- Centre for Advance Studies, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India
- Laboratory of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, UG and MUG, Abrahama 58 Street, 80-307 Gdańsk, Poland
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Kanwal S, Mahmood T. Occurrence of genetic modifications in core, 5'UTR and NS5b of HCV associated with viral response to treatment. Virol J 2014; 11:171. [PMID: 25270660 PMCID: PMC4289283 DOI: 10.1186/1743-422x-11-171] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Accepted: 09/05/2014] [Indexed: 12/22/2022] Open
Abstract
Background It is becoming progressively more understandable that genetic variability of viruses is a major challenge in translating the laboratory findings to clinic. Genetic variability is the underlying cause of variant viral proteins which are not targetable by host immunological machinery. Methods 500 patients were enrolled in study and amongst them, 451 patients were followed and categorized into two groups on the basis of their treatment response. Group 1 consisting of the 376 patients exhibited SVR while group 2 comprised 75 patients who were non-responders on the basis of viral load as evidenced by Real-Time PCR. Comparative sequence analysis was done between 75 non-responders and 75 responders (randomly picked from 376) by targeting three genomic regions, 5′UTR, core and NS5B and amplified products were directly sequenced and obtained sequences were cleaned, aligned and submitted to GenBank. Maximum Parsimony (MP) method was used for phylogenetic analysis and dendrograms were dragged using MEGA 5. Heterogeneity at nucleotide and amino acid level was determined using software BioEdit and DNAman while phosphorylation and N-linked glycosylation sites were determined using NetPhos 2.0 and SignalP-NN. Results Genotype 3 was prevalent in group 1 whereas non-responders indicated rare genotypes of Pakistan i.e. 4 and 5, genotype 6q and 6v were reported first time from Pakistan in this study. At nucleotide and amino acid level, the genetic distance and mutation, number of predicted N-phosphorylation and N-glycosylation sites was higher in group 2 as compared to group 1. Difference in percentage composition of individual amino acids was noted to be different between the two groups. Conclusions It can be concluded that heterogeneity both at nucleotide and amino acid level contributed in developing drug resistant phenotype. Moreover, occurrence of rare genotypes might hurdle the way to positive response of conventional treatment. Furthermore, prediction of phosphorylation and glycosylation sites could help in targeting the proper sites for drug designing.
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Affiliation(s)
| | - Tariq Mahmood
- Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan.
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Li W, Zhang Y, Kao CC. The classic swine fever virus (CSFV) core protein can enhance de novo-initiated RNA synthesis by the CSFV polymerase NS5B. Virus Genes 2014; 49:106-15. [DOI: 10.1007/s11262-014-1080-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 04/23/2014] [Indexed: 12/28/2022]
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Blackard JT, Ma G, Sengupta S, Martin CM, Powell EA, Shata MT, Sherman KE. Evidence of distinct populations of hepatitis C virus in the liver and plasma of patients co-infected with HIV and HCV. J Med Virol 2014; 86:1332-41. [PMID: 24788693 DOI: 10.1002/jmv.23968] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/08/2014] [Indexed: 12/15/2022]
Abstract
Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co-infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co-infected with HIV and HCV. Median intra-patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co-infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies.
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Affiliation(s)
- Jason T Blackard
- Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio
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15
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Khaliq S, Latief N, Jahan S. Role of different regions of the hepatitis C virus genome in the therapeutic response to interferon-based treatment. Arch Virol 2013; 159:1-15. [PMID: 23851652 DOI: 10.1007/s00705-013-1780-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2013] [Accepted: 05/28/2013] [Indexed: 12/21/2022]
Abstract
Hepatitis C virus (HCV) is considered a significant risk factor in HCV-induced liver diseases and development of hepatocellular carcinoma (HCC). Nucleotide substitutions in the viral genome result in its diversification into quasispecies, subtypes and distinct genotypes. Different genotypes vary in their infectivity and immune response due to these nucleotide/amino acid variations. The current combination treatment for HCV infection is pegylated interferon α (PEG-IFN-α) with ribavirin, with a highly variable response rate mainly depending upon the HCV genotype. Genotypes 2 and 3 are found to respond better than genotypes 1 and 4, which are more resistant to IFN-based therapies. Different studies have been conducted worldwide to explore the basis of this difference in therapy response, which identified some putative regions in the HCV genome, especially in Core and NS5a, and to some extent in the E2 region, containing specific sequences in different genotypes that act differently with respect to the IFN response. In the review, we try to summarize the role of HCV proteins and their nucleotide sequences in association with treatment outcome in IFN-based therapy.
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Affiliation(s)
- Saba Khaliq
- Department of Immunology, University of Health Sciences, Lahore, Pakistan,
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16
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Hussain A, Idrees M. The first complete genome sequence of HCV-1a from Pakistan and a phylogenetic analysis with complete genomes from the rest of the world. Virol J 2013; 10:211. [PMID: 23805872 PMCID: PMC3816317 DOI: 10.1186/1743-422x-10-211] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2013] [Accepted: 06/03/2013] [Indexed: 12/14/2022] Open
Abstract
Background Here, we report the first patient derived hepatitis C virus (HCV) complete genome from Pakistan as is not available from this region of the world. Findings Comprehensive evolutionary and phylogenetic analyses were conducted. The comparison was made in order to identify evolutionary and molecular phylogenetic relationships among HCV strains belonging to genotype 1a. The evolutionary divergence analysis for nucleotide and amino acid sequences, conducted by equal input model, suggested that evolutionary nucleotide and amino acid distances showed that the HCV Pakistani strain was genetically far from Denmark strain (0.29400 nt, 0.819646 aa) and near to German strain (0.06557 nt, 0.139449 aa), respectively. Conclusion The current study will help to understand phylogenetic relationship of circulating Pakistani isolates.
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Affiliation(s)
- Abrar Hussain
- Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan.
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17
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Koutsoudakis G, Forns X, Pérez-Del-Pulgar S. [The molecular biology of hepatitis C virus]. GASTROENTEROLOGIA Y HEPATOLOGIA 2013; 36:280-93. [PMID: 23490024 DOI: 10.1016/j.gastrohep.2012.11.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/09/2012] [Accepted: 11/13/2012] [Indexed: 12/12/2022]
Abstract
Since the discovery of the hepatitis C virus (HCV), a plethora of experimental models have evolved, allowing the virus's life cycle and the pathogenesis of associated liver diseases to be investigated. These models range from inoculation of cultured cells with serum from patients with hepatitis C to the use of surrogate models for the study of specific stages of the HCV life cycle: retroviral pseudoparticles for the study of HCV entry, replicons for the study of HCV replication, and the HCV cell culture model, which reproduces the entire life cycle (replication and production of infectious particles). The use of these tools has been and remains crucial to identify potential therapeutic targets in the different stages of the virus's life cycle and to screen new antiviral drugs. A clear example is the recent approval of two viral protease inhibitors (boceprevir and telaprevir) in combination with pegylated interferon and ribavirin for the treatment of chronic hepatitis C. This review analyzes the advances made in the molecular biology of HCV and highlights possible candidates as therapeutic targets for the treatment of HCV infection.
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Affiliation(s)
- George Koutsoudakis
- Servicio de Hepatología, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, España
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18
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Castilho MCB, Martins AN, Horbach IS, Perez RDM, Figueiredo FAF, Pinto PDTA, Nabuco LC, Lima DBD, Tanuri A, Porto LC, Ferreira Júnior ODC. Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients. Mem Inst Oswaldo Cruz 2012; 106:968-75. [PMID: 22241118 DOI: 10.1590/s0074-02762011000800011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 09/08/2011] [Indexed: 01/06/2023] Open
Abstract
Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.
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Affiliation(s)
- Magda Cristina Bernardino Castilho
- Laboratório de Histocompatibilidade e Criopreservação, Policlínica Piquet Carneiro, Departamento de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
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19
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Broecker F, Moelling K. Short hairpin-looped oligodeoxynucleotides reduce hepatitis C virus replication. Virol J 2012; 9:134. [PMID: 22823899 PMCID: PMC3508801 DOI: 10.1186/1743-422x-9-134] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Accepted: 07/23/2012] [Indexed: 11/10/2022] Open
Abstract
Background Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Standard therapy consists of a combination of interferon-alpha and ribavirin, but many patients respond poorly, especially those infected with HCV genotypes 1 and 4. Furthermore, standard therapy is associated with severe side-effects. Thus, alternative therapeutic approaches against HCV are needed. Findings Here, we studied the effect of a new class of antiviral agents against HCV, short, partially double-stranded oligodeoxynucleotides (ODNs), on viral replication. We targeted the 5’ nontranslated region (5’ NTR) of the HCV genome that has previously been shown as effective target for small interfering RNAs (siRNAs) in vitro. One of the investigated ODNs, ODN 320, significantly and efficiently reduced replication of HCV replicons in a sequence-, time- and dose-dependent manner. ODN 320 targets a genomic region highly conserved among different HCV genotypes and might thus be able to inhibit a broad range of genotypes and subtypes. Conclusions ODNs provide an additional approach for inhibition of HCV, might be superior to siRNAs in terms of stability and cellular delivery, and suitable against HCV resistant to standard therapy. This study underlines the potential of partially double-stranded ODNs as antiviral agents.
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Blackard JT, Ma G, Welge JA, Martin CM, Sherman KE, Taylor LE, Mayer KH, Jamieson DJ. Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization. J Med Virol 2012; 84:242-52. [PMID: 22170544 DOI: 10.1002/jmv.22269] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non-structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV-infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non-structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence.
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Affiliation(s)
- Jason T Blackard
- Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
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21
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RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2011; 1814:1325-32. [PMID: 21621653 DOI: 10.1016/j.bbapap.2011.05.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/22/2010] [Revised: 05/09/2011] [Accepted: 05/09/2011] [Indexed: 01/10/2023]
Abstract
The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1-6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg(2+) were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a>2a, 3a, 4a, 5a>1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.
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22
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Sequence variability of HCV Core region: Important predictors of HCV induced pathogenesis and viral production. INFECTION GENETICS AND EVOLUTION 2011; 11:543-56. [PMID: 21292033 DOI: 10.1016/j.meegid.2011.01.017] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/21/2010] [Revised: 01/17/2011] [Accepted: 01/21/2011] [Indexed: 02/07/2023]
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23
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Inoue Y, Aizaki H, Hara H, Matsuda M, Ando T, Shimoji T, Murakami K, Masaki T, Shoji I, Homma S, Matsuura Y, Miyamura T, Wakita T, Suzuki T. Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein. Virology 2010; 410:38-47. [PMID: 21093005 DOI: 10.1016/j.virol.2010.10.026] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2010] [Revised: 07/18/2010] [Accepted: 10/15/2010] [Indexed: 12/12/2022]
Abstract
To identify the host factors implicated in the regulation of hepatitis C virus (HCV) genome replication, we performed comparative proteome analyses of HCV replication complex (RC)-rich membrane fractions prepared from cells harboring genome-length bicistronic HCV RNA at the exponential and stationary growth phases. We found that the eukaryotic chaperonin T-complex polypeptide 1 (TCP1)-ring complex/chaperonin-containing TCP1 (TRiC/CCT) plays a role in the replication possibly through an interaction between subunit CCT5 and the viral RNA polymerase NS5B. siRNA-mediated knockdown of CCT5 suppressed RNA replication and production of the infectious virus. Gain-of-function activity was shown following co-transfection with whole eight TRiC/CCT subunits. HCV RNA synthesis was inhibited by an anti-CCT5 antibody in a cell-free assay. These suggest that recruitment of the chaperonin by the viral nonstructural proteins to the RC, which potentially facilitate folding of the RC component(s) into the mature active form, may be important for efficient replication of the HCV genome.
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Affiliation(s)
- Yasushi Inoue
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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24
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Blackard JT, Ma G, Limketkai BN, Welge JA, Dryer PD, Martin CM, Hiasa Y, Taylor LE, Mayer KH, Jamieson DJ, Sherman KE. Variability of the polymerase gene (NS5B) in hepatitis C virus-infected women. J Clin Microbiol 2010; 48:4256-9. [PMID: 20810773 PMCID: PMC3020841 DOI: 10.1128/jcm.01613-10] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2010] [Revised: 08/20/2010] [Accepted: 08/20/2010] [Indexed: 01/24/2023] Open
Abstract
There are limited data on diversity within the hepatitis C virus polymerase (NS5B). In concordance with its key functional role during the life cycle, NS5B intrapatient variability was low. Moreover, differences between NS5B nonsynonymous (dN) and synonymous (dS) mutation rates (dN - dS) were positively correlated with CD4 cell count, while nonsynonymous mutations were strongly correlated with reduced replication in vivo.
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Affiliation(s)
- Jason T Blackard
- Department of Internal Medicine, Division of Digestive Diseases, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
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25
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Sillanpää M, Melén K, Porkka P, Fagerlund R, Nevalainen K, Lappalainen M, Julkunen I. Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection. Virol J 2009; 6:84. [PMID: 19549310 PMCID: PMC2709157 DOI: 10.1186/1743-422x-6-84] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2009] [Accepted: 06/23/2009] [Indexed: 12/11/2022] Open
Abstract
Background The viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in Sf9 insect cells by using the baculovirus expression system. Results The recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3) recognised NS5B. Conclusion The data shows significant variation in the specificity in humoral immunity in chronic HCV patients.
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Affiliation(s)
- Maarit Sillanpää
- Department of Vaccination and Immune Protection, National Institute for Health and Welfare (THL), FI-00271 Helsinki, Finland.
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26
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Quezada EM, Kane CM. The Hepatitis C Virus NS5A Stimulates NS5B During In Vitro RNA Synthesis in a Template Specific Manner. Open Biochem J 2009; 3:39-48. [PMID: 19590581 PMCID: PMC2701273 DOI: 10.2174/1874091x00903010039] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2008] [Revised: 03/11/2009] [Accepted: 03/12/2009] [Indexed: 01/29/2023] Open
Abstract
The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previously shown to localize to the replisome and be necessary for viral replication. However, its role in replication has not been defined. Using an in vitro biochemical assay, we detected a stimulatory effect of NS5A on the NS5B replication reaction with minimal natural templates. NS5A stimulates replication by NS5B on two templates derived from the 3’ end of the RNA genome (4 fold ± 1.3 fold). A pre-incubation step with the two proteins prior to the replication reaction and substoichiometric levels of NS5A are required for detecting stimulation. With a template derived from the 3’end complementary to the RNA genome (the negative strand) no stimulation was observed. Furthermore, with a synthetic template that allows studying different phases of replication, NS5A stimulates NS5B during elongation. These findings suggest that NS5A stimulates NS5B during synthesis of the complementary (i.e., negative) strand of the RNA genome.
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Affiliation(s)
- Elizabeth M Quezada
- Department of Molecular and Cell Biology, University of California - Berkeley. Berkeley, CA 94720-3202, USA
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27
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Wagner R, Larson DP, Beno DWA, Bosse TD, Darbyshire JF, Gao Y, Gates BD, He W, Henry RF, Hernandez LE, Hutchinson DK, Jiang WW, Kati WM, Klein LL, Koev G, Kohlbrenner W, Krueger AC, Liu J, Liu Y, Long MA, Maring CJ, Masse SV, Middleton T, Montgomery DA, Pratt JK, Stuart P, Molla A, Kempf DJ. Inhibitors of Hepatitis C Virus Polymerase: Synthesis and Biological Characterization of Unsymmetrical Dialkyl-Hydroxynaphthalenoyl-benzothiadiazines. J Med Chem 2009; 52:1659-69. [DOI: 10.1021/jm8010965] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Rolf Wagner
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Daniel P. Larson
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - David W. A. Beno
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Todd D. Bosse
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - John F. Darbyshire
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Yi Gao
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Bradley D. Gates
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Wenping He
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Rodger F. Henry
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Lisa E. Hernandez
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | | | - Wen W. Jiang
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Warren M. Kati
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Larry L. Klein
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Gennadiy Koev
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - William Kohlbrenner
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - A. Chris Krueger
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Jinrong Liu
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Yaya Liu
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Michelle A. Long
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Clarence J. Maring
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Sherie V. Masse
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Tim Middleton
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Debra A. Montgomery
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - John K. Pratt
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Patricia Stuart
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Akhteruzzaman Molla
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
| | - Dale J. Kempf
- Global Pharmaceutical Research and Development, Abbott, Abbott Park, Illinois 60064
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28
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Ramachandran S, Xia GL, Ganova-Raeva LM, Nainan OV, Khudyakov Y. End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: performance under optimal and suboptimal conditions. J Virol Methods 2008; 151:217-224. [PMID: 18571738 DOI: 10.1016/j.jviromet.2008.05.005] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2008] [Revised: 04/15/2008] [Accepted: 05/08/2008] [Indexed: 12/17/2022]
Abstract
An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.
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Affiliation(s)
- Sumathi Ramachandran
- Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
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29
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Tellinghuisen TL, Evans MJ, von Hahn T, You S, Rice CM. Studying hepatitis C virus: making the best of a bad virus. J Virol 2007; 81:8853-67. [PMID: 17522203 PMCID: PMC1951464 DOI: 10.1128/jvi.00753-07] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
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30
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Romano PR, McCallus DE, Pachuk CJ. RNA interference-mediated prevention and therapy for hepatocellular carcinoma. Oncogene 2006; 25:3857-65. [PMID: 16799627 DOI: 10.1038/sj.onc.1209549] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and is on the increase worldwide. Hepatocellular carcinoma results from chronic liver disease and cirrhosis most commonly associated with chronic hepatitis B (HBV) or hepatitis C (HCV) infection. The highest incidences of HCC are found in China and Africa, where chronic HBV infection is the major risk component. In the United States, Europe and Japan, the significant increase in HCC and HCC-related deaths within the last three decades is mainly attributed to the rise in the number of HCV-infected individuals; smaller increases of HCC are associated with HBV. Given that HCV and HBV infection account for the majority of HCCs, therapeutic and prophylactic approaches to control or eliminate virus infection may prove effective in reducing the occurrence of HCC. Although anti-viral therapies exist for both HBV and HCV infections, they are ineffective for a significant number of patients. In addition, some treatments such as interferon therapy are dose limiting owing to toxic side effects. Clearly, new approaches are needed. RNA interference (RNAi)-based approaches may meet this need and have already shown promising preclinical results in cell culture and animal models. Although this paper focuses on the potential of RNAi as a prophylactic for HCC development, the potential use of RNAi-mediated approaches for HCC therapy will also be discussed.
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31
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Cai Z, Yi M, Zhang C, Luo G. Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase. J Virol 2005; 79:11607-17. [PMID: 16140738 PMCID: PMC1212605 DOI: 10.1128/jvi.79.18.11607-11617.2005] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2005] [Accepted: 06/14/2005] [Indexed: 11/20/2022] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.
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Affiliation(s)
- Zhaohui Cai
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose St., MN477, Lexington, KY 40536-0298, USA
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Hamano K, Sakamoto N, Enomoto N, Izumi N, Asahina Y, Kurosaki M, Ueda E, Tanabe Y, Maekawa S, Itakura J, Watanabe H, Kakinuma S, Watanabe M. Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy. J Gastroenterol Hepatol 2005; 20:1401-9. [PMID: 16105128 DOI: 10.1111/j.1440-1746.2005.04024.x] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND AND AIM Combination treatments of interferon-alpha (IFN) and ribavirin (RBV) are more effective than those of IFN alone in hepatitis C virus (HCV) infection. However, mechanisms of the action of the combination regimen are not well understood. To elucidate the viral genetic basis of IFN plus RBV combination therapy, genetic variabilities of HCV-1b were analyzed. METHODS We performed pair-wise comparisons of full-length HCV genomic sequences in three patients' sera before and after initiation of IFN plus RBV treatment. Subsequently, we analyzed amino acid sequences of the NS5B region, which codes for the viral RNA-dependent RNA polymerase, and compared these with the outcomes of the therapy in 81 patients. RESULTS Analysis of the entire HCV sequence in patients who received IFN plus RBV therapy did not show consistent amino acid changes between before and after the initiation of the therapy. NS5B sequence analyses revealed that mutations at positions 300-358 of NS5B, including polymerase motif B to E, occurred more frequently in a group of patients exhibiting a sustained viral response (SVR) or an end-of-treatment response (ETR) compared with a group of patients exhibiting a non-response (NR). Closer examination revealed that mutations at aa 309, 333, 338 and 355 of NS5B occurred significantly more frequently in the SVR plus ETR group than in the NR group (P = 0.0004). Multivariate analysis showed that the number of mutations at these four sites was an independent predictor of SVR plus ETR versus NR. CONCLUSIONS Particular amino acid changes in the NS5B region of HCV may correlate with outcomes of IFN plus RBV combination therapy.
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Affiliation(s)
- Kosei Hamano
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Japan
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Takahashi H, Yamaji M, Hosaka M, Kishine H, Hijikata M, Shimotohno K. Analysis of the 5' end structure of HCV subgenomic RNA replicated in a Huh7 cell line. Intervirology 2005; 48:104-11. [PMID: 15812182 DOI: 10.1159/000081736] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2004] [Accepted: 03/10/2004] [Indexed: 11/19/2022] Open
Abstract
OBJECTIVE Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5' end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate. METHODS HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5' end. RESULTS Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase. CONCLUSION It is suggested that the 5' end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5' end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5' end. This result indicates that the 5' end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.
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Affiliation(s)
- Hitoshi Takahashi
- Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan
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Lahser FC, Malcolm BA. A continuous nonradioactive assay for RNA-dependent RNA polymerase activity. Anal Biochem 2005; 325:247-54. [PMID: 14751259 DOI: 10.1016/j.ab.2003.10.034] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.
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Affiliation(s)
- Frederick C Lahser
- Department of Antiviral Therapeutics, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
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35
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Naka K, Ikeda M, Abe KI, Dansako H, Kato N. Mizoribine inhibits hepatitis C virus RNA replication: Effect of combination with interferon-α. Biochem Biophys Res Commun 2005; 330:871-9. [PMID: 15809077 DOI: 10.1016/j.bbrc.2005.03.062] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2005] [Indexed: 01/30/2023]
Abstract
Interferon (IFN)-alpha monotherapy, as well as the more effective combination therapy of IFN-alpha and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side effects such as anemia due to the administration of ribavirin present a problem for patients who are advanced in years. Using a recently developed reporter assay system in which genome-length dicistronic HCV RNA encoding Renilla luciferase gene was found to replicate efficiently, we found that mizoribine, an imidazole nucleoside, inhibited HCV RNA replication. The anti-HCV activity of mizoribine (IC50: approximately 100 microM) was similar to that of ribavirin. Using this genome-length HCV RNA replication monitor system, we were the first to demonstrate that the combination of IFN-alpha and ribavirin exhibited more effective anti-HCV activity than the use of IFN-alpha alone. Moreover, we found that the anti-HCV activity of mizoribine in co-treatment with IFN-alpha was at least equivalent to that of ribavirin. This effect was apparent in the presence of at least 5 microM mizoribine. Since mizoribine is currently used in several clinical applications and has not been associated with severe side effects, mizoribine is considered to be of potential use as a new anti-HCV reagent in combination with IFN-alpha.
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Affiliation(s)
- Kazuhito Naka
- Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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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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37
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Vuillermoz I, Khattab E, Sablon E, Ottevaere I, Durantel D, Vieux C, Trepo C, Zoulim F. Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy. J Med Virol 2005; 74:41-53. [PMID: 15258967 DOI: 10.1002/jmv.20144] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.
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Affiliation(s)
- I Vuillermoz
- INSERM UNIT 271, 151 Cours Albert Thomas, Lyon, France
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38
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Azzi A, Lin S. Human SARS-coronavirus RNA-dependent RNA polymerase: activity determinants and nucleoside analogue inhibitors. Proteins 2005; 57:12-4. [PMID: 15326590 PMCID: PMC7167687 DOI: 10.1002/prot.20194] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Affiliation(s)
- Arezki Azzi
- Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL). Québec City, Canada
| | - Sheng‐Xiang Lin
- Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL). Québec City, Canada
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Bartenschlager R, Frese M, Pietschmann T. Novel insights into hepatitis C virus replication and persistence. Adv Virus Res 2005; 63:71-180. [PMID: 15530561 DOI: 10.1016/s0065-3527(04)63002-8] [Citation(s) in RCA: 212] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the family Flaviviridae. A hallmark of HCV is its high propensity to establish a persistent infection that in many cases leads to chronic liver disease. Molecular studies of the virus became possible with the first successful cloning of its genome in 1989. Since then, the genomic organization has been delineated, and viral proteins have been studied in some detail. In 1999, an efficient cell culture system became available that recapitulates the intracellular part of the HCV life cycle, thereby allowing detailed molecular studies of various aspects of viral RNA replication and persistence. This chapter attempts to summarize the current state of knowledge in these most actively worked on fields of HCV research.
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Affiliation(s)
- Ralf Bartenschlager
- Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
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40
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Dutartre H, Boretto J, Guillemot JC, Canard B. A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B. J Biol Chem 2004; 280:6359-68. [PMID: 15561709 DOI: 10.1074/jbc.m410191200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/09/2022] Open
Abstract
Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.
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Affiliation(s)
- Hélène Dutartre
- CNRS and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d'Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
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Benzaghou I, Bougie I, Bisaillon M. Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase. J Biol Chem 2004; 279:49755-61. [PMID: 15375162 DOI: 10.1074/jbc.m409657200] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp(220), Asp(318), and Asp(319) for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.
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Affiliation(s)
- Ines Benzaghou
- Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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Boonrod K, Galetzka D, Nagy PD, Conrad U, Krczal G. Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nat Biotechnol 2004; 22:856-62. [PMID: 15195103 DOI: 10.1038/nbt983] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2004] [Accepted: 05/07/2004] [Indexed: 11/08/2022]
Abstract
Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of resistance against four plant viruses from different genera, three of which belong to the Tombusviridae family. Virus resistance based on antibodies to RdRps adds another tool to the repertoire for combating plant viruses.
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Affiliation(s)
- KaJohn Boonrod
- Centrum Grüne Gentechnik, Dienstleistungszentrum Ländlicher Raum - Rheinpfalz-, Breitenweg 71, D-67435 Neustadt, Germany
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Kumagai N, Takahashi N, Kinoshita M, Tsunematsu S, Tsuchimoto K, Saito H, Ishii H. Polymorphisms of NS5B protein relates to early clearance of hepatitis C virus by interferon plus ribavirin: a pilot study. J Viral Hepat 2004; 11:225-35. [PMID: 15117324 DOI: 10.1111/j.1365-2893.2004.00501.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Although randomized trials have shown enhancement of efficacy for combination therapy with interferon (IFN) alpha-2b and ribavirin compared with IFN monotherapy as first-line treatment for chronic hepatitis C, infection with genotype 1b and high viremia are still associated with significantly low response rates compared with non-1 genotypes and low viremia. We analysed amino acid sequences of the viral RNA-dependent RNA polymerase (RdRP) or nonstructural protein 5B (NS5B), responsible for ribavirin misincorporation into RNA products in patients with genotype 1b-related chronic hepatitis C and high viremia, and examined the relationship between such RdRp polymorphisms, and the initial decline in viral load induced by combination therapy with IFN-alpha and ribavirin. Substitution of glutamic acid to lysine at the 124th position (E124K) and of isoleucine to valine at the 85th position (I85V) were found to be closely associated with a potent decline of viral load and viral clearance at 8 weeks of treatment (five of five patients, coincidence rate 100%). In conclusion, our results suggest that the polymorphisms of E124K and I85V identified in NS5B protein are crucial for early viral clearance in patients with genotype 1b and high viremia by combination therapy with IFN and ribavirin, and that detection of amino acid sequence motifs might enable prediction of clinical efficacy.
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Affiliation(s)
- N Kumagai
- Research Center for Liver Diseases, the Kitasato Institute, Minato-ku, Tokyo, Japan.
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44
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Krönke J, Kittler R, Buchholz F, Windisch MP, Pietschmann T, Bartenschlager R, Frese M. Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J Virol 2004; 78:3436-46. [PMID: 15016866 PMCID: PMC371081 DOI: 10.1128/jvi.78.7.3436-3446.2004] [Citation(s) in RCA: 122] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.
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Affiliation(s)
- Jan Krönke
- Department of Molecular Virology, Hygiene Institute, University of Heidelberg, D-69120 Heidelberg, Germany
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45
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Cai Z, Liang TJ, Luo G. Effects of mutations of the initiation nucleotides on hepatitis C virus RNA replication in the cell. J Virol 2004; 78:3633-43. [PMID: 15016884 PMCID: PMC371060 DOI: 10.1128/jvi.78.7.3633-3643.2004] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2003] [Accepted: 12/12/2003] [Indexed: 11/20/2022] Open
Abstract
Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5' and 3' ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5' end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5' end dramatically reduced the efficiency of cell colony formation. Likewise, the 3'-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3' end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5'-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5' end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5'-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3'-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3' ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3' end. We also determined the 5'-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5' end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.
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Affiliation(s)
- Zhaohui Cai
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA
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Liu C, Chopra R, Swanberg S, Olland S, O'Connell J, Herrmann S. Elongation of synthetic RNA templates by hepatitis C virus NS5B polymerase. J Biol Chem 2003; 279:10738-46. [PMID: 14688285 DOI: 10.1074/jbc.m310062200] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Here we examine the ability of seven, 3'-related, short synthetic RNAs to serve as templates for the hepatitis C virus (HCV) polymerase, non-structural protein 5B (NS5B). These RNAs, termed HL, range from 8 to 16 nucleotides in length, each with ACC at the 3' terminus. Interestingly HL12 and longer templates have a predicted secondary structure. Those with one or two unpaired adenylates at the 5'-end of a stem were increased in size by one or two nucleotides, respectively, following incubation with NS5B and UTP. Using labeled template RNA and cold UTP, extension in size could be inhibited by addition of non-labeled template of the same size. This template elongation was not inhibited by cold linear HL10 template unless pGpG was added. Fluorescence anisotropy demonstrated HL14, a template with secondary structure, bound with an apparent K(d) of 22 nm. A linear template, HL10, plus pGpG primer was bound by NS5B with a K(d) of 45 nm, whereas HL10 alone bound with an apparent K(d) of 182 nm. The amplitude of the template extension product was increased by a brief preincubation at 4 degrees C followed by incubation at 23 or 30 degrees C. The nucleotide-mediated increase in size occurred for both templates that required a mismatch or bulge at the 3'-end as well as for those without the mismatch. These results suggest an NS5B active site pocket can readily accommodate short templates with four or five base stems and initiate copy-back replication in the presence of a one nucleotide mismatch.
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Affiliation(s)
- Cuihua Liu
- Wyeth Research, Cambridge, Massachusetts 02140, USA
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Ranjith-Kumar CT, Santos JL, Gutshall LL, Johnston VK, Lin-Goerke J, Kim MJ, Porter DJ, Maley D, Greenwood C, Earnshaw DL, Baker A, Gu B, Silverman C, Sarisky RT, Kao C. Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase. Virology 2003; 312:270-80. [PMID: 12919733 DOI: 10.1016/s0042-6822(03)00247-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.
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Affiliation(s)
- C T Ranjith-Kumar
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
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Shim J, Larson G, Lai V, Naim S, Wu JZ. Canonical 3'-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase. Antiviral Res 2003; 58:243-51. [PMID: 12767472 DOI: 10.1016/s0166-3542(03)00007-x] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.
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Affiliation(s)
- Jaehoon Shim
- Drug discovery, Ribapharm Corporation, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA
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Bougie I, Charpentier S, Bisaillon M. Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase. J Biol Chem 2003; 278:3868-75. [PMID: 12458224 DOI: 10.1074/jbc.m209785200] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.
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Affiliation(s)
- Isabelle Bougie
- Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Québec J1H 5N4, Canada
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Walker MP, Appleby TC, Zhong W, Lau JYN, Hong Z. Hepatitis C virus therapies: current treatments, targets and future perspectives. Antivir Chem Chemother 2003; 14:1-21. [PMID: 12790512 DOI: 10.1177/095632020301400101] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Chronic hepatitis C virus (HCV) infection is the cause of an emerging global epidemic of chronic liver disease. Current combination therapies are at best 80% efficacious and are often poorly tolerated. Strategies to improve the therapeutic response include the development of novel interferons, nucleoside analogues with reduced haemolysis compared with ribavirin and inosine 5'-monophosphate dehydrogenase inhibitors. Compounds in preclinical or early clinical trials include small molecules that inhibit virus-specific enzymes (such as the serine proteases, RNA polymerase and helicase) or interfere with translation (including anti-sense molecules, iRNA and ribozymes). Advances in understanding HCV replication, obtaining a sub-genomic replicon and contriving potential small animal models, in addition to solving crystallographic structures for the replication enzymes, have improved prospects for developing novel therapies. This review summarizes current and evolving treatments for chronic hepatitis C infection. In addition, progress in HCV targets and drug discovery tools valuable in the search for novel anti-HCV agents is detailed.
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