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Savino DF, Silva JV, da Silva Santos S, Lourenço FR, Giarolla J. How do physicochemical properties contribute to inhibitory activity of promising peptides against Zika Virus NS3 protease? J Mol Model 2024; 30:54. [PMID: 38289526 DOI: 10.1007/s00894-024-05843-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Accepted: 01/09/2024] [Indexed: 02/01/2024]
Abstract
CONTEXT AND RESULTS Flavivirus diseases' cycles, especially Dengue and Yellow Fever, can be observed all over Brazilian territory, representing a great health concern. Additionally, there are no drugs available in therapy. In this scenario, in silico methodologies were applied to obtain physicochemical properties, as well as to better understand the ligand-biological target interaction mode of 20 previously reported NS2B/NS3 protease inhibitors of Dengue virus. Since catalytic site of flavivirus hold similarities, such as the same catalytic triad (His51, Asp75 e Ser135), the ability of this series of molecules to fit in Zika NS3 domains can be achieved. We performed an exploratory data analysis, using statistical methodologies, such as PCA (Principal Component Analysis) and HCA (Hierarchical Component Analysis), to assist the comprehension of how physicochemical properties impact the interaction observed by the docking studies, as well as to build a correlation between the respective ranked characteristics. Based on these previous studies, peptides were selected for the dynamics simulations, which were useful to better understand the ligand-protein interactions. Information relating to, for instance, energy, ΔG, average number of hydrogen bonds and distance from Ser135 (one of the main amino acids in the catalytic pocket) were discussed. In this sense, peptides 15 (considering ΔG value and Hbond number), 7 (ΔG and energy) and 1, 6, 7 and 15 (the proximity to Ser135 throughout the dynamics simulation) were highlighted as promising. Those interesting results could contribute to future studies regarding Zika virus drug design, since this infection represents a great concern in neglected populations. METHODS The models were constructed in the ChemDraw software. The ligand parametrization was performed in the CHEM3D 17.0, UCSF Chimera. Docking simulations were carried out in the GOLD software, after the redocking validation. We used ASP as the function score. Additionally, for dynamics simulations we applied GROMACS software, exploring, mainly, free binding energy calculations. Exploratory analysis was carried out in Minitab 17.3.1 statistical software. Prior to the exploratory analysis, data of quantum chemical properties of the peptides were collected in Microsoft Excel spreadsheet and organized to obtain Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA).
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Affiliation(s)
- Débora Feliciano Savino
- Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo (USP), Professor Lineu Prestes Avenue, 580, Building 13, São Paulo, SP, 05508-900, Brazil
| | - João Vitor Silva
- Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo (USP), Professor Lineu Prestes Avenue, 580, Building 13, São Paulo, SP, 05508-900, Brazil
| | - Soraya da Silva Santos
- Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo (USP), Professor Lineu Prestes Avenue, 580, Building 13, São Paulo, SP, 05508-900, Brazil
| | - Felipe Rebello Lourenço
- Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo (USP), Professor Lineu Prestes Avenue, 580, Building 13, São Paulo, SP, 05508-900, Brazil
| | - Jeanine Giarolla
- Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo (USP), Professor Lineu Prestes Avenue, 580, Building 13, São Paulo, SP, 05508-900, Brazil.
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Zhu S, Tang Y, Diao Y. Development and biochemical characteristics of a monoclonal antibody against prM protein of Tembusu virus. Poult Sci 2023; 102:103065. [PMID: 37751643 PMCID: PMC10522996 DOI: 10.1016/j.psj.2023.103065] [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: 06/15/2023] [Revised: 08/16/2023] [Accepted: 08/22/2023] [Indexed: 09/28/2023] Open
Abstract
Tembusu virus (TMUV), a pathogenic member of the Flavivirus family, is an infectious diseases that seriously jeopardize duck health in 2010 in China. TMUV disease causes significant economic losses to the duck industry. This study aimed to prepare monoclonal antibodies against TMUV prM protein and to identify their epitopes. The 501bp prM gene was amplified to the pET-32a prokaryotic expression vector and expressed as a recombinant protein of size 38 KD in Escherichia coli. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-prM antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named B27, which stably secreted IgG1-antibody against TMUV prM with high antibody titers up to 1:1:3,276,800 was screened. This monoclonal antibody (mAb) is well specific and can be used for ELISA/Western-blot (WB)/indirect fluorescence assay (IFA) etc. The mAb B27 has poor neutralization ability and concentration dependence, with a maximum neutralization degree of 23.87% at antibody dilution 10-6. Next, we truncated prM gene and expressed the truncated protein to screen antigen epitopes. The mAb's linear antigen epitope of the TMUV prM protein was first identified and was accurate to 6 consecutive amino acids 59GYEPED64, which located in the pr protein. Bioinformatic analysis showed that this antigenic epitope was located on the surface of the antigen, which was conducive to the direct contact of antigen antibody and conformed to the properties of antigenic epitopes. In addition, its 6 amino acids are highly homologous among 27 published TMUV strains, indicating that its epitope is stable. This study will help to further understand the protein structure and the function of prM, and lay the foundation for establishing specific prM detection methods and the mechanistic study of TMUV prM protein.
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Affiliation(s)
- Siming Zhu
- College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong, 271018, China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, 271018, China; Shandong Provincial Engineering Technology Research Center of Animal Disease, Tai'an, Shandong, 271018, China
| | - Yi Tang
- College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong, 271018, China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, 271018, China; Shandong Provincial Engineering Technology Research Center of Animal Disease, Tai'an, Shandong, 271018, China.
| | - Youxiang Diao
- College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong, 271018, China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, 271018, China; Shandong Provincial Engineering Technology Research Center of Animal Disease, Tai'an, Shandong, 271018, China
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Manzato VDM, Di Santo C, Torquato RJS, Coelho C, Gallo G, Hardy L, Würtele M, Tanaka AS. Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease. Biochimie 2023; 214:96-101. [PMID: 37364769 DOI: 10.1016/j.biochi.2023.06.010] [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: 01/17/2023] [Revised: 06/20/2023] [Accepted: 06/23/2023] [Indexed: 06/28/2023]
Abstract
Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1-P4' was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1-P4' positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.
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Affiliation(s)
- Veronica de Moraes Manzato
- Department of Biochemistry, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
| | - Camila Di Santo
- Department of Biochemistry, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
| | - Ricardo Jose Soares Torquato
- Department of Biochemistry, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
| | - Camila Coelho
- Department of Science and Technology, Federal University of São Paulo (UNIFESP), São José dos Campos, SP, Brazil
| | - Gloria Gallo
- Department of Science and Technology, Federal University of São Paulo (UNIFESP), São José dos Campos, SP, Brazil
| | - Leon Hardy
- Department of Physics, University of South Florida, Tampa, USA
| | - Martin Würtele
- Department of Science and Technology, Federal University of São Paulo (UNIFESP), São José dos Campos, SP, Brazil
| | - Aparecida Sadae Tanaka
- Department of Biochemistry, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), RJ, Brazil.
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Fellenberg J, Dubrau D, Isken O, Tautz N. Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3. J Virol 2023; 97:e0057223. [PMID: 37695056 PMCID: PMC10537661 DOI: 10.1128/jvi.00572-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 07/18/2023] [Indexed: 09/12/2023] Open
Abstract
The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.
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Affiliation(s)
- Jonas Fellenberg
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Danilo Dubrau
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Olaf Isken
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Norbert Tautz
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
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Li R, Niu Z, Liu Y, Bai X, Wang D, Chen C. Crystal structure and cap binding analysis of the methyltransferase of langat virus. Antiviral Res 2022; 208:105459. [PMID: 36347437 DOI: 10.1016/j.antiviral.2022.105459] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 10/29/2022] [Accepted: 10/31/2022] [Indexed: 11/08/2022]
Abstract
Tick-borne encephalitis virus (TBEV) is a major dangerous human pathogen, as TBEV infection can cause serious illness that can lead to irreversible neurological sequelae and even death. Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serogroup, belongs to the family Flaviviridae, genus Flavivirus. Its nonstructural protein 5 (NS5) protein contains a methyltransferase (MTase) domain that can methylate RNA cap structures, which is critical for viral replication. We determined the structure of LGTV NS5 methyltransferase bound to S-adenosyl-L-homocysteine (SAH) at a 1.70 Å resolution. Sequence analysis and structural comparison of homologous MTases suggests that folds and structures are closely conserved throughout Flavivirus species and play important roles. This study provides the key structural information on LGTV MTase and the foundation for research on antiviral drugs targeting LGTV MTase.
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Affiliation(s)
- Ruixue Li
- School of Life Sciences, Tianjin University, Tianjin, 300072, China
| | - Ziping Niu
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China
| | - Yujie Liu
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China
| | - Xue Bai
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China
| | - Deping Wang
- Key Laboratory of Cellular Physiology at Shanxi Medical University, Ministry of Education, the Department of Physiology, Shanxi Medical University, Taiyuan, 030001, China.
| | - Chen Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China.
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Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLoS Pathog 2022; 18:e1010895. [PMID: 36215335 PMCID: PMC9616216 DOI: 10.1371/journal.ppat.1010895] [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: 06/09/2022] [Revised: 10/28/2022] [Accepted: 09/25/2022] [Indexed: 11/16/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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Jhan MK, Chen CL, Shen TJ, Tseng PC, Wang YT, Satria RD, Yu CY, Lin CF. Polarization of Type 1 Macrophages Is Associated with the Severity of Viral Encephalitis Caused by Japanese Encephalitis Virus and Dengue Virus. Cells 2021; 10:3181. [PMID: 34831405 PMCID: PMC8621422 DOI: 10.3390/cells10113181] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 11/09/2021] [Accepted: 11/11/2021] [Indexed: 12/20/2022] Open
Abstract
Infection with flaviviruses causes mild to severe diseases, including viral hemorrhagic fever, vascular shock syndrome, and viral encephalitis. Several animal models explore the pathogenesis of viral encephalitis, as shown by neuron destruction due to neurotoxicity after viral infection. While neuronal cells are injuries caused by inflammatory cytokine production following microglial/macrophage activation, the blockade of inflammatory cytokines can reduce neurotoxicity to improve the survival rate. This study investigated the involvement of macrophage phenotypes in facilitating CNS inflammation and neurotoxicity during flavivirus infection, including the Japanese encephalitis virus, dengue virus (DENV), and Zika virus. Mice infected with different flaviviruses presented encephalitis-like symptoms, including limbic seizure and paralysis. Histology indicated that brain lesions were identified in the hippocampus and surrounded by mononuclear cells. In those regions, both the infiltrated macrophages and resident microglia were significantly increased. RNA-seq analysis showed the gene profile shifting toward type 1 macrophage (M1) polarization, while M1 markers validated this phenomenon. Pharmacologically blocking C-C chemokine receptor 2 and tumor necrosis factor-α partly retarded DENV-induced M1 polarization. In summary, flavivirus infection, such as JEV and DENV, promoted type 1 macrophage polarization in the brain associated with encephalitic severity.
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MESH Headings
- Animals
- Animals, Suckling
- Cell Line
- Cell Polarity
- Dengue Virus/physiology
- Disease Models, Animal
- Encephalitis Virus, Japanese/physiology
- Encephalitis, Japanese/immunology
- Encephalitis, Japanese/pathology
- Encephalitis, Japanese/virology
- Encephalitis, Viral/immunology
- Encephalitis, Viral/pathology
- Encephalitis, Viral/virology
- Hippocampus/pathology
- Inflammation/pathology
- Macrophages/pathology
- Mice, Inbred ICR
- Neurotoxins/toxicity
- Receptors, CCR2/metabolism
- Severity of Illness Index
- Tumor Necrosis Factor-alpha/metabolism
- Mice
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Affiliation(s)
- Ming-Kai Jhan
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (M.-K.J.); (T.-J.S.)
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
| | - Chia-Ling Chen
- School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 110, Taiwan;
| | - Ting-Jing Shen
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (M.-K.J.); (T.-J.S.)
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
| | - Po-Chun Tseng
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
- Core Laboratory of Immune Monitoring, Office of Research & Development, Taipei Medical University, Taipei 110, Taiwan
| | - Yung-Ting Wang
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
| | - Rahmat Dani Satria
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
- International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
- Laboratory Medicine, Department of Clinical Pathology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
- Clinical Laboratory Installation, Dr. Sardjito Central General Hospital, Yogyakarta 55281, Indonesia
| | - Chia-Yi Yu
- National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli 350, Taiwan;
| | - Chiou-Feng Lin
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (M.-K.J.); (T.-J.S.)
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; (P.-C.T.); (Y.-T.W.); (R.D.S.)
- Core Laboratory of Immune Monitoring, Office of Research & Development, Taipei Medical University, Taipei 110, Taiwan
- International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
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Afaq S, Atiya A, Malik A, Alwabli AS, Alzahrani DA, Al-Solami HM, Alzahrani O, Alam Q, Kamal MA, Abulfaraj AA, Alhebshi AM, Tarique M. Analysis of methyltransferase (MTase) domain from Zika virus (ZIKV). Bioinformation 2020; 16:229-235. [PMID: 32308265 PMCID: PMC7147495 DOI: 10.6026/97320630016229] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Revised: 02/15/2020] [Accepted: 02/20/2020] [Indexed: 11/23/2022] Open
Abstract
A comprehensive analysis of methyltransferase (MTase) from Zika virus (ZIKV) is of interest in the development of drugs and biomarkers in the combat and care of ZIKA fever with impulsive joint pain and conjunctivitis. MTase sequence is homologous in several viral species. We analyzed the MTase domain from ZIKV using Bioinformatics tools such as SMART, PROSITE, PFAM, PANTHER, and InterProScan to glean insights on the sequence to structure to function data. We document inclusive information on MTase from ZIKV for application in the design of drugs and biomarkers to fight against the disease.
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Affiliation(s)
- Sarah Afaq
- Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia
| | - Akhtar Atiya
- Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia
| | - Arshi Malik
- Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia
| | - Afaf S Alwabli
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
| | - Dhafer A Alzahrani
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
| | - Habeeb M Al-Solami
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
| | - Othman Alzahrani
- Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Kingdom of Saudi Arabia
| | - Qamre Alam
- Medical Genomics Research Department, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Ministry of National Guard Health Affairs, Riyadh, Kingdom of Saudi Arabia
| | - Mohammad Azhar Kamal
- University of Jeddah, Faculty of Science, Department of Biochemistry, Jeddah, Kingdom of Saudi Arabia
- University of Jeddah Center for Science and Medical Research (UJC-SMR), Jeddah, Kingdom of Saudi Arabia
| | - Aala A Abulfaraj
- Department of Biological Sciences, College of Sciences and Arts-Rabigh Campus, King Abdulaziz University, Jeddah 21589, Saudi Arabia
| | - Alawiah M Alhebshi
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
| | - Mohammed Tarique
- Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India
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Abstract
Dengue virus (DENV) belongs to the family Flaviviridae, genus Flavivirus. It is a single-stranded positive-sense ribonucleic acid virus with 10,700 bases. The genus Flavivirus includes other arthropod borne viruses such as yellow fever virus, West Nile virus, Zika virus, tick-borne encephalitis virus. It infects ~50–200 million people annually, putting over 3.6 billion people living in tropical regions at risk and causing ~20,000 deaths annually. The expansion of dengue is attributed to factors such as the modern dynamics of climate change, globalization, travel, trade, socioeconomics, settlement, and also viral evolution. There are four antigenically different serotypes of DENV based on the differences in their viral structural and nonstructural proteins. DENV infection causes a spectrum of illness ranging from asymptomatic to dengue fever to severe dengue shock syndrome. Infection with one serotype confers lifelong immunity against that serotype, but heterologus infection leads to severe dengue hemorrhagic fever due to antibody-dependent enhancement. Diagnosis of dengue infections is based mainly on serological detection of either antigen in acute cases or antibodies in both acute and chronic infection. Viral detection and real-time PCR detection though helpful is not feasible in resource poor setup. Treatment of dengue depends on symptomatic management along with fluid resuscitation and may require platelet transfusion. Although vaccine development is in late stages of development, developing a single vaccine against four serotypes often causes serious challenges to researchers; hence, the main stay of prevention is vector control and management.
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10
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Roder AE, Vazquez C, Horner SM. The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein. PLoS Pathog 2019; 15:e1007163. [PMID: 30730994 PMCID: PMC6382253 DOI: 10.1371/journal.ppat.1007163] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Revised: 02/20/2019] [Accepted: 01/05/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.
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Affiliation(s)
- Allison E Roder
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
| | - Christine Vazquez
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
| | - Stacy M Horner
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
- Department of Medicine, Duke University Medical Center, Durham, NC, United States of America
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11
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Sharma KK, Marzinek JK, Tantirimudalige SN, Bond PJ, Wohland T. Single-molecule studies of flavivirus envelope dynamics: Experiment and computation. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2018; 143:38-51. [PMID: 30223001 DOI: 10.1016/j.pbiomolbio.2018.09.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Revised: 09/06/2018] [Accepted: 09/11/2018] [Indexed: 12/11/2022]
Abstract
Flaviviruses are simple enveloped viruses exhibiting complex structural and functional heterogeneities. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology approaches. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that are employed to investigate flaviviruses. In particular, we review how (i) time-resolved Förster resonance energy transfer (trFRET) was applied to probe dengue envelope conformations; (ii) FRET-fluorescence correlation spectroscopy to investigate dengue envelope intrinsic dynamics and (iii) single particle tracking to follow the path of dengue viruses in cells. We also discuss how such methods may be supported by molecular dynamics (MD) simulations over a range of spatio-temporal scales, to provide complementary data on the structure and dynamics of flaviviral systems. We describe recent improvements in multiscale MD approaches that allowed the simulation of dengue particle envelopes in near-atomic resolution. We hope this review is an incentive for setting up and applying similar single-molecule studies and combine them with MD simulations to investigate structural dynamics of entire flavivirus particles over the nanosecond-to-millisecond time-scale and follow viruses during infection in cells over milliseconds to minutes.
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Affiliation(s)
- Kamal Kant Sharma
- Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
| | - Jan K Marzinek
- Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore
| | - Sarala Neomi Tantirimudalige
- Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
| | - Peter J Bond
- Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore.
| | - Thorsten Wohland
- Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Department of Chemistry, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117557, Singapore.
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12
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Duan X, Anwar MI, Xu Z, Ma L, Yuan G, Chen Y, Liu X, Xia J, Zhou Y, Li YP. Adaptive mutation F772S-enhanced p7-NS4A cooperation facilitates the assembly and release of hepatitis C virus and is associated with lipid droplet enlargement. Emerg Microbes Infect 2018; 7:143. [PMID: 30087320 PMCID: PMC6081454 DOI: 10.1038/s41426-018-0140-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Revised: 06/17/2018] [Accepted: 06/23/2018] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and liver cancer worldwide. Adaptive mutations play important roles in the development of the HCV replicon and its infectious clones. We and others have previously identified the p7 mutation F772S and the co-presence of NS4A mutations in infectious HCV full-length clones and chimeric recombinants. However, the underlying mechanism of F772S function remains incompletely understood. Here, we investigated the functional role of F772S using an efficient JFH1-based reporter virus with Core-NS2 from genotype 2a strain J6, and we designated J6-p7/JFH1-4A according to the strain origin of the p7 and NS4A sequences. We found that replacing JFH1-4A with J6-4A (wild-type or mutated NS4A) or genotype 2b J8-4A severely attenuated the viability of J6-p7/JFH1-4A. However, passage-recovered viruses that contained J6-p7 all acquired F772S. Introduction of F772S efficiently rescued the viral spread and infectivity titers of J6-p7/J6-4A, which reached the levels of the original J6-p7/JFH1-4A and led to a concomitant increase in RNA replication, assembly and release of viruses with J6-specific p7 and NS4A. These data suggest that an isolate-specific cooperation existed between p7 and NS4A. NS4A exchange- or substitution-mediated viral attenuation was attributed to the RNA sequence, and no p7-NS4A protein interaction was detected. Moreover, we found that F772S-enhanced p7-NS4A cooperation was associated with the enlargement of intracellular lipid droplets. This study therefore provides new insights into the mechanisms of adaptive mutations and facilitates studies on the HCV life cycle and virus–host interaction.
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Affiliation(s)
- Xiaobing Duan
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China.,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China.,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Muhammad Ikram Anwar
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China.,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China.,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Zhanxue Xu
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China.,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China.,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Ling Ma
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China.,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China.,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Guosheng Yuan
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Yiyi Chen
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China.,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China.,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Xi Liu
- Department of Infectious Diseases, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 519000, China
| | - Jinyu Xia
- Department of Infectious Diseases, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 519000, China
| | - Yuanping Zhou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Yi-Ping Li
- Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 501180, China. .,Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 501180, China. .,Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Sun Yat-Sen University, Guangzhou, 510080, China. .,Program in Pathobiology, The Fifth Affiliated Hospital and Zhongshan School of Medicine, Sun Yat-sen University, Zhuhai, 519000, China.
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13
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Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus. J Vet Res 2018; 62:17-26. [PMID: 29978123 PMCID: PMC5957457 DOI: 10.2478/jvetres-2018-0003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Accepted: 03/08/2018] [Indexed: 11/20/2022] Open
Abstract
Introduction Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. Material and Methods Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. Results The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. Conclusion Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.
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Yao X, Ling Y, Guo S, Wu W, He S, Zhang Q, Zou M, Nandakumar KS, Chen X, Liu S. Tatanan A from the Acorus calamus L. root inhibited dengue virus proliferation and infections. PHYTOMEDICINE : INTERNATIONAL JOURNAL OF PHYTOTHERAPY AND PHYTOPHARMACOLOGY 2018; 42:258-267. [PMID: 29655694 DOI: 10.1016/j.phymed.2018.03.018] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/21/2017] [Revised: 01/08/2018] [Accepted: 03/08/2018] [Indexed: 06/08/2023]
Abstract
BACKGROUND Acorus calamus l. (Acoraceae) is a well-known traditional Chinese medicinal plant, whose root are historically mainly used to treat neurodegenerative diseases, and for cholera treatment. This datum strongly indicates the antimicrobial activity of A. calamus. PURPOSE Our goal is to find the active constituents of A. calamus to treat dengue virus (DENV) infections, and to study the effects and mechanisms of these active substances. METHODS The root of A. calamus was extracted by ethanol. Mosquito larva C6/36 cells were used for DENV2 replication and transfection host. Mouse kidney fibroblast cells (BHK-21) were used as a host cell to study the infection ability of the virus. DENV2-induced cytopathic effect (CPE) and plaque assay were used to evaluate the inhibitory effect of A. calamus extracts on DENV2 infectivity inhibition. The levels of E and NS1 protein expression were measured by real-time PCR and western blot assays. RESULTS 12 compounds were isolated from ethanol extract of A. calamus root, tatanan A showed the best anti-DENV ability among these 12 compounds, which significantly alleviated DENV2-induced CPE and cytotoxicity effects, with an EC50 of 3.9 µM. In addition, RNA replication assay further confirmed the antivirus ability of tatanan A. Time-addition assay showed that tatanan A affected the early stage of viral RNA replication, which in turn inhibited mRNA and protein levels of DENV2. CONCLUSIONS These results demonstrated the anti-DENV2 effect of tatanan A, in inhibiting DENV2 RNA replication and infections. In summary, tatanan A was found to be a novel natural DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.
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Affiliation(s)
- Xingang Yao
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
| | - Yun Ling
- Department of Pharmaceutical and Life Sciences, Jiujiang University, Jiujiang 332005, PR China.
| | - Songxin Guo
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
| | - Wenyu Wu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, PR China
| | - Shijun He
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
| | - Qing Zhang
- Department of Pharmaceutical and Life Sciences, Jiujiang University, Jiujiang 332005, PR China
| | - Min Zou
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
| | - Kutty Selva Nandakumar
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
| | - Xiaoguang Chen
- School of Public Health, Southern Medical University, Southern Medical University, Guangzhou 510515, PR China
| | - Shuwen Liu
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China.
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15
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Antiviral Activity of Thiazolide Derivatives Against Dengue Virus in Huh-7 Cell Line. Jundishapur J Microbiol 2017. [DOI: 10.5812/jjm.62467] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
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16
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Wang X, Ma D, Huang X, Li L, Li D, Zhao Y, Qiu L, Pan Y, Chen J, Xi J, Shan X, Sun Q. Complete genome analysis of dengue virus type 3 isolated from the 2013 dengue outbreak in Yunnan, China. Virus Res 2017. [PMID: 28648850 DOI: 10.1016/j.virusres.2017.06.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
In the past few decades, dengue has spread rapidly and is an emerging disease in China. An unexpected dengue outbreak occurred in Xishuangbanna, Yunnan, China, resulting in 1331 patients in 2013. In order to obtain the complete genome information and perform mutation and evolutionary analysis of causative agent related to this largest outbreak of dengue fever. The viruses were isolated by cell culture and evaluated by genome sequence analysis. Phylogenetic trees were then constructed by Neighbor-Joining methods (MEGA6.0), followed by analysis of nucleotide mutation and amino acid substitution. The analysis of the diversity of secondary structure for E and NS1 protein were also performed. Then selection pressures acting on the coding sequences were estimated by PAML software. The complete genome sequences of two isolated strains (YNSW1, YNSW2) were 10,710 and 10,702 nucleotides in length, respectively. Phylogenetic analysis revealed both strain were classified as genotype II of DENV-3. The results indicated that both isolated strains of Xishuangbanna in 2013 and Laos 2013 stains (KF816161.1, KF816158.1, LC147061.1, LC147059.1, KF816162.1) were most similar to Bangladesh (AY496873.2) in 2002. After comparing with the DENV-3SS (H87) 62 amino acid substitutions were identified in translated regions, and 38 amino acid substitutions were identified in translated regions compared with DENV-3 genotype II stains Bangladesh (AY496873.2). 27(YNSW1) or 28(YNSW2) single nucleotide changes were observed in structural protein sequences with 7(YNSW1) or 8(YNSW2) non-synonymous mutations compared with AY496873.2. Of them, 4 non-synonymous mutations were identified in E protein sequences with (2 in the β-sheet, 2 in the coil). Meanwhile, 117(YNSW1) or 115 (YNSW2) single nucleotide changes were observed in non-structural protein sequences with 31(YNSW1) or 30 (YNSW2) non-synonymous mutations. Particularly, 14 single nucleotide changes were observed in NS1 sequences with 4/14 non-synonymous substitutions (4 in the coil). Selection pressure analysis revealed no positive selection in the amino acid sites of the genes encoding for structural and non-structural proteins. This study may help understand the intrinsic geographical relatedness of dengue virus 3 and contributes further to research on their infectivity, pathogenicity and vaccine development.
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Affiliation(s)
- Xiaodan Wang
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Dehong Ma
- Xishuangbanna Dai Autonomous Prefecture People's Hospital, Xishuangbanna, 666100, PR China
| | - Xinwei Huang
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Lihua Li
- Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Duo Li
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Yujiao Zhao
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Lijuan Qiu
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Yue Pan
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Junying Chen
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Juemin Xi
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China
| | - Xiyun Shan
- Xishuangbanna Dai Autonomous Prefecture People's Hospital, Xishuangbanna, 666100, PR China.
| | - Qiangming Sun
- Institute of Medical Biology, Chinese Academy of Medical Sciences, PR China; Peking Union Medical College, Kunming, 650118, PR China; Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, PR China.
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17
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Dubrau D, Tortorici MA, Rey FA, Tautz N. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis. PLoS Pathog 2017; 13:e1006134. [PMID: 28151973 PMCID: PMC5308820 DOI: 10.1371/journal.ppat.1006134] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 02/14/2017] [Accepted: 12/16/2016] [Indexed: 01/20/2023] Open
Abstract
The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. Many positive-strand RNA viruses replicate without transcribing subgenomic RNAs otherwise often used to temporally coordinate the expression of proteins involved either in genome replication (early) or virion formation (late). Instead, the RNA genomes of the Flaviviridae are translated into a single polyprotein. Their nonstructural proteins (NS), while not present in the virions, are known to be crucially involved in RNA replication and virion formation. The important question how the same proteins form specific complexes required for fundamentally different aspects of the viral replication cycle is not solved yet. For pestiviruses the mature NS3/4A complex is an essential component of the viral RNA-replicase but is incapable of participating in virion morphogenesis which in turn depends on uncleaved NS2-3 in complex with NS4A. However, a gain of function mutation in NS3 enabled the NS3/4A complex to function in virion assembly. Using structure guided mutagenesis in combination with functional studies we identified the interface between NS3 and the C-terminal NS4A region as a module critical for the decision whether a NS3/4A complex serves in RNA replication or as a packaging component. Thus, we propose that subtle changes in local protein interactions represent decisive switches in viral complex formation pathways.
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Affiliation(s)
- Danilo Dubrau
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - M. Alejandra Tortorici
- Institut Pasteur, Unité de Virologie Structurale, Paris, France
- CNRS UMR 3569 Virologie, Paris, France
| | - Félix A. Rey
- Institut Pasteur, Unité de Virologie Structurale, Paris, France
- CNRS UMR 3569 Virologie, Paris, France
| | - Norbert Tautz
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
- * E-mail:
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18
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Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production. J Virol 2017; 91:JVI.02124-16. [PMID: 27928005 DOI: 10.1128/jvi.02124-16] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Accepted: 11/29/2016] [Indexed: 12/26/2022] Open
Abstract
Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3'X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3'X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3'X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3'X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3'X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells. IMPORTANCE The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.
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19
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Zhang C, Feng T, Cheng J, Li Y, Yin X, Zeng W, Jin X, Li Y, Guo F, Jin T. Structure of the NS5 methyltransferase from Zika virus and implications in inhibitor design. Biochem Biophys Res Commun 2016; 492:624-630. [PMID: 27866982 DOI: 10.1016/j.bbrc.2016.11.098] [Citation(s) in RCA: 54] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 11/16/2016] [Indexed: 11/28/2022]
Abstract
Recent outbreak of flavivirus Zika virus (ZIKV) in America has urged the basic as well as translational studies of this important human pathogen. The nonstructural protein 5 (NS5) of the flavivirus has an N-terminal methyltransferase (MTase) domain that plays critical roles in viral RNA genome capping. The null mutant of NS5 MTase is lethal for virus. Therefore, NS5 is a potential drug target for the treatment of Zika virus infection. In this study, we determined crystal structures of the ZIKV MTase in complex with GTP and RNA cap analogue 7meGpppA. Structural analyses revealed highly conserved GTP/cap-binding pocket and S-adenosylmethionine (SAM)-binding pocket. Two conformations of the second base of the cap were identified, which suggests the flexibility of RNA conformation. In addition, the ligand-binding pockets identified a continuous region of hotspots suitable for drug design. Docking calculation shows that the Dengue virus inhibitor compound 10 may bind to the ZIKV MTase.
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Affiliation(s)
- Caiying Zhang
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Tingting Feng
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Jinbo Cheng
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Yajuan Li
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Xueying Yin
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Weihong Zeng
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Xiangyu Jin
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Yuelong Li
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China
| | - Feng Guo
- School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Tengchuan Jin
- Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, 230027, China.
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20
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Chemical diversity and antiviral potential in the pantropical Diospyros genus. Fitoterapia 2016; 112:9-15. [DOI: 10.1016/j.fitote.2016.04.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Revised: 04/20/2016] [Accepted: 04/22/2016] [Indexed: 01/02/2023]
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21
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Characterization of α-taxilin as a novel factor controlling the release of hepatitis C virus. Biochem J 2015; 473:145-55. [PMID: 26527738 DOI: 10.1042/bj20150717] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2015] [Accepted: 11/02/2015] [Indexed: 01/07/2023]
Abstract
Although it is well established that the release of HCV (hepatitis C virus) occurs through the secretory pathway, many aspects concerning the control of this process are not yet fully understood. α-Taxilin was identified as a novel binding partner of syntaxin-4 and of other members of the syntaxin family, which are part of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and so are involved in intracellular vesicle traffic. Since α-taxilin prevents t-SNARE (target SNARE) formation by binding exclusively to free syntaxin-4, it exerts an inhibitory effect on the vesicular transport. HCV-replicating Huh7.5 cells and HCV-infected primary human hepatocytes and liver samples of patients suffering from chronic HCV contain significantly less α-taxilin compared with the controls. HCV impairs the expression of α-taxilin via NS5A-dependent interruption of the Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signal transduction cascade. Moreover, the half-life of α-taxilin is significantly reduced in HCV-replicating cells. Whereas modulation of α-taxilin expression does not significantly affect genome replication, the overexpression of α-taxilin prevents the release of HCV. In contrast with this, silencing of α-taxilin expression leads to increased release of infectious viral particles. This is due to the negative effect of α-taxilin on t-SNARE formation that leads to impaired vesicular trafficking. Accordingly, overexpression of the t-SNARE component syntaxin-4 increases release of HCV, whereas silencing leads to an impaired release. These data identify α-taxilin as a novel factor that controls the release of HCV and reveal the mechanism by which HCV controls the activity of α-taxilin.
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22
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Abstract
BACKGROUND Infection is one of the main risk factors for cancer. OBJECTIVES Epidemiology, pathogenesis, and disease burden of infection-related cancers were reviewed by infectious agents. FINDINGS Chronic infection with Epstein-Barr virus, hepatitis B and C viruses, Kaposi sarcoma herpes virus, human immunodeficiency virus (HIV) type 1, human papillomavirus (HPV), human T-cell lymphotropic virus type 1, Helicobacter pylori, Clonorchis sinensis, Opisthorchis viverrini, and Schistosoma haematobium are associated with nasopharyngeal carcinoma; lymphoma and leukemia, including non-Hodgkin lymphoma, Hodgkin lymphoma, and Burkitt lymphoma; hepatocellular carcinoma; Kaposi sarcoma; oropharyngeal carcinoma; cervical carcinoma and carcinoma of other anogential sites; adult T-cell leukemia/lymphoma; gastric carcinoma; cholangiocarcinoma; and urinary bladder cancer. In 2008, approximately 2 million new cancer cases (16%) worldwide were attributable to infection. If these infections could be prevented and/or treated, it is estimated that there would be about 23% fewer cancers in less developed regions of the world, and about 7% fewer cancers in more developed regions. CONCLUSION Widespread application of existing public health methods for the prevention of infection, such as vaccination, safer injection practices, quality-assured screening of all donated blood and blood components, antimicrobial treatments, and safer sex practices, including minimizing one's lifetime number of sexual partners and condom use, could have a substantial effect on the future burden of cancer worldwide.
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Hussain A, Idrees M, Asif M, Ali L, Rasool M. Phylogenetic and 2D/3D Analysis of HCV 1a NS4A Gene/Protein in Pakistani Isolates. HEPATITIS MONTHLY 2015; 15:e19936. [PMID: 26288631 PMCID: PMC4532788 DOI: 10.5812/hepatmon.15(6)2015.19936] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/03/2014] [Revised: 07/19/2014] [Accepted: 09/11/2014] [Indexed: 12/11/2022]
Abstract
BACKGROUND The nonstructural protein NS4A of hepatitis C virus is composed of 54 amino acids. This small size protein has vital role in many cellular functions. The most important reported function is being a cofactor of viral enzymes serine protease and helicase. OBJECTIVES The objective of this study was to analyze the phylogenetic variation, its impact in terms of translation and any functional change in protein structure at primary 2D/3D structure using computational tools from Pakistani patients isolates. MATERIALS AND METHODS Patient sera infected with Hepatitis C virus, genotype 1A, were obtained from Molecular Diagnostics lab, CEMB, University of the Punjab Lahore by using BD Vacutainer collection tubes (Becton Dickenson). RESULTS Phylogenetic analysis of the gene revealed that Pakistani 1a HCV strains are in the start of third cluster and there is a difference between inter Pakistani isolates at primary, secondary and tertiary levels. CONCLUSIONS Mutations were present in the central domain of NS4A (amino acids 21 - 34).
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Affiliation(s)
- Abrar Hussain
- Department of Biotechnology and Informtics, BUITEMS, Quetta, Pakistan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Muhammad Idrees
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
- Corresponding Author: Muhammad Idrees, National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. Tel: +92-3214769212, E-mail:
| | - Muhammad Asif
- Department of Biotechnology and Informtics, BUITEMS, Quetta, Pakistan
| | - Liaqat Ali
- Division of Infectious Diseases, Department of Internal Medicine II, University Hospital Freiburg, Freiburg, Germany
- Faculty of Biology, Albert Ludwigs University of Freiburg, Freiburg, Germany
| | - Mahmood Rasool
- Center of Excellence in Genomic Medicine Research, (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia
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Kazakov T, Yang F, Ramanathan HN, Kohlway A, Diamond MS, Lindenbach BD. Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins. PLoS Pathog 2015; 11:e1004817. [PMID: 25875808 PMCID: PMC4395149 DOI: 10.1371/journal.ppat.1004817] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2015] [Accepted: 03/18/2015] [Indexed: 02/07/2023] Open
Abstract
Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.
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Affiliation(s)
- Teymur Kazakov
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Feng Yang
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Harish N. Ramanathan
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Andrew Kohlway
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
| | - Michael S. Diamond
- Departments of Medicine, Molecular Microbiology, and Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America
| | - Brett D. Lindenbach
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
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25
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Isken O, Langerwisch U, Jirasko V, Rehders D, Redecke L, Ramanathan H, Lindenbach BD, Bartenschlager R, Tautz N. A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication. PLoS Pathog 2015; 11:e1004736. [PMID: 25774920 PMCID: PMC4361677 DOI: 10.1371/journal.ppat.1004736] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2014] [Accepted: 02/06/2015] [Indexed: 12/22/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.
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Affiliation(s)
- Olaf Isken
- Institute of Virology and Cell Biology, University of Lübeck, Germany
| | | | - Vlastimil Jirasko
- Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Dirk Rehders
- Joint Laboratory for Structural Biology of Infection and Inflammation of the University of Hamburg and the University of Lübeck, DESY, Hamburg, Germany
| | - Lars Redecke
- Joint Laboratory for Structural Biology of Infection and Inflammation of the University of Hamburg and the University of Lübeck, DESY, Hamburg, Germany
| | - Harish Ramanathan
- Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, United States of America
| | - Brett D. Lindenbach
- Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, United States of America
| | - Ralf Bartenschlager
- Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Norbert Tautz
- Institute of Virology and Cell Biology, University of Lübeck, Germany
- * E-mail:
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26
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Flavivirus reverse genetic systems, construction techniques and applications: a historical perspective. Antiviral Res 2014; 114:67-85. [PMID: 25512228 PMCID: PMC7173292 DOI: 10.1016/j.antiviral.2014.12.007] [Citation(s) in RCA: 95] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2014] [Revised: 11/26/2014] [Accepted: 12/03/2014] [Indexed: 12/20/2022]
Abstract
The study of flaviviruses, which cause some of the most important emerging tropical and sub-tropical human arbovirus diseases, has greatly benefited from the use of reverse genetic systems since its first development for yellow fever virus in 1989. Reverse genetics technology has completely revolutionized the study of these viruses, making it possible to manipulate their genomes and evaluate the direct effects of these changes on their biology and pathogenesis. The most commonly used reverse genetics system is the infectious clone technology. Whilst flavivirus infectious clones provide a powerful tool, their construction as full-length cDNA molecules in bacterial vectors can be problematic, laborious and time consuming, because they are often unstable, contain unwanted induced substitutions and may be toxic for bacteria due to viral protein expression. The incredible technological advances that have been made during the past 30years, such as the use of PCR or new sequencing methods, have allowed the development of new approaches to improve preexisting systems or elaborate new strategies that overcome these problems. This review summarizes the evolution and major technical breakthroughs in the development of flavivirus reverse genetics technologies and their application to the further understanding and control of these viruses and their diseases.
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27
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Daep CA, Muñoz-Jordán JL, Eugenin EA. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neurovirol 2014; 20:539-60. [PMID: 25287260 PMCID: PMC4331079 DOI: 10.1007/s13365-014-0285-z] [Citation(s) in RCA: 132] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Revised: 08/01/2014] [Accepted: 08/26/2014] [Indexed: 10/24/2022]
Abstract
The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.
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Affiliation(s)
- Carlo Amorin Daep
- Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
- Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
| | - Jorge L. Muñoz-Jordán
- Centers for Disease Control and Prevention Dengue Branch, 1324 Cañada Street, San Juan, PR 00971
| | - Eliseo Alberto Eugenin
- Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
- Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
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28
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Abstract
Hepatitis C virus (HCV) is a major global health burden accounting for around 170 million chronic infections worldwide. Although highly potent direct-acting antiviral drugs to treat chronic hepatitis C have been approved recently, owing to their high costs and limited availability and a large number of undiagnosed infections, the burden of disease is expected to rise in the next few years. In addition, HCV is an excellent paradigm for understanding the tight link between a pathogen and host cell pathways, most notably lipid metabolism. HCV extensively remodels intracellular membranes to establish its cytoplasmic replication factory and also usurps components of the intercellular lipid transport system for production of infectious virus particles. Here, we review the molecular mechanisms of viral replicase function, cellular pathways employed during HCV replication factory biogenesis, and viral, as well as cellular, determinants of progeny virus production.
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29
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Li C, Li Y, Shen L, Huang J, Sun Y, Luo Y, Zhao B, Wang C, Yuan J, Qiu HJ. The role of noncoding regions of classical swine fever virus C-strain in its adaptation to the rabbit. Virus Res 2014; 183:117-22. [DOI: 10.1016/j.virusres.2014.02.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 02/06/2014] [Accepted: 02/07/2014] [Indexed: 11/16/2022]
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30
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Teo CSH, Chu JJH. Cellular vimentin regulates construction of dengue virus replication complexes through interaction with NS4A protein. J Virol 2014; 88:1897-913. [PMID: 24284321 PMCID: PMC3911532 DOI: 10.1128/jvi.01249-13] [Citation(s) in RCA: 112] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2013] [Accepted: 11/20/2013] [Indexed: 01/23/2023] Open
Abstract
Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.
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Affiliation(s)
- Catherine Su Hui Teo
- Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore
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31
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Griffiths MJ, Turtle L, Solomon T. Japanese encephalitis virus infection. HANDBOOK OF CLINICAL NEUROLOGY 2014; 123:561-76. [PMID: 25015504 DOI: 10.1016/b978-0-444-53488-0.00026-2] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Affiliation(s)
- Michael J Griffiths
- Institute of Infection and Global Health, University of Liverpool, Liverpool, UK; Alder Hey Children's NHS Foundation Trust, Liverpool, UK.
| | - Lance Turtle
- Institute of Infection and Global Health, University of Liverpool, Liverpool, UK
| | - Tom Solomon
- Institute of Infection and Global Health, University of Liverpool, Liverpool, UK; Walton Centre NHS Foundation Trust, Liverpool, UK; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, UK
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32
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Hundt J, Li Z, Liu Q. Post-translational modifications of hepatitis C viral proteins and their biological significance. World J Gastroenterol 2013; 19:8929-8939. [PMID: 24379618 PMCID: PMC3870546 DOI: 10.3748/wjg.v19.i47.8929] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Accepted: 12/04/2013] [Indexed: 02/06/2023] Open
Abstract
Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.
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Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain. J Virol 2013; 88:628-42. [PMID: 24173222 DOI: 10.1128/jvi.02052-13] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.
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Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor. Antimicrob Agents Chemother 2013; 57:6034-49. [PMID: 24060868 DOI: 10.1128/aac.01176-13] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.
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35
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In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons. Antiviral Res 2013; 100:439-45. [PMID: 24013001 DOI: 10.1016/j.antiviral.2013.08.018] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2012] [Revised: 08/22/2013] [Accepted: 08/25/2013] [Indexed: 12/18/2022]
Abstract
Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.
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36
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Macarthur KL, Smolic R, Smolic MV, Wu CH, Wu GY. Update on the Development of Anti-Viral Agents Against Hepatitis C. J Clin Transl Hepatol 2013; 1:9-21. [PMID: 26357602 PMCID: PMC4521270 DOI: 10.14218/jcth.2013.007xx] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2013] [Revised: 05/14/2013] [Accepted: 05/14/2013] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infects nearly 170 million people worldwide and causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The search for a drug regimen that maximizes efficacy and minimizes side effects is quickly evolving. This review will discuss a wide range of drug targets currently in all phases of development for the treatment of HCV. Direct data from agents in phase III/IV clinical trials will be presented, along with reported side-effect profiles. The mechanism of action of all treatments and resistance issues are highlighted. Special attention is given to available trial data supporting interferon-free treatment regimens. HCV has become an increasingly important public health concern, and it is important for physicians to stay up to date on the rapidly growing novel therapeutic options.
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Affiliation(s)
| | | | | | - Catherine H. Wu
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA
| | - George Y. Wu
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA
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Abstract
Genome replication is a crucial step in the life cycle of any virus. HCV is a positive strand RNA virus and requires a set of nonstructural proteins (NS3, 4A, 4B, 5A, and 5B) as well as cis-acting replication elements at the genome termini for amplification of the viral RNA. All nonstructural proteins are tightly associated with membranes derived from the endoplasmic reticulum and induce vesicular membrane alterations designated the membranous web, harboring the viral replication sites. The viral RNA-dependent RNA polymerase NS5B is the key enzyme of RNA synthesis. Structural, biochemical, and reverse genetic studies have revealed important insights into the mode of action of NS5B and the mechanism governing RNA replication. Although a comprehensive understanding of the regulation of RNA synthesis is still missing, a number of important viral and host determinants have been defined. This chapter summarizes our current knowledge on the role of viral and host cell proteins as well as cis-acting replication elements involved in the biogenesis of the membranous web and in viral RNA synthesis.
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Affiliation(s)
- Volker Lohmann
- Department of Infectious Diseases, University of Heidelberg, Heidelberg, Germany.
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38
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Moradpour D, Penin F. Hepatitis C virus proteins: from structure to function. Curr Top Microbiol Immunol 2013; 369:113-42. [PMID: 23463199 DOI: 10.1007/978-3-642-27340-7_5] [Citation(s) in RCA: 149] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Great progress has been made over the past years in elucidating the structure and function of the hepatitis C virus (HCV) proteins, most of which are now actively being pursued as antiviral targets. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease, the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a master organizer of replication complex formation while NS5A is a zinc-containing phosphoprotein involved in the regulation of HCV RNA replication versus particle production. Core to NS2 make up the assembly module while NS3 to NS5B represent the replication module (replicase). However, HCV proteins exert multiple functions during the viral life cycle, and these may be governed by different structural conformations and/or interactions with viral and/or cellular partners. Remarkably, each viral protein is anchored to intracellular membranes via specific determinants that are essential to protein function in the cell. This review summarizes current knowledge of the structure and function of the HCV proteins and highlights recent advances in the field.
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Affiliation(s)
- Darius Moradpour
- Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
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Galli A, Scheel TKH, Prentoe JC, Mikkelsen LS, Gottwein JM, Bukh J. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. J Gen Virol 2013; 94:2221-2235. [PMID: 23907394 DOI: 10.1099/vir.0.053868-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.
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Affiliation(s)
- Andrea Galli
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Troels K H Scheel
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick C Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Lotte S Mikkelsen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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Blum HE. Hepatitis C and Hepatocellular Carcinoma. VIRAL HEPATITIS 2013:353-361. [DOI: 10.1002/9781118637272.ch24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Yamanaka A, Thongrungkiat S, Ramasoota P, Konishi E. Genetic and evolutionary analysis of cell-fusing agent virus based on Thai strains isolated in 2008 and 2012. INFECTION GENETICS AND EVOLUTION 2013; 19:188-94. [PMID: 23871775 DOI: 10.1016/j.meegid.2013.07.012] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/15/2013] [Revised: 06/25/2013] [Accepted: 07/09/2013] [Indexed: 12/30/2022]
Abstract
Increasing attention is being devoted to ecological and evolutionary relationships between insect-specific flaviviruses and globally important human-pathogenic flaviviruses such as dengue viruses. One such insect flavivirus, cell-fusing agent virus (CFAV), remains poorly investigated. In this study, we isolated 13 and 16 CFAV strains from Aedes aegypti mosquitoes collected in Thailand in 2008 and 2012, respectively, and performed genetic and evolutionary analyses based on gene regions encoding the envelope protein (E) and nonstructural proteins 3 (NS3) and 5 (NS5). Consistent with previously reported CFAV strains, E, NS3 and NS5 regions comprised 1,290, 1,761 and 2,664 nucleotides, respectively. Nucleotide and amino acid identities of these three regions were >98% among the 29 isolates, and approximately 95-96% and 96-99%, respectively, between the isolates and previously reported CFAV strains. When amino acid sequences from representative strains of six insect-specific and seven mosquito-borne flaviviruses were compared, average identities of 14.9%, 31.8% and 44.3% were calculated for E, NS3 and NS5 regions, respectively. Phylogenetic analysis based on nucleotide and amino acid data indicated that the Thai CFAV isolates of the current study were distinct from previously reported CFAV strains from Indonesia and Puerto Rico. Analysis of each gene region consistently uncovered a clade made up of nearly the same subset of Thai CFAV isolates; this result, and the isolation of CFAV from mosquitoes reared from larvae, suggest that the virus is maintained by vertical transmission and conserved in a particular environment without considerable evolutionary alteration. The most recent common ancestor of the Thai CFAV isolates in this study was dated to 11-27 years ago, and is estimated to have diverged 46-86 years ago from previously reported CFAV strains. Superinfection with CFAV of Aedes mosquitoes carrying dengue viruses present in Thailand for over 50 years has most likely taken place.
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Affiliation(s)
- Atsushi Yamanaka
- BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchahewi, Bangkok 10440, Thailand.
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42
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Reiss S, Harak C, Romero-Brey I, Radujkovic D, Klein R, Ruggieri A, Rebhan I, Bartenschlager R, Lohmann V. The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A. PLoS Pathog 2013; 9:e1003359. [PMID: 23675303 PMCID: PMC3649985 DOI: 10.1371/journal.ppat.1003359] [Citation(s) in RCA: 101] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2012] [Accepted: 03/28/2013] [Indexed: 12/11/2022] Open
Abstract
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis. Hepatitis C virus (HCV) infections affect about 170 million people worldwide and often result in severe chronic liver disease. HCV is a positive-strand RNA virus inducing massive rearrangements of intracellular membranes to generate the sites of genome replication, designated the membranous web. The complex biogenesis of the membranous web is still poorly understood, but requires the concerted action of several viral nonstructural proteins and cellular factors. Recently, we and others identified the lipid kinase phosphatidylinositol-4 kinase III alpha (PI4KIIIα), catalyzing the synthesis of phosphatidylinositol 4-phosphate (PI4P), as an essential host factor involved in the formation of the membranous web. In this study, we characterized the virus-host interaction in greater detail using a genetic approach. We identified a highly conserved region in the viral phosphoprotein NS5A crucial for the interaction with PI4KIIIα. Surprisingly, we found that PI4KIIIα, despite being a lipid kinase, appeared to regulate the phosphorylation status of NS5A, thus contributing to viral replication. Our results furthermore suggest that the morphology of the membranous web is regulated by NS5A phosphorylation, providing novel insights into the complex regulation of viral RNA replication.
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Affiliation(s)
- Simon Reiss
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
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ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes. Antimicrob Agents Chemother 2013; 57:3168-77. [PMID: 23629709 DOI: 10.1128/aac.02630-12] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.
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Gu M, Rice CM. Structures of hepatitis C virus nonstructural proteins required for replicase assembly and function. Curr Opin Virol 2013; 3:129-36. [PMID: 23601958 DOI: 10.1016/j.coviro.2013.03.013] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2012] [Revised: 03/08/2013] [Accepted: 03/20/2013] [Indexed: 02/07/2023]
Abstract
Approximately 3% of the world population is infected with hepatitis C virus (HCV), causing a serious public health burden. Like other positive-strand RNA viruses, HCV assembles replicase complexes in association with cellular membranes and produces progeny RNA genomes through negative-strand intermediates. The viral proteins required for RNA replication are nonstructural (NS) proteins NS3 to NS5B. Owing to many obstacles and limitations in structural characterization of proteins and complexes with multiple transmembrane segments, attempts to understand the assembly and action of the HCV replicase complex have been challenging. Nevertheless, great progress has been made in obtaining structural information for several replicase components, providing insights into some aspects of the viral genome replication machinery.
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Affiliation(s)
- Meigang Gu
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, United States.
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Holler TP, Parkinson T, Pryde DC. Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase. Expert Opin Drug Discov 2013; 4:293-314. [PMID: 23489127 DOI: 10.1517/17460440902762802] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND Chronic hepatitis C virus (HCV) infection is a main cause of cirrhosis of the liver and hepatocellular carcinoma. The standard of care is a combination of pegylated interferon with ribavirin, a regimen that has undesirable side effects and is frequently ineffective. Compounds targeting HCV protease and polymerase are in late-stage clinical trials and have been extensively reviewed elsewhere. OBJECTIVE To review and evaluate the progress towards finding novel HCV antivirals targeting HCV proteins beyond the already precedented NS3 protease and NS5B polymerase. METHODS Searches of CAplus and Medline databases were combined with information from key conferences. This review focuses on NS2/3 serine protease, NS3 helicase activity and the non-structural proteins 4A, 4B and 5A. CONCLUSIONS Use of the replicon model of HCV replication and biochemical assays of specific targets has allowed screening of vast libraries of compounds, but resulted in clinical candidates from only NS4A and NS5A. The field is hindered by a lack of good chemical matter that inhibits the remaining enzymes from HCV, and a lack of understanding of the functions of non-structural proteins 4A, 4B and 5A in the replication of HCV.
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Affiliation(s)
- Tod P Holler
- Associate Research Fellow Pfizer Global Research and Development, Antiviral Biology, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK +44 130 464 6387 ; +44 130 465 1819 ;
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Ceelen L, Lieveld M, Forsyth R, Vinken M. The HepaRG cell line: a valuable in vitro tool for hepatitis virus infection studies. Hepatol Int 2013. [PMID: 26201773 DOI: 10.1007/s12072-013-9428-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Hepatitis virus infections, mainly hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, constitute a major problem for public health since they have a worldwide distribution and because they are associated with hepatocellular carcinoma and death. Current anti-HBV vaccines seem to be effective in the majority of people. However, an important issue waiting to be tackled nowadays is how to cure patients with chronic hepatitis B. Moreover, no vaccine is available today for the prevention of HCV infection. Therefore, the use of adequate in vitro infection systems is a prerequisite for the molecular understanding of the infection events of these viruses, which could result in the development of novel powerful therapeutics. In this respect, the HepaRG cell line exhibits a hepatocyte-like morphology and displays drug metabolism capacity similar to that of primary hepatocytes. HepaRG cells have yet been proven to be a useful tool in the study of viral infections, particularly for deciphering the mechanism of HBV entry into hepatocytes.
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Affiliation(s)
| | | | | | - Mathieu Vinken
- Department of Toxicology, Faculty of Medicine and Pharmacy, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
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Small molecule inhibitors of the hepatitis C virus-encoded NS5A protein. Virus Res 2012; 170:1-14. [PMID: 23009750 DOI: 10.1016/j.virusres.2012.09.007] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2012] [Revised: 09/13/2012] [Accepted: 09/13/2012] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) is a modern-day pandemic; 2-3% of the world's population are thought to be infected with the virus and are subsequently at risk of developing end-stage liver diseases. The traditional standard of care (SOC) for HCV-infected patients has been limited to a regimen of pegylated-interferon alpha (pegIFN) and ribavirin; displaying low cure rates in a majority of patients and severe side effects. However, in 2011 the first direct-acting antivirals (DAA) were licensed to treat HCV-infected patients in combination with SOC, which served to elevate treatment response rates. The HCV drug development pipeline is currently populated with many additional and improved DAAs; primarily molecules that target the virus-encoded protease or polymerase enzymes. These molecules are being evaluated both in combination with the traditional SOC and together with other DAAs as all-oral pegIFN-free regimens with the ultimate goal of developing multiple DAA-containing HCV therapies that do not rely on an pegIFN backbone. A recent addition to the arsenal of HCV inhibitors in development is represented by an entirely new DAA class; molecules that target the HCV-encoded non-enzymatic NS5A protein. NS5A is essential for HCV propagation and, although its actual functions are largely unknown, it is likely a key regulator of viral genome replication and virion assembly. The protein is exquisitely sensitive to small molecule-mediated inhibition; NS5A-targeting molecules are probably the most potent antiviral molecules ever discovered and exhibit a number of other attractive drug-like properties, including activity against many HCV genotypes/subtypes and once-daily dosing potential. Although their mechanism of action is unclear, NS5A-targeting molecules are already proving their utility in clinical evaluation; particularly as components of pegIFN-sparring DAA combination regimens. This review will aim to amalgamate our current understanding and knowledge of NS5A-targeting molecules; their discovery, properties, applications, and insight into their future impact as components of all-oral pegIFN-free DAA combination therapies to combat HCV infection.
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A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses. PLoS One 2012; 7:e44965. [PMID: 23028707 PMCID: PMC3441746 DOI: 10.1371/journal.pone.0044965] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Accepted: 08/16/2012] [Indexed: 12/17/2022] Open
Abstract
The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses.
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Ndjomou J, Kolli R, Mukherjee S, Shadrick WR, Hanson AM, Sweeney NL, Bartczak D, Li K, Frankowski KJ, Schoenen FJ, Frick DN. Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation. Antiviral Res 2012; 96:245-55. [PMID: 22940425 DOI: 10.1016/j.antiviral.2012.08.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2012] [Revised: 07/19/2012] [Accepted: 08/17/2012] [Indexed: 01/22/2023]
Abstract
The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
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Affiliation(s)
- Jean Ndjomou
- Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer St., Milwaukee, WI 53211, United States
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A meta-analysis of the existing knowledge of immunoreactivity against hepatitis C virus (HCV). PLoS One 2012; 7:e38028. [PMID: 22675428 PMCID: PMC3364976 DOI: 10.1371/journal.pone.0038028] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2012] [Accepted: 04/29/2012] [Indexed: 02/06/2023] Open
Abstract
Approximately 3% of the world population is infected by HCV, which represents a major global health challenge. Almost 400 different scientific reports present immunological data related to T cell and antibody epitopes derived from HCV literature. Analysis of all HCV-related epitope hosted in the Immune Epitope Database (IEDB), a repository of freely accessible immune epitope data, revealed more than 1500 and 1900 distinct T cell and antibody epitopes, respectively. The inventory of all data revealed specific trends in terms of the host and the HCV genotypes from which sequences were derived. Upon further analysis we found that this large number of epitopes reflects overlapping structures, and homologous sequences derived from different HCV isolates. To access and visualize this information we developed a novel strategy that assembles large sets of epitope data, maps them onto reference genomes and displays the frequency of positive responses. Compilation of the HCV immune reactivity from hundreds of different studies, revealed a complex and thorough picture of HCV immune epitope data to date. The results pinpoint areas of more intense reactivity or research activities at the level of antibody, CD4 and CD8 responses for each of the individual HCV proteins. In general, the areas targeted by the different effector immune functions were distinct and antibody reactivity was positively correlated with hydrophilicity, while T cell reactivity correlated with hydrophobicity. At the sequence level, epitopes frequently recognized by both T cell and B cell correlated with low variability, and our analysis thus highlighted areas of potential interest for practical applications. The human reactivity was further analyzed to pinpoint differential patterns of reactivity associated with acute versus chronic infection, to reveal the apparent impact of glycosylation on T cell, but not antibody responses, and to highlight a paucity of studies involved antibody epitopes associated with virus neutralization.
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