|
1
|
Goonawardane N, Yin C, Roberts GC, Zothner C, Harris M. A key role for hepatitis C virus NS5A serine 225 phosphorylation revealed by super-resolution microscopy. Sci Rep 2025; 15:9567. [PMID: 40113977 PMCID: PMC11926191 DOI: 10.1038/s41598-025-93812-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Accepted: 03/10/2025] [Indexed: 03/22/2025] Open
Abstract
NS5A is a multi-functional phosphoprotein that plays a key role in hepatitis C virus (HCV) genome replication and assembly. The consequences of NS5A phosphorylation for HCV biology remain largely undefined. We previously identified serine 225 (S225) as a major phosphorylation site within the low complexity sequence 1 (LCSI) of NS5A and used a phosphoablatant mutant (S225A) to define the role of this phosphorylation event in genome replication, NS5A-host interactions and sub-cellular localisation. In this study, we investigate this further by raising an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was predominantly in the hyper-phosphorylated NS5A species. Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI, we obtained evidence that is consistent with bidirectional hierarchical phosphorylation initiated by phosphorylation at S225. Using super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive punctae in HCV-infected cells; pS225 was present on the surface of these punctae, close to lipid droplets. Although S225 phosphorylation was not specifically affected by treatment with the NS5A-targeting direct acting antiviral agent daclatasvir, this resulted in the condensation of NS5A-positive punctae into larger structures, recapitulating the S225A phenotype. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.
Collapse
Affiliation(s)
- Niluka Goonawardane
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Chunhong Yin
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
- Infectious Disease Control Institute, Shandong Center for Disease Control and Prevention, Shandong Provincial Key Laboratory of Infectious Disease Prevention and Control, Jinan, 250014, Shandong, People's Republic of China
| | - Grace C Roberts
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Carsten Zothner
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
| |
Collapse
|
|
2
|
Ke PY, Yeh CT. Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy. Pathogens 2024; 13:980. [PMID: 39599533 PMCID: PMC11597459 DOI: 10.3390/pathogens13110980] [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: 10/09/2024] [Revised: 10/30/2024] [Accepted: 11/07/2024] [Indexed: 11/29/2024] Open
Abstract
Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host-virus interactions.
Collapse
Affiliation(s)
- Po-Yuan Ke
- Department of Biochemistry and Molecular Biology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
| | - Chau-Ting Yeh
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
| |
Collapse
|
|
3
|
Liu J, Ito M, Liu L, Nakashima K, Satoh S, Konno A, Suzuki T. Involvement of ribosomal protein L17 and Y-box binding protein 1 in the assembly of hepatitis C virus potentially via their interaction with the 3' untranslated region of the viral genome. J Virol 2024; 98:e0052224. [PMID: 38899899 PMCID: PMC11265288 DOI: 10.1128/jvi.00522-24] [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: 03/19/2024] [Accepted: 05/27/2024] [Indexed: 06/21/2024] Open
Abstract
The 3' untranslated region (3'UTR) of the hepatitis C virus (HCV) RNA genome, which contains a highly conserved 3' region named the 3'X-tail, plays an essential role in RNA replication and promotes viral IRES-dependent translation. Although our previous work has found a cis-acting element for genome encapsidation within 3'X, there is limited information on the involvement of the 3'UTR in particle formation. In this study, proteomic analyses identified host cell proteins that bind to the 3'UTR containing the 3'X region but not to the sequence lacking the 3'X. Further characterization showed that RNA-binding proteins, ribosomal protein L17 (RPL17), and Y-box binding protein 1 (YBX1) facilitate the efficient production of infectious HCV particles in the virus infection cells. Using small interfering RNA (siRNA)-mediated gene silencing in four assays that distinguish between the various stages of the HCV life cycle, RPL17 and YBX1 were found to be most important for particle assembly in the trans-packaging assay with replication-defective subgenomic RNA. In vitro assays showed that RPL17 and YBX1 bind to the 3'UTR RNA and deletion of the 3'X region attenuates their interaction. Knockdown of RPL17 or YBX1 resulted in reducing the amount of HCV RNA co-precipitating with the viral Core protein by RNA immunoprecipitation and increasing the relative distance in space between Core and double-stranded RNA by confocal imaging, suggesting that RPL17 and YBX1 potentially affect HCV RNA-Core interaction, leading to efficient nucleocapsid assembly. These host factors provide new clues to understanding the molecular mechanisms that regulate HCV particle formation. IMPORTANCE Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.
Collapse
Affiliation(s)
- Jie Liu
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masahiko Ito
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Liang Liu
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kenji Nakashima
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Shinya Satoh
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Alu Konno
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Tetsuro Suzuki
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| |
Collapse
|
|
4
|
Golikov MV, Bartosch B, Smirnova OA, Ivanova ON, Ivanov AV. Plasma-Like Culture Medium for the Study of Viruses. mBio 2023; 14:e0203522. [PMID: 36515528 PMCID: PMC9973327 DOI: 10.1128/mbio.02035-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Viral infections attract more and more attention, especially after the emergence of novel zoonotic coronaviruses and the monkeypox virus over the last 2 decades. Research on viruses is based to a great extent on mammalian cell lines that are permissive to the respective viruses. These cell lines are usually cultivated according to the protocols established in the 1950s to 1970s, although it is clear that classical media have a significant imprint on cell growth, phenotype, and especially metabolism. So, recently in the field of biochemistry and metabolomics novel culture media have been developed that resemble human blood plasma. As perturbations in metabolic and redox pathways during infection are considered significant factors of viral pathogenesis, these novel medium formulations should be adapted by the virology field. So far, there are only scarce data available on viral propagation efficiencies in cells cultivated in plasma-like media. But several groups have presented convincing data on the use of such media for cultivation of uninfected cells. The aim of the present review is to summarize the current state of research in the field of plasma-resembling culture media and to point out the influence of media on various cellular processes in uninfected cells that may play important roles in viral replication and pathogenesis in order to sensitize virology research to the use of such media.
Collapse
Affiliation(s)
- Mikhail V. Golikov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Birke Bartosch
- Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Lyon, France
| | - Olga A. Smirnova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Olga N. Ivanova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Alexander V. Ivanov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| |
Collapse
|
|
5
|
Kang SM, Park JY, Han HJ, Song BM, Tark D, Choi BS, Hwang SB. Hepatitis C Virus Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity. Mol Cells 2022; 45:702-717. [PMID: 35993162 PMCID: PMC9589372 DOI: 10.14348/molcells.2022.0018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Revised: 05/23/2022] [Accepted: 05/23/2022] [Indexed: 11/27/2022] Open
Abstract
Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.
Collapse
Affiliation(s)
- Sang-Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
| | - Ji-Young Park
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
- Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea
| | - Hee-Jeong Han
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Byeong-Min Song
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Byeong-Sun Choi
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
| |
Collapse
|
|
6
|
Sequential Phosphorylation of Hepatitis C Virus NS5A Protein Requires the ATP-Binding Domain of NS3 Helicase. J Virol 2022; 96:e0010722. [PMID: 35293767 DOI: 10.1128/jvi.00107-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.
Collapse
|
|
7
|
Hasanshahi Z, Hashempour A, Ghasabi F, Moayedi J, Musavi Z, Dehghani B, Sharafi H, Joulaei H. First report on molecular docking analysis and drug resistance substitutions to approved HCV NS5A and NS5B inhibitors amongst Iranian patients. BMC Gastroenterol 2021; 21:443. [PMID: 34819046 PMCID: PMC8612383 DOI: 10.1186/s12876-021-01988-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Accepted: 10/22/2021] [Indexed: 12/13/2022] Open
Abstract
Background NS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors. Methods Viral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools. Results Several amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency. Conclusions Strong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines. Supplementary Information The online version contains supplementary material available at 10.1186/s12876-021-01988-y.
Collapse
Affiliation(s)
- Zahra Hasanshahi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ava Hashempour
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Farzane Ghasabi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Javad Moayedi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Zahra Musavi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Behzad Dehghani
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Heidar Sharafi
- Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran.,Middle East Liver Diseases (MELD) Center, Tehran, Iran
| | - Hassan Joulaei
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| |
Collapse
|
|
8
|
Saito A, Shofa M, Ode H, Yumiya M, Hirano J, Okamoto T, Yoshimura SH. How Do Flaviviruses Hijack Host Cell Functions by Phase Separation? Viruses 2021; 13:v13081479. [PMID: 34452345 PMCID: PMC8402827 DOI: 10.3390/v13081479] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Revised: 07/21/2021] [Accepted: 07/26/2021] [Indexed: 11/17/2022] Open
Abstract
Viral proteins interact with different sets of host cell components throughout the viral life cycle and are known to localize to the intracellular membraneless organelles (MLOs) of the host cell, where formation/dissolution is regulated by phase separation of intrinsically disordered proteins and regions (IDPs/IDRs). Viral proteins are rich in IDRs, implying that viruses utilize IDRs to regulate phase separation of the host cell organelles and augment replication by commandeering the functions of the organelles and/or sneaking into the organelles to evade the host immune response. This review aims to integrate current knowledge of the structural properties and intracellular localizations of viral IDPs to understand viral strategies in the host cell. First, the properties of viral IDRs are reviewed and similarities and differences with those of eukaryotes are described. The higher IDR content in viruses with smaller genomes suggests that IDRs are essential characteristics of viral proteins. Then, the interactions of the IDRs of flaviviruses with the MLOs of the host cell are investigated with emphasis on the viral proteins localized in the nucleoli and stress granules. Finally, the possible roles of viral IDRs in regulation of the phase separation of organelles and future possibilities for antiviral drug development are discussed.
Collapse
Affiliation(s)
- Akatsuki Saito
- Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan;
- Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan
- Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki 889-1692, Japan
- Correspondence: (A.S.); (T.O.); (S.H.Y.)
| | - Maya Shofa
- Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan;
- Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki 889-1692, Japan
| | - Hirotaka Ode
- Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya 460-0001, Japan;
| | - Maho Yumiya
- Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; (M.Y.); (J.H.)
| | - Junki Hirano
- Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; (M.Y.); (J.H.)
| | - Toru Okamoto
- Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; (M.Y.); (J.H.)
- Center for Infectious Diseases Education and Research, Osaka University, Osaka 565-0871, Japan
- Correspondence: (A.S.); (T.O.); (S.H.Y.)
| | - Shige H. Yoshimura
- Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan
- Correspondence: (A.S.); (T.O.); (S.H.Y.)
| |
Collapse
|
|
9
|
Gallardo-Flores CE, Colpitts CC. Cyclophilins and Their Roles in Hepatitis C Virus and Flavivirus Infections: Perspectives for Novel Antiviral Approaches. Pathogens 2021; 10:902. [PMID: 34358052 PMCID: PMC8308494 DOI: 10.3390/pathogens10070902] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 07/07/2021] [Accepted: 07/15/2021] [Indexed: 12/19/2022] Open
Abstract
Cyclophilins are cellular peptidyl-prolyl isomerases that play an important role in viral infections, with demonstrated roles in the replication of hepatitis C virus (HCV) and other viruses in the Flaviviridae family, such as dengue virus (DENV) and yellow fever virus (YFV). Here, we discuss the roles of cyclophilins in HCV infection and provide a comprehensive overview of the mechanisms underlying the requirement for cyclophilins during HCV replication. Notably, cyclophilin inhibitor therapy has been demonstrated to be effective in reducing HCV replication in chronically infected patients. While the roles of cyclophilins are relatively well-understood for HCV infection, cyclophilins are more recently emerging as host factors for flavivirus infection as well, providing potential new therapeutic avenues for these viral infections which currently lack antiviral therapies. However, further studies are required to elucidate the roles of cyclophilins in flavivirus replication. Here, we review the current knowledge of the role of cyclophilins in HCV infection to provide a conceptual framework to understand how cyclophilins may contribute to other viral infections, such as DENV and YFV. Improved understanding of the roles of cyclophilins in viral infection may open perspectives for the development of cyclophilin inhibitors as effective antiviral therapeutics for HCV and related viruses.
Collapse
Affiliation(s)
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada;
| |
Collapse
|
|
10
|
Li HC, Yang CH, Lo SY. Hepatitis C Viral Replication Complex. Viruses 2021; 13:v13030520. [PMID: 33809897 PMCID: PMC8004249 DOI: 10.3390/v13030520] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 03/18/2021] [Accepted: 03/19/2021] [Indexed: 12/16/2022] Open
Abstract
The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.
Collapse
Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 97004, Taiwan;
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 97004, Taiwan
- Correspondence: ; Tel.: +886-3-8565301 (ext. 2322)
| |
Collapse
|
|
11
|
Liang Y, Zhang G, Li Q, Han L, Hu X, Guo Y, Tao W, Zhao X, Guo M, Gan T, Tong Y, Xu Y, Zhou Z, Ding Q, Wei W, Zhong J. TRIM26 is a critical host factor for HCV replication and contributes to host tropism. SCIENCE ADVANCES 2021; 7:7/2/eabd9732. [PMID: 33523994 PMCID: PMC7793585 DOI: 10.1126/sciadv.abd9732] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Accepted: 11/13/2020] [Indexed: 05/04/2023]
Abstract
Hepatitis C virus (HCV) remains a major human pathogen that requires better understanding of virus-host interactions. In this study, we performed a genome-wide CRISPR-Cas9 screening and identified TRIM26, an E3 ligase, as a critical HCV host factor. Deficiency of TRIM26 specifically impairs HCV genome replication. Mechanistic studies showed that TRIM26 interacts with HCV-encoded NS5B protein and mediates its K27-linked ubiquitination at residue K51, and thus promotes the NS5B-NS5A interaction. Moreover, mouse TRIM26 does not support HCV replication because of its unique six-amino acid insert that prevents its interaction with NS5B. Ectopic expression of human TRIM26 in a mouse hepatoma cell line that has been reconstituted with other essential HCV host factors promotes HCV infection. In conclusion, we identified TRIM26 as a host factor for HCV replication and a new determinant of host tropism. These results shed light on HCV-host interactions and may facilitate the development of an HCV animal model.
Collapse
Affiliation(s)
- Yisha Liang
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Guigen Zhang
- Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics (ICG), Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.
- Institute of Human Virology, Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510000, China
| | - Qiheng Li
- Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics (ICG), Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Lin Han
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiaoyou Hu
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yu Guo
- Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics (ICG), Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Wanyin Tao
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaomin Zhao
- Center for Infectious Diseases Research, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Mingzhe Guo
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Tianyu Gan
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yimin Tong
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yongfen Xu
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhuo Zhou
- Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics (ICG), Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Qiang Ding
- Center for Infectious Diseases Research, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Wensheng Wei
- Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics (ICG), Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.
| | - Jin Zhong
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China.
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| |
Collapse
|
|
12
|
Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A. J Virol 2020; 94:JVI.00420-20. [PMID: 32699091 DOI: 10.1128/jvi.00420-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Accepted: 07/15/2020] [Indexed: 02/07/2023] Open
Abstract
Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.
Collapse
|
|
13
|
Li YP, Yang Y, Wang MQ, Zhang X, Wang WJ, Li M, Wu FP, Dang SS. Facial and bilateral lower extremity edema due to drug-drug interactions in a patient with hepatitis C virus infection and benign prostate hypertrophy: A case report. World J Clin Cases 2020; 8:3372-3376. [PMID: 32874995 PMCID: PMC7441249 DOI: 10.12998/wjcc.v8.i15.3372] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Revised: 06/07/2020] [Accepted: 07/16/2020] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND New direct-acting antivirals (DAAs)-based anti-hepatitis C virus (HCV) therapies are highly effective in patients with HCV infection. However, safety data are lacking regarding HCV treatment with DAAs and drugs for comorbidities. CASE SUMMARY Herein, we reported a case of HCV-infection in a 46-year-old man with benign prostatic hypertrophy. The patient received sofosbuvir/velpatasvir as well as methadone maintenance therapy for drug abuse. The viral load became negative at week 1 post treatment. He developed facial and bilateral lower extremity edema 48 h after starting receiving tamsulosin. Edema disappeared 10 d after treatment with oral furosemide and spironolactone. CONCLUSION In conclusion, this is the first case of an acute edema in the course of treatment with new DAAs, methadone and tamsulosin. These agents are useful in clinical management of patients with HCV infection, particularly in men with benign prostatic hypertrophy. Clinicians should be aware of potential drug-drug interactions in this subset of patients.
Collapse
Affiliation(s)
- Ya-Ping Li
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Ying Yang
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Mu-Qi Wang
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Xin Zhang
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Wen-Jun Wang
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Mei Li
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Feng-Ping Wu
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| | - Shuang-Suo Dang
- Department of Infectious Diseases, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
| |
Collapse
|
|
14
|
HCV-2a NS5A downregulates viral translation predominantly through domain I. Biochem Biophys Res Commun 2020; 529:77-84. [PMID: 32560823 DOI: 10.1016/j.bbrc.2020.05.177] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 05/25/2020] [Indexed: 11/21/2022]
Abstract
Hepatitis C virus (HCV) non-structural protein NS5A is a multifunctional protein with critical roles in viral replication and assembly. We previously showed that HCV-1b NS5A downregulates viral translation only in the presence of the poly(U/UC) tract in 3'UTR. As NS5A of different HCV genotypes may have different functions or carry out the same functions through genotype-specific mechanisms, we investigated the effect of HCV-2a NS5A on viral translation. We found that HCV-2a NS5A downregulates RNA translation of both HCV-2a and -1b, whereas the effect of HCV-1b NS5A is limited to HCV-1b only. In addition, individual regions of 3'UTR are not required for HCV-2a NS5A to downregulate viral RNA translation. We also found that HCV-2a NS5A inhibits capped mRNA translation. Mapping experiments showed that the translation downregulation by HCV-2a NS5A is predominantly mediated by domain I. Furthermore, we found that the integrity of serine-146 residue plays an important role in translation downregulation by NS5A. Our results increased our understanding on genotype-specific functions of HCV NS5A.
Collapse
|
|
15
|
Tabata K, Neufeldt CJ, Bartenschlager R. Hepatitis C Virus Replication. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a037093. [PMID: 31570388 DOI: 10.1101/cshperspect.a037093] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Replication and amplification of the viral genome is a key process for all viruses. For hepatitis C virus (HCV), a positive-strand RNA virus, amplification of the viral genome requires the synthesis of a negative-sense RNA template, which is in turn used for the production of new genomic RNA. This process is governed by numerous proteins, both host and viral, as well as distinct lipids and specific RNA elements within the positive- and negative-strand RNAs. Moreover, this process requires specific changes to host cell ultrastructure to create microenvironments conducive to viral replication. This review will focus on describing the processes and factors involved in facilitating or regulating HCV genome replication.
Collapse
Affiliation(s)
- Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Christopher J Neufeldt
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany.,Division of Virus-Associated Carcinogenesis, German Cancer Research Center, 69120 Heidelberg, Germany.,German Center for Infection Research, Heidelberg Partner Site, 69120 Heidelberg, Germany
| |
Collapse
|
|
16
|
Goonawardane N, Yin C, Harris M. Phenotypic analysis of mutations at residue 146 provides insights into the relationship between NS5A hyperphosphorylation and hepatitis C virus genome replication. J Gen Virol 2020; 101:252-264. [PMID: 31821131 PMCID: PMC7416608 DOI: 10.1099/jgv.0.001366] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Accepted: 11/19/2019] [Indexed: 12/23/2022] Open
Abstract
The hepatitis C virus genotype 2a isolate, JFH-1, exhibits much more efficient genome replication than other isolates. Although basic replication mechanisms must be conserved, this raises the question of whether the regulation of replication might exhibit isolate- and/or genotype-specific characteristics. Exemplifying this, the phenotype of NS5A hyperphosphorylation is genotype-dependent; in genotype 1b a loss of hyperphosphorylation correlates with an enhancement of replication. In contrast, the replication of JFH-1 is not regulated by hyperphosphorylation. We previously identified a novel phosphorylation site in JFH-1 NS5A: S146. A phosphomimetic substitution (S146D) had no effect on replication but correlated with a loss of hyperphosphorylation. In genotype 1b, residue 146 is alanine and we therefore investigated whether the substitution of A146 with a phosphorylatable (S), or phosphomimetic, residue would recapitulate the JFH-1 phenotype, decoupling hyperphosphorylation from replication. This was not the case, as A146D exhibited both a loss of hyperphosphorylation and a reduction in replication, accompanied by a perinuclear restriction of replication complexes, reductions in lipid droplet and PI4P lipid accumulation, and a disruption of NS5A dimerization. In contrast, the S232I culture-adaptive mutation in the low-complexity sequence I (LCSI) also exhibited a loss of hyperphosphorylation, but was associated with an increase in replication. Taken together, these data imply that hyperphosphorylation does not directly regulate replication. In contrast, the loss of hyperphosphorylation is a consequence of perturbing genome replication and NS5A function. Furthermore, we show that mutations in either domain I or LCSI of NS5A can disrupt hyperphosphorylation, demonstrating that multiple parameters influence the phosphorylation status of NS5A.
Collapse
Affiliation(s)
- Niluka Goonawardane
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
- Present address: Experimental Medicine, Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research, South Parks Road, University of Oxford, Oxford, OX1 3SY, UK
| | - Chunhong Yin
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
- Present address: Tsinghua-Peking Center for Life Sciences, School of Medicine, Tsinghua University, Beijing 100084, PR China
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| |
Collapse
|
|
17
|
Kukhanova MK, Karpenko IL, Ivanov AV. DEAD-box RNA Helicase DDX3: Functional Properties and Development of DDX3 Inhibitors as Antiviral and Anticancer Drugs. Molecules 2020; 25:1015. [PMID: 32102413 PMCID: PMC7070539 DOI: 10.3390/molecules25041015] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2020] [Revised: 02/05/2020] [Accepted: 02/21/2020] [Indexed: 12/11/2022] Open
Abstract
This short review is focused on enzymatic properties of human ATP-dependent RNA helicase DDX3 and the development of antiviral and anticancer drugs targeting cellular helicases. DDX3 belongs to the DEAD-box proteins, a large family of RNA helicases that participate in all aspects of cellular processes, such as cell cycle progression, apoptosis, innate immune response, viral replication, and tumorigenesis. DDX3 has a variety of functions in the life cycle of different viruses. DDX3 helicase is required to facilitate both the Rev-mediated export of unspliced/partially spliced human immunodeficiency virus (HIV) RNA from nucleus and Tat-dependent translation of viral genes. DDX3 silencing blocks the replication of HIV, HCV, and some other viruses. On the other hand, DDX displays antiviral effect against Dengue virus and hepatitis B virus through the stimulation of interferon beta production. The role of DDX3 in different types of cancer is rather controversial. DDX3 acts as an oncogene in one type of cancer, but demonstrates tumor suppressor properties in other types. The human DDX3 helicase is now considered as a new attractive target for the development of novel pharmaceutical drugs. The most interesting inhibitors of DDX3 helicase and the mechanisms of their actions as antiviral or anticancer drugs are discussed in this short review.
Collapse
Affiliation(s)
- Marina K. Kukhanova
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, 119991 Moscow, Russia;
| | | | - Alexander V. Ivanov
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, 119991 Moscow, Russia;
| |
Collapse
|
|
18
|
Rabaan AA, Al-Ahmed SH, Bazzi AM, Alfouzan WA, Alsuliman SA, Aldrazi FA, Haque S. Overview of hepatitis C infection, molecular biology, and new treatment. J Infect Public Health 2019; 13:773-783. [PMID: 31870632 DOI: 10.1016/j.jiph.2019.11.015] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2018] [Revised: 07/08/2019] [Accepted: 11/18/2019] [Indexed: 12/13/2022] Open
Abstract
The World Health Organization estimates that 71 million people worldwide have chronic hepatitis C viral infection. A major challenge is overall lack of public awareness of hepatitis C, particularly among infected people of their infection status. Chronic hepatitis C infection is associated with advanced liver disease, is the main cause of hepatocellular carcinoma and causes many extra-hepatic manifestations. The existence of seven viral genotypes complicates targeting of treatment. Recent years have seen the approval of many direct acting antivirals targeted at hepatitis C virus non-structural proteins. These have revolutionized therapy as they allow achievement of extremely high sustained virologic responses. Of great significance is the development of pan-genotypic drug combinations, including the NS3/4A-NS5A inhibitor combinations sofosbuvir-velpatasvir and glecaprevir-pibrentasvir. However, resistance-associated mutations can result in failure of these treatments in a small number of patients. This, combined with the high costs of treatment, highlights the importance of continued research into effective anti-hepatitis C therapies, for example aimed at viral entry. Recent developments include identification of the potential of low-cost anti-histamines for repurposing as inhibitors of hepatitis C viral entry. In this review we focus on molecular biology of hepatitis C virus, and the new developments in hepatitis C treatment.
Collapse
Affiliation(s)
- Ali A Rabaan
- Molecular Diagnostic Laboratory, Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia.
| | - Shamsah H Al-Ahmed
- Specialty Paediatric Medicine, Qatif Central Hospital, Qatif, Saudi Arabia
| | - Ali M Bazzi
- Microbiology Laboratory, Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia
| | - Wadha A Alfouzan
- Department of Microbiology, Faculty of Medicine, Kuwait University, Safat 13110, Kuwait; Faculty of Medicine, Kuwait University, Dasma 35153, Kuwait
| | - Shahab A Alsuliman
- Internal Medicine and Infectious Disease Department, Dammam Medical Complex, Dammam, Saudi Arabia
| | - Fatimah A Aldrazi
- Infection Control Department, Dammam Medical Complex, Dammam, Saudi Arabia
| | - Shafiul Haque
- Research and Scientific Studies Unit, College of Nursing & Allied Health Sciences, Jazan University, Saudi Arabia
| |
Collapse
|
|
19
|
Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States. J Virol 2019; 93:JVI.01028-19. [PMID: 31511391 DOI: 10.1128/jvi.01028-19] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 09/08/2019] [Indexed: 12/19/2022] Open
Abstract
The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle.IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.
Collapse
|
|
20
|
Yamashita M, Kuwahara M. The critical role of Bach2 in regulating type 2 chronic airway inflammation. Int Immunol 2019. [PMID: 29529253 DOI: 10.1093/intimm/dxy020] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Although Bach2 (broad complex-tramtrack-bric a brac and Cap'n'collar homology 2) plays an important role in regulating Th2 cell differentiation and type 2 immune responses, the underlying molecular mechanisms remain unclear. Our current studies demonstrate that Bach2 associates with Batf (basic leucine zipper transcription factor ATF-like) family transcription factors and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the interferon regulatory factor 4 (Irf4)-containing Batf complex to activator protein 1 (AP-1) motifs in the Th2 cytokine gene locus and suppresses Th2 cytokine production and/or Th2 cell differentiation. The deletion of Batf ameliorated the spontaneous development of type 2 airway inflammation that is found in mice with Bach2 deficiency specifically in T cells. Interestingly, Bach2 regulates Batf and Batf3 expression via two distinct pathways. First, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. Second, Bach2 suppresses interleukin 4 (IL-4)-induced augmentation of Batf and Batf3 expression through the regulation of IL-4 production. These findings suggest that IL-4 and Batf family transcription factors form a positive feedback amplification loop to induce Th2 cell differentiation and that Bach2-Batf interactions block the formation of this amplification loop. Furthermore, we found that reductions in Bach2 confer an innate immunological function on CD4 T cells to induce antigen-independent cytokine production. Some Bach2-deficient lung CD4 T cells showed characteristic features similar to pathogenic Th2 cells, including IL-33 receptor expression and IL-33-dependent Th2 cytokine production. These results suggest a critical role for Bach2 in regulating Th2 cell differentiation and the subsequent onset of chronic type 2 inflammation.
Collapse
Affiliation(s)
- Masakatsu Yamashita
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, Japan.,Translational Research Center, Ehime University Hospital, Shitsukawa, Toon, Ehime, Japan.,Division of Immune Regulation, Department of Proteo-Inovation, Proteo-Science Center, Ehime University, Toon City, Ehime, Japan
| | - Makoto Kuwahara
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, Japan.,Translational Research Center, Ehime University Hospital, Shitsukawa, Toon, Ehime, Japan.,Division of Immune Regulation, Department of Proteo-Inovation, Proteo-Science Center, Ehime University, Toon City, Ehime, Japan
| |
Collapse
|
|
21
|
Wang M, Wang Y, Liu Y, Wang H, Xin X, Li J, Hao Y, Han L, Yu F, Zheng C, Shen C. SPSB2 inhibits hepatitis C virus replication by targeting NS5A for ubiquitination and degradation. PLoS One 2019; 14:e0219989. [PMID: 31344133 PMCID: PMC6657855 DOI: 10.1371/journal.pone.0219989] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2019] [Accepted: 07/04/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) replication involves many viral and host factors. Host factor SPRY domain- and SOCS box-containing protein 2(SPSB2) belongs to SPSB family, and it recruits target proteins by the SPRY domain and forms E3 ubiquitin ligase complexes by the SOCS box. As an adaptor protein, it can regulate the host’s response to infection, but little is known about whether SPSB2 plays a role in HCV replication. In the present study, we found that HCV infection significantly upregulated the mRNA and protein levels of SPSB2 in HCVcc-infected cells. Exogenous expression of SPSB2 in hepatoma cells decreased HCV RNA and protein levels which depended on the SOCS box, while knockdown of endogenous SPSB2 increased HCV RNA and protein levels. Additionally, we demonstrated that SPSB2 interacted with HCV structural protein E1 and nonstructural protein protein 5A (NS5A) via the C-terminal portion of the SPSB2 SPRY domain. Furthermore, SPSB2 induced NS5A ubiquitination and mediated NS5A degradation. Collectively, this study discovered host factor SPSB2 significantly inhibits HCV replication by interacting and degrading NS5A.
Collapse
Affiliation(s)
- Mingzhen Wang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yu Wang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yuehong Liu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Hailong Wang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Xiu Xin
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Jiadai Li
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yao Hao
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Lingling Han
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Fang Yu
- Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan, China
| | - Congyi Zheng
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
- China Center for Type Culture Collection, Wuhan University, Wuhan, China
| | - Chao Shen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
- China Center for Type Culture Collection, Wuhan University, Wuhan, China
- * E-mail:
| |
Collapse
|
|
22
|
Pan TC, Lo CW, Chong WM, Tsai CN, Lee KY, Chen PY, Liao JC, Yu MJ. Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus. J Proteome Res 2019; 18:2813-2825. [PMID: 31199160 DOI: 10.1021/acs.jproteome.9b00130] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.
Collapse
Affiliation(s)
- Ting-Chun Pan
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Chieh-Wen Lo
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Weng Man Chong
- Institute of Atomic and Molecular Sciences , Academia Sinica , Taipei 10617 , Taiwan
| | - Chia-Ni Tsai
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Kuan-Ying Lee
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Pin-Yin Chen
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Jung-Chi Liao
- Institute of Atomic and Molecular Sciences , Academia Sinica , Taipei 10617 , Taiwan
| | - Ming-Jiun Yu
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| |
Collapse
|
|
23
|
Klinker S, Stindt S, Gremer L, Bode JG, Gertzen CGW, Gohlke H, Weiergräber OH, Hoffmann S, Willbold D. Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication. J Biol Chem 2019; 294:7388-7402. [PMID: 30862675 DOI: 10.1074/jbc.ra119.007656] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2019] [Revised: 03/11/2019] [Indexed: 12/23/2022] Open
Abstract
The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.
Collapse
Affiliation(s)
- Stefan Klinker
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf
| | - Sabine Stindt
- the Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf
| | - Lothar Gremer
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf.,the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Johannes G Bode
- the Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf
| | - Christoph G W Gertzen
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich.,the John von Neumann Institute for Computing (NIC) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, 52425 Jülich, and.,the Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Holger Gohlke
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich.,the John von Neumann Institute for Computing (NIC) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, 52425 Jülich, and.,the Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Oliver H Weiergräber
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Silke Hoffmann
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Dieter Willbold
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf, .,the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| |
Collapse
|
|
24
|
Schenk C, Meyrath M, Warnken U, Schnölzer M, Mier W, Harak C, Lohmann V. Characterization of a Threonine-Rich Cluster in Hepatitis C Virus Nonstructural Protein 5A and Its Contribution to Hyperphosphorylation. J Virol 2018; 92:JVI.00737-18. [PMID: 30258001 PMCID: PMC6258934 DOI: 10.1128/jvi.00737-18] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 09/20/2018] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein with key functions in regulating viral RNA replication and assembly. Two phosphoisoforms are discriminated by their different apparent molecular weights: a basally phosphorylated (p56) and a hyperphosphorylated (p58) variant. The precise mechanisms governing p58 synthesis and specific functions of the isoforms are poorly understood. Our study aimed at a deeper understanding of determinants involved in p58 synthesis. We analyzed two variants of p56 and p58 of isolate JFH-1 separately by mass spectrometry using an expression model and thereby identified a threonine-rich phosphopeptide exclusively found in the hyperphosphorylated variant. Individual exchange of possible phosphoacceptor sites to phosphoablatant or -mimetic residues had little impact on HCV replication or assembly in cell culture. A phosphospecific antibody recognizing pT242 revealed that this position was indeed phosphorylated only in p58 and depended on casein kinase Iα. Importantly, phosphoablative mutations at positions T244 and S247 abrogated pT242 detection without substantial effects on global p58 levels, whereas mutations in the preceding serine-rich cluster dramatically reduced total p58 levels but had minor impact on pT242 levels, suggesting the existence of distinct subspecies of hyperphosphorylated NS5A. Mass spectrometry analyses of different genotypes showed variable phosphorylation patterns across NS5A and suggested that the threonine-rich region is also phosphorylated at T242 in gt4a and at S249 in gt1a, gt1b, and gt4a. Our data therefore indicate that p58 is not a single homogenously phosphorylated protein species but rather a population of various phosphoisoforms, with high variability between genotypes.IMPORTANCE Hepatitis C virus infections affect 71 million people worldwide and cause severe chronic liver disease. Recently, efficient antiviral therapies have been established, with inhibitors of nonstructural protein NS5A as a cornerstone. NS5A is a central regulator of HCV replication and assembly but is still enigmatic in its molecular functions. It exists in two phosphoisoforms, p56 and p58. We identified a phosphopeptide exclusively found in p58 and analyzed the determinants involved in phosphorylation of this region. We found evidence for very different phosphorylation patterns resulting in p58. These results challenge the concept of p58 being a homogenous species of NS5A molecules phosphorylated at the same positions and argues for at least two independently phosphorylated variants showing the same electrophoretic mobility, likely serving different functions.
Collapse
Affiliation(s)
- Christian Schenk
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Max Meyrath
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Uwe Warnken
- Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Martina Schnölzer
- Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Walter Mier
- Department of Nuclear Medicine, Heidelberg University Hospital, Heidelberg, Germany
| | - Christian Harak
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| |
Collapse
|
|
25
|
Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories. Proc Natl Acad Sci U S A 2018; 115:E12015-E12023. [PMID: 30509975 DOI: 10.1073/pnas.1717944115] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.
Collapse
|
|
26
|
Ohashi H, Nishioka K, Nakajima S, Kim S, Suzuki R, Aizaki H, Fukasawa M, Kamisuki S, Sugawara F, Ohtani N, Muramatsu M, Wakita T, Watashi K. The aryl hydrocarbon receptor-cytochrome P450 1A1 pathway controls lipid accumulation and enhances the permissiveness for hepatitis C virus assembly. J Biol Chem 2018; 293:19559-19571. [PMID: 30381393 DOI: 10.1074/jbc.ra118.005033] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2018] [Revised: 10/24/2018] [Indexed: 12/12/2022] Open
Abstract
Viruses hijack and modify host cell functions to maximize viral proliferation. Hepatitis C virus (HCV) reorganizes host cell metabolism to produce specialized membrane structures and to modify organelles such as double-membrane vesicles and enlarged lipid droplets (LDs), thereby enabling virus replication and assembly. However, the molecular bases of these host-HCV interactions are largely unknown. Here, using a chemical screen, we demonstrate that the benzamide derivative flutamide reduces the host capacity to produce infectious HCV. Flutamide disrupted the formation of enlarged LDs in HCV-infected cells, thereby abolishing HCV assembly. We also report that aryl hydrocarbon receptor (AhR), a known flutamide target, plays a key role in mediating LD accumulation and HCV production. This AhR function in lipid production was also observed in HCV-uninfected Huh-7 cells and primary human hepatocytes, suggesting that AhR signaling regulates lipid accumulation independently of HCV infection. We further observed that a downstream activity, that of cytochrome P450 1A1 (CYP1A1), was the primary regulator of AhR-mediated lipid production. Specifically, blockade of AhR-induced CYP1A1 up-regulation counteracted LD overproduction, and overproduction of CYP1A1, but not of CYP1B1, in AhR-inactivated cells restored lipid accumulation. Of note, HCV infection up-regulated the AhR-CYP1A1 pathway, resulting in the accumulation of enlarged LDs. In conclusion, we demonstrate that the AhR-CYP1A1 pathway has a significant role in lipid accumulation, a hallmark of HCV infection that maximizes progeny virus production. Our chemical-genetic analysis reveals a new strategy and lead compounds to control hepatic lipid accumulation as well as HCV infection.
Collapse
Affiliation(s)
- Hirofumi Ohashi
- From the Department of Virology II and.,the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | - Kazane Nishioka
- From the Department of Virology II and.,the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | - Syo Nakajima
- From the Department of Virology II and.,the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | - Sulyi Kim
- From the Department of Virology II and
| | | | | | - Masayoshi Fukasawa
- the Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
| | - Shinji Kamisuki
- the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | - Fumio Sugawara
- the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | - Naoko Ohtani
- the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and
| | | | | | - Koichi Watashi
- From the Department of Virology II and .,the Tokyo University of Science Graduate School of Science and Technology, Noda 278-8510, Japan, and.,CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan
| |
Collapse
|
|
27
|
Cozza G, Fortuna M, Meggio F, Sarno S, Kubbutat MHG, Totzke F, Schaechtele C, Pinna LA, Olsufyeva EN, Preobrazhenskaya MN. Hydrophobic Derivatives of Glycopeptide Antibiotics as Inhibitors of Protein Kinases. BIOCHEMISTRY. BIOKHIMIIA 2018; 83:1222-1230. [PMID: 30472959 PMCID: PMC7088347 DOI: 10.1134/s0006297918100073] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 12/26/2017] [Revised: 05/17/2018] [Indexed: 01/01/2023]
Abstract
As key regulators of cell signaling, protein kinases (PKs) are attractive targets for therapeutic intervention in a variety of diseases. Herein, we report for the first time the inhibitory activity of polycyclic peptides, particularly, derivatives of glycopeptide antibiotics teicoplanin and eremomycin, against a panel of 12 recombinant human protein kinases and two protein kinases (CK1 and CK2) isolated from rat liver. Several of the investigated compounds inhibited various PKs with IC50 values below 10 μM and caused >90% suppression of the enzyme activity at 10 µM concentration. Kinetic analysis of the protein kinase CK2α inhibition by the teicoplanin aglycon analogue (7) demonstrated the non-competitive mechanism of inhibition (with regard to ATP). Interestingly, the inhibitory activity of some investigated compounds correlated with the earlier described antiviral activity against HIV, HCV, and other corona- and flaviviruses.
Collapse
Affiliation(s)
- G Cozza
- Department of Molecular Medicine, University of Padova, Padova, 35131, Italy
| | - M Fortuna
- Department of Biological Chemistry, University of Padova, Padova, 35131, Italy
| | - F Meggio
- Department of Biological Chemistry, University of Padova, Padova, 35131, Italy
| | - S Sarno
- Department of Biomedical Sciences, University of Padova, Padova, 35131, Italy
| | | | - F Totzke
- ProQinase GmbH, Freiburg, 79106, Germany
| | | | - L A Pinna
- Center for Neuroscience Research Neuroscience Institute, Padova, 35131, Italy
| | - E N Olsufyeva
- Gause Institute of New Antibiotics, Moscow, 119021, Russia.
| | | |
Collapse
|
|
28
|
Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A. J Virol 2018; 92:JVI.01295-18. [PMID: 30089697 DOI: 10.1128/jvi.01295-18] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2018] [Accepted: 07/30/2018] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.
Collapse
|
|
29
|
Morikawa K, Nakamura A, Shimazaki T, Sakamoto N. Safety and efficacy of elbasvir/grazoprevir for the treatment of chronic hepatitis C: current evidence. DRUG DESIGN DEVELOPMENT AND THERAPY 2018; 12:2749-2756. [PMID: 30233138 PMCID: PMC6132225 DOI: 10.2147/dddt.s133697] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Treatments for hepatitis C virus (HCV) have advanced greatly, becoming more efficacious with fewer adverse events, due to the availability of direct-acting antiviral agents, which target specific steps in the HCV life cycle. Recently, a combination regimen consisting of the HCV nonstructural protein 5A inhibitor elbasvir (EBR) and the HCV NS3/4A protease inhibitor grazoprevir (GZR) was approved for the treatment of patients with chronic HCV and genotypes (Gts) 1 and 4 in various countries. In Phase III trials, the combination of EBR/GZR (fixed-dose combination table or single agent) for 12 or 16 weeks of treatment with or without ribavirin resulted in a high sustained virological response at 12 weeks in treatment-naïve and treatment-experienced patients with HCV Gt 1a, 1b, 4, or 6, including special populations, such as individuals with advanced chronic kidney disease, HCV-HIV coinfection, and compensated cirrhosis. In this review, we focus on the mode of action, pharmacokinetics, clinical applications, efficacy, and safety profile of EBR/GZR, including special populations who have been considered refractory from the extensive evidence of clinical trials.
Collapse
Affiliation(s)
- Kenichi Morikawa
- Department of Gastroenterology and Hepatology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo, Japan,
| | - Akihisa Nakamura
- Department of Gastroenterology and Hepatology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo, Japan,
| | - Tomoe Shimazaki
- Department of Gastroenterology and Hepatology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo, Japan,
| | - Naoya Sakamoto
- Department of Gastroenterology and Hepatology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo, Japan,
| |
Collapse
|
|
30
|
Shanmugam S, Nichols AK, Saravanabalaji D, Welsch C, Yi M. HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A. PLoS Pathog 2018; 14:e1007177. [PMID: 30036383 PMCID: PMC6072203 DOI: 10.1371/journal.ppat.1007177] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Revised: 08/02/2018] [Accepted: 06/25/2018] [Indexed: 12/12/2022] Open
Abstract
The HCV NS5A protein plays multiple roles during viral replication, including viral genome replication and virus particle assembly. The crystal structures of the NS5A N-terminal domain indicated the potential existence of the NS5A dimers formed via at least two or more distinct dimeric interfaces. However, it is unknown whether these different forms of NS5A dimers are involved in its numerous functions. To address this question, we mutated the residues lining the two different NS5A dimer interfaces and determined their effects on NS5A self-interaction, NS5A-cyclophilin A (CypA) interaction, HCV RNA replication and infectious virus production. We found that the mutations targeting either of two dimeric interfaces disrupted the NS5A self-interaction in cells. The NS5A dimer-interrupting mutations also inhibited both viral RNA replication and infectious virus production with some genotypic differences. We also determined that reduced NS5A self-interaction was associated with altered NS5A-CypA interaction, NS5A hyperphosphorylation and NS5A subcellular localization, providing the mechanistic bases for the role of NS5A self-interaction in multiple steps of HCV replication. The NS5A oligomers formed via different interfaces are likely its functional form, since the residues at two different dimeric interfaces played similar roles in different aspects of NS5A functions and, consequently, HCV replication. In conclusion, this study provides novel insight into the functional significance of NS5A self-interaction in different steps of the HCV replication, potentially, in the form of oligomers formed via multiple dimeric interfaces. HCV NS5A is a multifunctional protein involved in both viral RNA replication and infectious virus production, and is a target of one of the most potent antivirals available to date. However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A functions during HCV life cycles. In this study, we have provided evidence that surface-exposed NS5A residues involved in two different dimeric interactions in crystal structures are indeed involved in NS5A self-interactions in cells. We also showed that these NS5A residues play critical role in HCV RNA replication and infectious virus production by regulating NS5A hyperphosphorylation, its subcellular localization and its interaction with host protein CypA. Overall, our data support the functional significance of “NS5A oligomers” formed via multiple interfaces in HCV replication. We speculate that the NS5A inhibitors exploited the NS5A oligomer-dependent functions during HCV replication, rather than targeting individual NS5A, which consequently resulted in their high potency.
Collapse
Affiliation(s)
- Saravanabalaji Shanmugam
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Alyssa K. Nichols
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Dhanaranjani Saravanabalaji
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Christoph Welsch
- Department of Internal Medicine I, Goethe University, Frankfurt/Main, Germany
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
- * E-mail:
| |
Collapse
|
|
31
|
Bessa LM, Schneider R, Hanoulle X. NMR and circular dichroism data for domain 2 of the HCV NS5A protein phosphorylated by the Casein Kinase II. Data Brief 2018; 17:325-333. [PMID: 29876401 PMCID: PMC5988295 DOI: 10.1016/j.dib.2018.01.038] [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: 06/01/2017] [Revised: 10/04/2017] [Accepted: 01/16/2018] [Indexed: 11/22/2022] Open
Abstract
The Hepatitis C Virus (HCV)1 nonstructural 5A protein (NS5A) is a phosphoprotein (Evans et al., 2004; Ross-Thriepland and Harris, 2014) [1], [2] composed of an N-terminal well-structured domain and two C-terminal intrinsically disordered domains (Moradpour et al., 2007; Bartenschlager et al., 2013; Badillo et al., 2017) [3], [4], [5]. So far, no precise molecular function has been identified for this viral protein (Ross-Thriepland and Harris, 2015) [6] which is required for viral replication (Tellinghuisen et al., 2008) [7]. In this article, we present datasets of NMR and circular dichroism analyses of the domain 2 of the HCV NS5A protein (NS5A-D2) phosphorylated in vitro by the Casein Kinase II (CKII) (Dal Pero et al., 2007; Clemens et al., 2015; Masak et al., 2014; Kim et al., 2014) [8], [9], [10], [11]. We describe the in vitro phosphorylation of the serine 288 (pS288) of NS5A-D2 by CKII and report the circular dichroism spectrum of the phosphorylated domain (NS5-D2_CKII). This data article also contains the 1H, 15N and 13C NMR chemical shift assignments (HN, N, Cα, Cβ and C’) for the phosphorylated NS5A-D2 domain, and an assigned 1H,15N-HSQC spectrum is shown. The NMR data have been acquired on an 800 MHz spectrometer. These NMR data have been used to calculate both the 1H,15N combined chemical shift perturbations (CSP) induced by the phosphorylation of pS288 and the secondary structural propensity (SSP) scores that describe the structural tendencies in this intrinsically disordered domain. The circular dichroism spectrum and the SSP scores of NS5A-D2_CKII have been compared with those of unphosphorylated NS5A-D2 [12,13].
Collapse
|
|
32
|
Shi G, Suzuki T. Molecular Basis of Encapsidation of Hepatitis C Virus Genome. Front Microbiol 2018; 9:396. [PMID: 29563905 PMCID: PMC5845887 DOI: 10.3389/fmicb.2018.00396] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Accepted: 02/21/2018] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV), a major etiologic agent of human liver diseases, is a positive-sense single-stranded RNA virus and is classified in the Flaviviridae family. Although research findings for the assembly of HCV particles are accumulating due to development of HCV cell culture system, the mechanism(s) by which the HCV genome becomes encapsidated remains largely unclear. In general, viral RNA represents only a small fraction of the RNA molecules in the cells infected with RNA viruses, but the viral genomic RNA is considered to selectively packaged into virions. It was recently demonstrated that HCV RNAs containing 3' end of the genome are selectively incorporated into virus particles during the assembly process and the 3' untranslated region functions as a cis-acting element for RNA packaging. Here, we discuss the molecular basis of RNA encapsidation of HCV and classical flaviviruses, contrast with the packaging mechanism of HIV-1.
Collapse
Affiliation(s)
- Guoli Shi
- Antiviral Immunity and Resistance Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States
| | - Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| |
Collapse
|
|
33
|
Aeginetia indica Decoction Inhibits Hepatitis C Virus Life Cycle. Int J Mol Sci 2018; 19:ijms19010208. [PMID: 29315273 PMCID: PMC5796157 DOI: 10.3390/ijms19010208] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Revised: 01/03/2018] [Accepted: 01/05/2018] [Indexed: 12/16/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is still a global epidemic despite the introduction of several highly effective direct-acting antivirals that are tagged with sky-high prices. The present study aimed to identify an herbal decoction that ameliorates HCV infection. Among six herbal decoctions tested, the Aeginetia indica decoction had the most profound effect on the HCV reporter activity in infected Huh7.5.1 liver cells in a dose- and time-dependent manner. The Aeginetia indica decoction exerted multiple inhibitory effects on the HCV life cycle. Pretreatment of the cells with the Aeginetia indica decoction prior to HCV infection reduced the HCV RNA and non-structural protein 3 (NS3) protein levels in the infected cells. The Aeginetia indica decoction reduced HCV internal ribosome entry site-mediated protein translation activity. It also reduced the HCV RNA level in the infected cells in association with reduced NS5A phosphorylation at serine 235, a predominant phosphorylation event indispensable to HCV replication. Thus, the Aeginetia indica decoction inhibits HCV infection, translation, and replication. Mechanistically, the Aeginetia indica decoction probably reduced HCV replication via reducing NS5A phosphorylation at serine 235.
Collapse
|
|
34
|
Sugiyama R, Murayama A, Nitta S, Yamada N, Tasaka-Fujita M, Masaki T, Aly HH, Shiina M, Ryo A, Ishii K, Wakita T, Kato T. Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling. Oncotarget 2017; 9:5627-5640. [PMID: 29464023 PMCID: PMC5814163 DOI: 10.18632/oncotarget.23562] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Accepted: 10/27/2017] [Indexed: 02/06/2023] Open
Abstract
The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.
Collapse
Affiliation(s)
- Ryuichi Sugiyama
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Sayuri Nitta
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.,Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Norie Yamada
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Megumi Tasaka-Fujita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Takahiro Masaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Present address: Department of Laboratory Medicine, The Jikei University School of Medicine, Nishi-shinbashi, Minato-ku, Tokyo, Japan
| | - Hussein Hassan Aly
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Masaaki Shiina
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Gastroenterology and Hepatology, Shin-Yurigaoka General Hospital, Kawasaki, Japan
| | - Akihide Ryo
- Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Japan
| | - Koji Ishii
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| |
Collapse
|
|
35
|
Goonawardane N, Ross-Thriepland D, Harris M. Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein. J Gen Virol 2017; 99:62-72. [PMID: 29139348 PMCID: PMC5882090 DOI: 10.1099/jgv.0.000975] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.
Collapse
Affiliation(s)
- Niluka Goonawardane
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Douglas Ross-Thriepland
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.,Present address: AstraZeneca, Cambridge Biomedical Campus, Cambridge, CB20AA, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| |
Collapse
|
|
36
|
Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions. J Virol 2017; 91:JVI.00805-17. [PMID: 28615203 PMCID: PMC5553161 DOI: 10.1128/jvi.00805-17] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 06/03/2017] [Indexed: 12/31/2022] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.
Collapse
|
|
37
|
Hsu SC, Lo CW, Pan TC, Lee KY, Yu MJ. Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication. J Virol 2017; 91:e00194-17. [PMID: 28446668 PMCID: PMC5487554 DOI: 10.1128/jvi.00194-17] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Accepted: 04/17/2017] [Indexed: 12/30/2022] Open
Abstract
The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235.IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.
Collapse
Affiliation(s)
- Shih-Chin Hsu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Chieh-Wen Lo
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Ting-Chun Pan
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Kuan-Ying Lee
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Ming-Jiun Yu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
| |
Collapse
|
|
38
|
Badillo A, Receveur-Brechot V, Sarrazin S, Cantrelle FX, Delolme F, Fogeron ML, Molle J, Montserret R, Bockmann A, Bartenschlager R, Lohmann V, Lippens G, Ricard-Blum S, Hanoulle X, Penin F. Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A. Biochemistry 2017; 56:3029-3048. [PMID: 28535337 DOI: 10.1021/acs.biochem.7b00212] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the effect of D2 mutations conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors on the structure of the recombinant D2 Con1 mutants and their binding to CypA. Although resistance mutations D320E and R318W could induce some local and/or global folding perturbation, which could thus affect the kinetics of conformer interconversions, they do not significantly affect the kinetics of CypA/D2 interaction measured by surface plasmon resonance (SPR). The combination of all our data led us to build a model of the overall structure of NS5A, which provides a useful template for further investigations of the structural and functional features of this enigmatic protein.
Collapse
Affiliation(s)
- Aurelie Badillo
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | | | - Stéphane Sarrazin
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - François-Xavier Cantrelle
- University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 000 Lille, France
| | - Frédéric Delolme
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Marie-Laure Fogeron
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Jennifer Molle
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Roland Montserret
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Anja Bockmann
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg , Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg , Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
| | - Guy Lippens
- University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 000 Lille, France
| | - Sylvie Ricard-Blum
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| | - Xavier Hanoulle
- University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 000 Lille, France
| | - François Penin
- Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Labex Ecofect, Université de Lyon, 69367 Lyon, France
| |
Collapse
|
|
39
|
Abstract
PURPOSE OF REVIEW Direct-acting antiviral agents (DAAs) have markedly improved the prognosis of hepatitis C virus (HCV)-genotype 3 (GT3), a highly prevalent infection worldwide. However, in patients with hepatic fibrosis, cirrhosis, or hepatocellular carcinoma (HCC), GT3 infection presents a treatment challenge compared with other genotypes. The dependence of the HCV life cycle on host lipid metabolism suggests the possible utility of targeting host cellular factors for combination anti-HCV therapy. We discuss current and emergent DAA regimens for HCV-GT3 treatment. We then summarize recent research findings on the reliance of HCV entry, replication, and virion assembly on host lipid metabolism. RECENT FINDINGS Current HCV treatment guidelines recommend the use of daclatasvir plus sofosbuvir (DCV/SOF) or sofosbuvir plus velpatasvir (SOF/VEL) for the management of GT3 based upon clinical efficacy [≥88% overall sustained virological response (SVR)] and tolerability. Potential future DAA options, such as SOF/VEL co-formulated with GS-9857, also look promising in treating cirrhotic GT3 patients. However, HCV resistance to DAAs will likely continue to impact the therapeutic efficacy of interferon-free treatment regimens. Disruption of HCV entry by targeting required host cellular receptors shows potential in minimizing HCV resistance and broadening therapeutic options for certain subpopulations of GT3 patients. The use of cholesterol biosynthesis and transport inhibitors may also improve health outcomes for GT3 patients when used synergistically with DAAs. Due to the morbidity and mortality associated with HCV-GT3 infection compared to other genotypes, efforts should be made to address current limitations in the therapeutic prevention and management of HCV-GT3 infection.
Collapse
|
|
40
|
Abstract
Chronic infection with hepatitis C virus (HCV) is a global public health burden. It has been only several decades since this virus was first identified. In the meantime, a lot of progress has been made in the fight against HCV. Although the development of pegylated interferon (PEG-IFN) and its combination with ribavirin (RBV) has significantly increased effectiveness of IFN-based treatment, candidate patients must be assessed for eligibility prior to the treatment due to side effects of the regimens and the rates of sustained virological response (SVR) were only around 50%. In 2011, the protease inhibitor (PI) Telaprevir was firstly approved as a direct-acting antiviral (DAA) for hepatitis C. The second generation of PIs was subsequently introduced and, by adding PI to Peg-IFN/RBV, the SVR rates were found to be raised to up to 80%. Further, with the recent approval of the NS5A inhibitors and the NS5B polymerase inhibitors and with the SVR rates reaching 90% or greater using IFN-free, DAA combination regimens, it is now expected that the majority of patients with chronic hepatitis C can be cured of infection in the near future.
Collapse
|
|
41
|
Wang H, Tai AW. Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging. J Hepatol 2017; 66:55-66. [PMID: 27599826 PMCID: PMC5167665 DOI: 10.1016/j.jhep.2016.08.018] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/29/2016] [Revised: 08/22/2016] [Accepted: 08/26/2016] [Indexed: 01/22/2023]
Abstract
BACKGROUND & AIMS Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation. METHODS Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time. RESULTS We found that HCV replication organelles are continuously generated de novo at spatially distinct sites from preformed ones. This process is accompanied by accumulated intracellular membrane alteration, increased cholesterol delivery, NS5A phosphorylation, and positive-strand RNA content, and by eventual association with HCV core protein around lipid droplets. Generation of spatially segregated foci requires viral NS5A and the host factors phosphatidylinositol 4-kinase and oxysterol-binding protein, while association of foci with lipid droplets requires cholesterol. CONCLUSIONS Our results reveal that HCV replication organelles are not static structures, but instead are continuously generated and dynamically change in composition and possibly also in function. LAY SUMMARY Hepatitis C virus replication membrane structures are continuously generated at spatially distinct sites. New replication organelles are different in composition, and possibly also in function, compared to old replication organelles.
Collapse
Affiliation(s)
- Hongliang Wang
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan
| | - Andrew W. Tai
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan
,Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan
,Medicine Service, Ann Arbor Veterans Administration Health System, Ann Arbor, Michigan
,Correspondence: Andrew W. Tai, University of Michigan, 6520 MSRB I SPC 5682, 1150 W Medical Center Dr, Ann Arbor, MI 48109-5682, Tel: (734) 764-2804, FAX: (734) 763-2535,
| |
Collapse
|
|
42
|
Abstract
Viruses use synthetic mechanism and organelles of the host cells to facilitate their replication and make new viruses. Host's ATP provides necessary energy. Hepatitis C virus (HCV) is a major cause of liver disease. Like other positive-strand RNA viruses, the HCV genome is thought to be synthesized by the replication complex, which consists of viral- and host cell-derived factors, in tight association with structurally rearranged vesicle-like cytoplasmic membranes. The virus-induced remodeling of subcellular membranes, which protect the viral RNA from nucleases in the cytoplasm, promotes efficient replication of HCV genome. The assembly of HCV particle involves interactions between viral structural and nonstructural proteins and pathways related to lipid metabolisms in a concerted fashion. Association of viral core protein, which forms the capsid, with lipid droplets appears to be a prerequisite for early steps of the assembly, which are closely linked with the viral genome replication. This review presents the recent progress in understanding the mechanisms for replication and assembly of HCV through its interactions with organelles or distinct organelle-like structures.
Collapse
Affiliation(s)
- Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, 431-3192, Japan.
| |
Collapse
|
|
43
|
Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat Microbiol 2016; 2:16247. [PMID: 27991882 DOI: 10.1038/nmicrobiol.2016.247] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Accepted: 11/04/2016] [Indexed: 12/30/2022]
Abstract
With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.
Collapse
|
|
44
|
Lee KY, Chen YH, Hsu SC, Yu MJ. Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases. PLoS One 2016; 11:e0166763. [PMID: 27875595 PMCID: PMC5119781 DOI: 10.1371/journal.pone.0166763] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Accepted: 11/03/2016] [Indexed: 12/14/2022] Open
Abstract
Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.
Collapse
Affiliation(s)
- Kuan-Ying Lee
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Yi-Hung Chen
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Shih-Chin Hsu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Ming-Jiun Yu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| |
Collapse
|
|
45
|
Kwon J, Kim DH, Park JM, Park YH, Hwang YH, Wu HG, Shin KH, Kim IA. Targeting Phosphatidylinositol 4-Kinase IIIα for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent. Int J Radiat Oncol Biol Phys 2016; 96:867-876. [DOI: 10.1016/j.ijrobp.2016.08.007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2016] [Revised: 08/05/2016] [Accepted: 08/10/2016] [Indexed: 12/13/2022]
|
|
46
|
Miyamura T, Lemon SM, Walker CM, Wakita T. The HCV Replicase Complex and Viral RNA Synthesis. HEPATITIS C VIRUS I 2016. [PMCID: PMC7122888 DOI: 10.1007/978-4-431-56098-2_8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Replication of hepatitis C virus (HCV) is tightly linked to membrane alterations designated the membranous web, harboring the viral replicase complex. In this chapter we describe the morphology and 3D architecture of the HCV-induced replication organelles, mainly consisting of double membrane vesicles, which are generated by a concerted action of the nonstructural proteins NS3 to NS5B. Recent studies have furthermore identified a number of host cell proteins and lipids contributing to the biogenesis of the membranous web, which are discussed in this chapter. Viral RNA synthesis is tightly associated with these membrane alterations and mainly driven by the viral RNA dependent RNA polymerase NS5B. We summarize our current knowledge of the structure and function of NS5B, the role of cis-acting replication elements at the termini of the genome in regulating RNA synthesis and the contribution of additional viral and host factors to viral RNA synthesis, which is still ill defined.
Collapse
Affiliation(s)
- Tatsuo Miyamura
- National Institute of Infectious Diseases, Tokyo, Tokyo Japan
| | - Stanley M. Lemon
- Departments of Medicine and Microbiology & Immunology , The University of North Carolina, Chapel Hill, North Carolina USA
| | - Christopher M. Walker
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio USA
| | - Takaji Wakita
- National Institute of Infectious Diseases, Tokyo, Tokyo Japan
| |
Collapse
|
|
47
|
Chahine EB, Sucher AJ, Hemstreet BA. Sofosbuvir/Velpatasvir: The First Pangenotypic Direct-Acting Antiviral Combination for Hepatitis C. Ann Pharmacother 2016; 51:44-53. [PMID: 27609942 DOI: 10.1177/1060028016668897] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
OBJECTIVES To review the pharmacology, efficacy, and safety of sofosbuvir/velpatasvir in the treatment of patients with hepatitis C virus (HCV) infection. DATA SOURCES A literature search through PubMed was conducted (June 2008 to August 2016) using the terms GS-5816, velpatasvir, and sofosbuvir. References from retrieved articles and the prescribing information were reviewed for any additional material. STUDY SELECTION/DATA EXTRACTION The literature search was limited to human studies published in English. Phase I, II, and III studies of sofosbuvir/velpatasvir for HCV were identified. DATA SYNTHESIS Sofosbuvir/velpatasvir is indicated for adult patients with chronic HCV genotype 1 through 6. It is given without ribavirin in patients with or without compensated cirrhosis and with ribavirin in patients who have decompensated cirrhosis. The ASTRAL-1 study demonstrated that sofosbuvir 400 mg plus velpatasvir 100 mg for 12 weeks was effective at achieving high sustained virological response (SVR12) rates in patients with HCV genotype 1, 2, 4, 5, or 6. The ASTRAL-2 and ASTRAL-3 studies demonstrated that the same regimen was effective at achieving high SVR12 rates in patients with HCV genotype 2 or 3. The ASTRAL-4 study demonstrated that the same regimen plus ribavirin was effective at achieving high SVR12 rate in patients with decompensated cirrhosis. The most common adverse reactions (≥10% of patients) associated with sofosbuvir/velpatasvir were headache and fatigue. CONCLUSIONS Sofosbuvir/velpatasvir is safe and effective to treat HCV genotypes 1, 2, 3, 4, 5, and 6 in patients with or without compensated cirrhosis. The addition of ribavirin is recommended in patients with decompensated cirrhosis.
Collapse
|
|
48
|
Kuwahara M, Ise W, Ochi M, Suzuki J, Kometani K, Maruyama S, Izumoto M, Matsumoto A, Takemori N, Takemori A, Shinoda K, Nakayama T, Ohara O, Yasukawa M, Sawasaki T, Kurosaki T, Yamashita M. Bach2-Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop. Nat Commun 2016; 7:12596. [PMID: 27581382 PMCID: PMC5025763 DOI: 10.1038/ncomms12596] [Citation(s) in RCA: 74] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2016] [Accepted: 07/08/2016] [Indexed: 12/17/2022] Open
Abstract
Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2–Batf complex antagonizes the recruitment of the Batf–Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2–Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf–Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2–Batf interactions are required to prevent an excessive Th2 response. Bach2 limits T cell effector functions. Here the authors show that Bach2–Batf complex antagonizes the recruitment of the Batf–Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production, and describe mechanisms of negative feedback by which Bach2 restricts Baft-mediated Th2 response.
Collapse
Affiliation(s)
- Makoto Kuwahara
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.,Department of Translational Immunology, Translational Research Center, Ehime University Hospital, Shitsukawa, Toon, Ehime 791-0295, Japan.,Division of Immune Regulation, Department of Proteo-Inovation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan
| | - Wataru Ise
- Department of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Mizuki Ochi
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.,Division of Cell-Free Sciences, Department of Proteo-Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan
| | - Junpei Suzuki
- Department of Translational Immunology, Translational Research Center, Ehime University Hospital, Shitsukawa, Toon, Ehime 791-0295, Japan.,Department of Hematology, Clinical Immunology and Infectious Diseases, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
| | - Kohei Kometani
- Laboratory for Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences, 1-7-22 suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
| | - Saho Maruyama
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
| | - Maya Izumoto
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
| | - Akira Matsumoto
- Department of Infection and Host Defenses, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
| | - Nobuaki Takemori
- Division of Proteomics, Department of Proteo-Medicine, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan
| | - Ayako Takemori
- Division of Proteomics, Department of Proteo-Medicine, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan
| | - Kenta Shinoda
- Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 Japan
| | - Toshinori Nakayama
- Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 Japan
| | - Osamu Ohara
- Human DNA Analysis Group, Department of Technology Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
| | - Masaki Yasukawa
- Department of Hematology, Clinical Immunology and Infectious Diseases, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
| | - Tatsuya Sawasaki
- Division of Cell-Free Sciences, Department of Proteo-Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan
| | - Tomohiro Kurosaki
- Department of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.,Laboratory for Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences, 1-7-22 suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
| | - Masakatsu Yamashita
- Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.,Department of Translational Immunology, Translational Research Center, Ehime University Hospital, Shitsukawa, Toon, Ehime 791-0295, Japan.,Division of Immune Regulation, Department of Proteo-Inovation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan
| |
Collapse
|
|
49
|
Sólyom Z, Ma P, Schwarten M, Bosco M, Polidori A, Durand G, Willbold D, Brutscher B. The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation. Biophys J 2016; 109:1483-96. [PMID: 26445449 DOI: 10.1016/j.bpj.2015.06.040] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2015] [Revised: 06/01/2015] [Accepted: 06/16/2015] [Indexed: 12/16/2022] Open
Abstract
Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.
Collapse
Affiliation(s)
- Zsófia Sólyom
- Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France
| | - Peixiang Ma
- Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France; Institute of Complex Systems-6 Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany
| | - Melanie Schwarten
- Institute of Complex Systems-6 Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany
| | - Michaël Bosco
- Institut des Biomolécules Max Mousseron, UMR 5247, Centre National de Recherche Scientifique, École Nationale Supérieure de Chimie de Montpellier, Université Montpellier, Montpellier, France; Equipe Chimie Bioorganique et Systèmes Amphiphiles, Avignon Université, Avignon, France
| | - Ange Polidori
- Institut des Biomolécules Max Mousseron, UMR 5247, Centre National de Recherche Scientifique, École Nationale Supérieure de Chimie de Montpellier, Université Montpellier, Montpellier, France; Equipe Chimie Bioorganique et Systèmes Amphiphiles, Avignon Université, Avignon, France
| | - Grégory Durand
- Institut des Biomolécules Max Mousseron, UMR 5247, Centre National de Recherche Scientifique, École Nationale Supérieure de Chimie de Montpellier, Université Montpellier, Montpellier, France; Equipe Chimie Bioorganique et Systèmes Amphiphiles, Avignon Université, Avignon, France
| | - Dieter Willbold
- Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Institute of Complex Systems-6 Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
| | - Bernhard Brutscher
- Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France.
| |
Collapse
|
|
50
|
Meyers NL, Fontaine KA, Kumar GR, Ott M. Entangled in a membranous web: ER and lipid droplet reorganization during hepatitis C virus infection. Curr Opin Cell Biol 2016; 41:117-24. [PMID: 27240021 DOI: 10.1016/j.ceb.2016.05.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Revised: 05/04/2016] [Accepted: 05/05/2016] [Indexed: 12/19/2022]
Abstract
Hepatitis C virus (HCV) is a major cause of liver disease worldwide. To establish and maintain chronic infection, HCV extensively rearranges cellular organelles to generate distinct compartments for viral RNA replication and virion assembly. Here, we review our current knowledge of how HCV proliferates and remodels ER-derived membranes while preserving and expanding associated lipid droplets during viral infection. Unraveling the molecular mechanisms responsible for HCV-induced membrane reorganization will enhance our understanding of the HCV life-cycle, the associated liver pathology, and the biology of the ER:lipid droplet interface in general.
Collapse
Affiliation(s)
- Nathan L Meyers
- Gladstone Institutes, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94941, United States
| | - Krystal A Fontaine
- Gladstone Institutes, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94941, United States
| | - G Renuka Kumar
- Gladstone Institutes, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94941, United States
| | - Melanie Ott
- Gladstone Institutes, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94941, United States.
| |
Collapse
|