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Yamauchi K, Maekawa S, Osawa L, Komiyama Y, Nakakuki N, Takada H, Muraoka M, Suzuki Y, Sato M, Takano S, Enomoto N. Single-molecule sequencing of the whole HCV genome revealed envelope deletions in decompensated cirrhosis associated with NS2 and NS5A mutations. J Gastroenterol 2024; 59:1021-1036. [PMID: 39225750 DOI: 10.1007/s00535-024-02146-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 08/16/2024] [Indexed: 09/04/2024]
Abstract
BACKGROUND Defective hepatitis C virus (HCV) genomes with deletion of the envelope region have been occasionally reported by short-read sequencing analyses. However, the clinical and virological details of such deletion HCV have not been fully elucidated. METHODS We developed a highly accurate single-molecule sequencing system for full-length HCV genes by combining the third-generation nanopore sequencing with rolling circle amplification (RCA) and investigated the characteristics of deletion HCV through the analysis of 21 patients chronically infected with genotype-1b HCV. RESULT In 5 of the 21 patients, a defective HCV genome with approximately 2000 bp deletion from the E1 to NS2 region was detected, with the read frequencies of 34-77%, suggesting the trans-complementation of the co-infecting complete HCV. Deletion HCV was found exclusively in decompensated cirrhosis (5/12 patients), and no deletion HCV was observed in nine compensated patients. Comparing the amino acid substitutions between the deletion and complete HCV (DAS, deletion-associated substitutions), the deletion HCV showed higher amino acid mutations in the ISDR (interferon sensitivity-determining region) in NS5A, and also in the TMS (transmembrane segment) 3 to H (helix) 2 region of NS2. CONCLUSIONS Defective HCV genome with deletion of envelope genes is associated with decompensated cirrhosis. The deletion HCV seems susceptible to innate immunity, such as endogenous interferon with NS5A mutations, escaping from acquired immunity with deletion of envelope proteins with potential modulation of replication capabilities with NS2 mutations. The relationship between these mutations and liver damage caused by HCV deletion is worth investigating.
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Affiliation(s)
- Kozue Yamauchi
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Shinya Maekawa
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
| | - Leona Osawa
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Yasuyuki Komiyama
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Natsuko Nakakuki
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Hitomi Takada
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Masaru Muraoka
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Yuichiro Suzuki
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Mitsuaki Sato
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Shinichi Takano
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Nobuyuki Enomoto
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
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2
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Goh L, Kerkar N. Hepatitis C Virus and Molecular Mimicry. Pathogens 2024; 13:527. [PMID: 39057754 PMCID: PMC11280050 DOI: 10.3390/pathogens13070527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 06/04/2024] [Accepted: 06/20/2024] [Indexed: 07/28/2024] Open
Abstract
This review delves into the interactions between hepatitis C virus (HCV) and the host immune system, shedding light on how by using the mechanism of molecular mimicry, the virus strategically evades the immune system, resulting in a cascade of diverse complications. HCV, notorious for its ability to persistently infect hepatocytes, employs molecular mimicry to resemble host proteins, thereby avoiding immune detection and mounting an effective defense. This mimicry also triggers systemic autoimmune responses that lead to various sequelae. The objective of this review is to comprehensively explore the role of HCV-induced molecular mimicry, which not only facilitates viral survival but is also instrumental in developing autoimmune and inflammatory disorders. By mimicking host proteins, HCV triggers an immune response that inadvertently attacks the host, fostering the development of autoimmune and other inflammatory disorders. Understanding the nuanced mechanisms of HCV-mediated molecular mimicry provides crucial insights into the multifaceted sequelae of viral infections on host immune responses. Unravelling these complexities is paramount for advancing therapeutic strategies that not only target the virus directly but also mitigate the secondary autoimmune and inflammatory complications induced by HCV.
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Affiliation(s)
- Lynette Goh
- KK Women’s and Children’s Hospital, Singapore 229899, Singapore
| | - Nanda Kerkar
- Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA;
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3
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Bunz M, Eisele M, Hu D, Ritter M, Kammerloher J, Lampl S, Schindler M. CD81 suppresses NF-κB signaling and is downregulated in hepatitis C virus expressing cells. Front Cell Infect Microbiol 2024; 14:1338606. [PMID: 38357447 PMCID: PMC10864554 DOI: 10.3389/fcimb.2024.1338606] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2023] [Accepted: 01/15/2024] [Indexed: 02/16/2024] Open
Abstract
The tetraspanin CD81 is one of the main entry receptors for Hepatitis C virus, which is a major causative agent to develop liver cirrhosis and hepatocellular carcinoma (HCC). Here, we identify CD81 as one of few surface proteins that are downregulated in HCV expressing hepatoma cells, discovering a functional role of CD81 beyond mediating HCV entry. CD81 was downregulated at the mRNA level in hepatoma cells that replicate HCV. Kinetics of HCV expression were increased in CD81-knockout cells and accompanied by enhanced cellular growth. Furthermore, loss of CD81 compensated for inhibition of pro-survival TBK1-signaling in HCV expressing cells. Analysis of functional phenotypes that could be associated with pro-survival signaling revealed that CD81 is a negative regulator of NF-κB. Interaction of the NF-κB subunits p50 and p65 was increased in cells lacking CD81. Similarly, we witnessed an overall increase in the total levels of phosphorylated and cellular p65 upon CD81-knockout in hepatoma cells. Finally, translocation of p65 in CD81-negative hepatoma cells was markedly induced upon stimulation with TNFα or PMA. Altogether, CD81 emerges as a regulator of pro-survival NF-κB signaling. Considering the important and established role of NF-κB for HCV replication and tumorigenesis, the downregulation of CD81 by HCV and the associated increase in NF-κB signaling might be relevant for viral persistence and chronic infection.
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Affiliation(s)
- Maximilian Bunz
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Mona Eisele
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Dan Hu
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Michael Ritter
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Julia Kammerloher
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
- Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany
| | - Sandra Lampl
- Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany
| | - Michael Schindler
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
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4
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Barik S. Suppression of Innate Immunity by the Hepatitis C Virus (HCV): Revisiting the Specificity of Host-Virus Interactive Pathways. Int J Mol Sci 2023; 24:16100. [PMID: 38003289 PMCID: PMC10671098 DOI: 10.3390/ijms242216100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2023] [Revised: 10/31/2023] [Accepted: 11/06/2023] [Indexed: 11/26/2023] Open
Abstract
The hepatitis C virus (HCV) is a major causative agent of hepatitis that may also lead to liver cancer and lymphomas. Chronic hepatitis C affects an estimated 2.4 million people in the USA alone. As the sole member of the genus Hepacivirus within the Flaviviridae family, HCV encodes a single-stranded positive-sense RNA genome that is translated into a single large polypeptide, which is then proteolytically processed to yield the individual viral proteins, all of which are necessary for optimal viral infection. However, cellular innate immunity, such as type-I interferon (IFN), promptly thwarts the replication of viruses and other pathogens, which forms the basis of the use of conjugated IFN-alpha in chronic hepatitis C management. As a countermeasure, HCV suppresses this form of immunity by enlisting diverse gene products, such as HCV protease(s), whose primary role is to process the large viral polyprotein into individual proteins of specific function. The exact number of HCV immune suppressors and the specificity and molecular mechanism of their action have remained unclear. Nonetheless, the evasion of host immunity promotes HCV pathogenesis, chronic infection, and carcinogenesis. Here, the known and putative HCV-encoded suppressors of innate immunity have been reviewed and analyzed, with a predominant emphasis on the molecular mechanisms. Clinically, the knowledge should aid in rational interventions and the management of HCV infection, particularly in chronic hepatitis.
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Affiliation(s)
- Sailen Barik
- EonBio, 3780 Pelham Drive, Mobile, AL 36619, USA
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5
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Malekshahi A, Alamdary A, Safarzadeh A, Khavandegar A, Nikoo HR, Safavi M, Ajorloo M, Bahavar A, Ajorloo M. Potential roles of core and core+1 proteins during the chronic phase of hepatitis C virus infection. Future Virol 2023. [DOI: 10.2217/fvl-2022-0117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/16/2023]
Abstract
The HCV Core protein is a multifunctional protein that interacts with many viral and cellular proteins. In addition to the encapsidation of the viral genome, it can disturb various cellular pathways and impede antiviral cellular responses such as interferon (IFN) production. The Core protein can also disrupt the functions of immune cells against HCV. The Core protein helps viral infection persistency by interfering with apoptosis. The Core+1 protein plays a significant role in inducing chronic HCV infection through diverse mechanisms. We review some of the mechanisms by which Core and Core+1 proteins facilitate HCV infection to chronic infection. These proteins could be considered for designing more sufficient treatments and effective vaccines against HCV.
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Affiliation(s)
- Asra Malekshahi
- Student Research Committee, Lorestan University of Medical Sciences, Khorramabad, Iran
| | - Ashkan Alamdary
- Department of Biology, Science & Research Branch, Islamic Azad University, Tehran, Iran
| | - Ali Safarzadeh
- Department of Biology, University of Padova, Padova, Italy
| | - Armin Khavandegar
- Student Research Committee, Alborz University of Medical Sciences, Karaj, Iran
| | - Hadi Razavi Nikoo
- Infectious Disease Research Center, Golestan University of Medical Sciences, Gorgan, Iran
- Department of Microbiology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
| | - Mahshid Safavi
- Student Research Committee, Alborz University of Medical Sciences, Karaj, Iran
| | - Mobina Ajorloo
- Student Research Committee, Alborz University of Medical Sciences, Karaj, Iran
| | - Atefeh Bahavar
- Department of Microbiology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
| | - Mehdi Ajorloo
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
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6
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Chen S, Harris M. NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles. PLoS Pathog 2023; 19:e1010812. [PMID: 36795772 PMCID: PMC9977016 DOI: 10.1371/journal.ppat.1010812] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Revised: 03/01/2023] [Accepted: 02/02/2023] [Indexed: 02/17/2023] Open
Abstract
Hepatitis C virus NS5A is a multifunctional phosphoprotein comprised of three domains (DI, DII and DIII). DI and DII have been shown to function in genome replication, whereas DIII has a role in virus assembly. We previously demonstrated that DI in genotype 2a (JFH1) also plays a role in virus assembly, exemplified by the P145A mutant which blocked infectious virus production. Here we extend this analysis to identify two other conserved and surface exposed residues proximal to P145 (C142 and E191) that exhibited no defect in genome replication but impaired virus production. Further analysis revealed changes in the abundance of dsRNA, the size and distribution of lipid droplets (LD) and the co-localisation between NS5A and LDs in cells infected with these mutants, compared to wildtype. In parallel, to investigate the mechanism(s) underpinning this role of DI, we assessed the involvement of the interferon-induced double-stranded RNA-dependent protein kinase (PKR). In PKR-silenced cells, C142A and E191A exhibited levels of infectious virus production, LD size and co-localisation between NS5A and LD that were indistinguishable from wildtype. Co-immunoprecipitation and in vitro pulldown experiments confirmed that wildtype NS5A domain I (but not C142A or E191A) interacted with PKR. We further showed that the assembly phenotype of C142A and E191A was restored by ablation of interferon regulatory factor-1 (IRF1), a downstream effector of PKR. These data suggest a novel interaction between NS5A DI and PKR that functions to evade an antiviral pathway that blocks virus assembly through IRF1.
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Affiliation(s)
- Shucheng Chen
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
- * E-mail:
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7
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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.
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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
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8
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Corbet GA, Burke JM, Bublitz GR, Tay JW, Parker R. dsRNA-induced condensation of antiviral proteins modulates PKR activity. Proc Natl Acad Sci U S A 2022; 119:e2204235119. [PMID: 35939694 PMCID: PMC9388085 DOI: 10.1073/pnas.2204235119] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 07/01/2022] [Indexed: 12/21/2022] Open
Abstract
Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.
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Affiliation(s)
- Giulia A. Corbet
- Department of Biochemistry, University of Colorado, Boulder, CO 80309
| | - James M. Burke
- Department of Biochemistry, University of Colorado, Boulder, CO 80309
| | - Gaia R. Bublitz
- Department of Biochemistry, University of Colorado, Boulder, CO 80309
| | | | - Roy Parker
- Department of Biochemistry, University of Colorado, Boulder, CO 80309
- BioFrontiers Institute, Boulder, CO 80309
- Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789
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9
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Han J, Kyu Lee M, Jang Y, Cho WJ, Kim M. Repurposing of cyclophilin A inhibitors as broad-spectrum antiviral agents. Drug Discov Today 2022; 27:1895-1912. [PMID: 35609743 PMCID: PMC9123807 DOI: 10.1016/j.drudis.2022.05.016] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2021] [Revised: 03/30/2022] [Accepted: 05/18/2022] [Indexed: 12/28/2022]
Abstract
Cyclophilin A (CypA) is linked to diverse human diseases including viral infections. With the worldwide emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2), drug repurposing has been highlighted as a strategy with the potential to speed up antiviral development. Because CypA acts as a proviral component in hepatitis C virus, coronavirus and HIV, its inhibitors have been suggested as potential treatments for these infections. Here, we review the structure of cyclosporin A and sanglifehrin A analogs as well as synthetic micromolecules inhibiting CypA; and we discuss their broad-spectrum antiviral efficacy in the context of the virus lifecycle.
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Affiliation(s)
- Jinhe Han
- College of Pharmacy, Chonnam National University, Gwangju, 61186, Republic of Korea
| | - Myoung Kyu Lee
- Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea
| | - Yejin Jang
- Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea
| | - Won-Jea Cho
- College of Pharmacy, Chonnam National University, Gwangju, 61186, Republic of Korea.
| | - Meeheyin Kim
- Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea; Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea.
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10
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Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview. Int J Mol Sci 2022; 23:ijms23105475. [PMID: 35628287 PMCID: PMC9141274 DOI: 10.3390/ijms23105475] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 05/11/2022] [Accepted: 05/13/2022] [Indexed: 02/05/2023] Open
Abstract
Hepatitis C virus (HCV) infection remains a major global health burden, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that detect pathogen-associated molecular patterns and activate downstream signaling to induce proinflammatory cytokine and chemokine production. An increasing number of studies have suggested the importance of TLR responses in the outcome of HCV infection. However, the exact role of innate immune responses, including TLR response, in controlling chronic HCV infection remains to be established. A proper understanding of the TLR response in HCV infection is essential for devising new therapeutic approaches against HCV infection. In this review, we discuss the progress made in our understanding of the host innate immune response to HCV infection, with a particular focus on the TLR response. In addition, we discuss the mechanisms adopted by HCV to avoid immune surveillance mediated by TLRs.
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11
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Davidson S, Yu CH, Steiner A, Ebstein F, Baker PJ, Jarur-Chamy V, Hrovat Schaale K, Laohamonthonkul P, Kong K, Calleja DJ, Harapas CR, Balka KR, Mitchell J, Jackson JT, Geoghegan ND, Moghaddas F, Rogers KL, Mayer-Barber KD, De Jesus AA, De Nardo D, Kile BT, Sadler AJ, Poli MC, Krüger E, Goldbach Mansky R, Masters SL. Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24. Sci Immunol 2022; 7:eabi6763. [PMID: 35148201 PMCID: PMC11036408 DOI: 10.1126/sciimmunol.abi6763] [Citation(s) in RCA: 42] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor-induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum-associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.
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Affiliation(s)
- Sophia Davidson
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Chien-Hsiung Yu
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Annemarie Steiner
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
- Institute of Structural Biology, University Hospital Bonn, Bonn 53127, Germany
| | - Frédéric Ebstein
- University Medicine Greifswald, Institute of Medical Biochemistry and Molecular Biology, Greifswald 17475, Germany
| | - Paul J. Baker
- Inflammation and Innate Immunity Unit, Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA
| | - Valentina Jarur-Chamy
- Immunogenetics and Translational Immunology Program. Facultad de Medicina, Universidad del Desarrollo Clínica Alemana, Santiago, Chile
| | - Katja Hrovat Schaale
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Pawat Laohamonthonkul
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Klara Kong
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Dale J. Calleja
- Ubiquitin Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Cassandra R. Harapas
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Katherine R. Balka
- Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - Jacob Mitchell
- Translational Autoinflammatory Disease Studies (TADS), Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA
| | - Jacob T. Jackson
- Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Niall D. Geoghegan
- Centre for Dynamic Imaging, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Fiona Moghaddas
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Kelly L. Rogers
- Centre for Dynamic Imaging, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
| | - Katrin D. Mayer-Barber
- Inflammation and Innate Immunity Unit, Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA
| | - Adriana A. De Jesus
- Translational Autoinflammatory Disease Studies (TADS), Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA
| | - Dominic De Nardo
- Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - Benjamin T. Kile
- Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
- Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia 5000, Australia
| | - Anthony J. Sadler
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
- Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia
| | - M. Cecilia Poli
- Immunogenetics and Translational Immunology Program. Facultad de Medicina, Universidad del Desarrollo Clínica Alemana, Santiago, Chile
- Division of Pediatric Immunology, Allergy, and Rheumatology, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Elke Krüger
- University Medicine Greifswald, Institute of Medical Biochemistry and Molecular Biology, Greifswald 17475, Germany
| | - Raphaela Goldbach Mansky
- Translational Autoinflammatory Disease Studies (TADS), Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA
| | - Seth L. Masters
- Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
- Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
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12
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Lee J, Ou JHJ. Hepatitis C virus and intracellular antiviral response. Curr Opin Virol 2022; 52:244-249. [PMID: 34973476 PMCID: PMC8844188 DOI: 10.1016/j.coviro.2021.12.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Accepted: 12/09/2021] [Indexed: 02/03/2023]
Abstract
To establish successful infection in cells, it is essential for hepatitis C virus (HCV) to overcome intracellular antiviral responses. The host cell mechanism that fights against the virus culminates in the production of interferons (IFNs), IFN-stimulated genes (ISGs) and pro-inflammatory cytokines as well as the induction of autophagy and apoptosis. HCV has developed multiple means to disrupt the host signaling pathways that lead to these antiviral responses. HCV impedes signaling pathways initiated by pattern-recognition receptors (PRRs), usurps and uses the antiviral autophagic response to enhance its replication, alters mitochondrial dynamics and metabolism to prevent cell death and attenuate IFN response, and dysregulates inflammasomal response to cause IFN resistance and immune tolerance. These effects of HCV allow HCV to successful replicate and persist in its host cells.
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13
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Inflammation and Liver Cell Death in Patients with Hepatitis C Viral Infection. Curr Issues Mol Biol 2021; 43:2022-2035. [PMID: 34889885 PMCID: PMC8929145 DOI: 10.3390/cimb43030139] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 11/05/2021] [Accepted: 11/11/2021] [Indexed: 01/03/2023] Open
Abstract
Hepatitis C virus (HCV)-induced liver disease contributes to chronic hepatitis. The immune factors identified in HCV include changes in the innate and adaptive immune system. The inflammatory mediators, known as "inflammasome", are a consequence of the metabolic products of cells and commensal or pathogenic bacteria and viruses. The only effective strategy to prevent disease progression is eradication of the viral infection. Immune cells play a pivotal role during liver inflammation, triggering fibrogenesis. The present paper discusses the potential role of markers in cell death and the inflammatory cascade leading to the severity of liver damage. We aim to present the clinical parameters and laboratory data in a cohort of 88 HCV-infected non-cirrhotic and 25 HCV cirrhotic patients, to determine the characteristic light microscopic (LM) and transmission electron microscopic (TEM) changes in their liver biopsies and to present the link between the severity of liver damage and the serum levels of cytokines and caspases. A matched HCV non-infected cohort was used for the comparison of serum inflammatory markers. We compared the inflammation in HCV individuals with a control group of 280 healthy individuals. We correlated the changes in inflammatory markers in different stages of the disease and the histology. We concluded that the serum levels of cytokine, chemokine, and cleaved caspase markers reveal the inflammatory status in HCV. Based upon the information provided by the changes in biomarkers the clinician can monitor the severity of HCV-induced liver damage. New oral well-tolerated treatment regimens for chronic hepatitis C patients can achieve cure rates of over 90%. Therefore, using the noninvasive biomarkers to monitor the evolution of the liver damage is an effective personalized medicine procedure to establish the severity of liver injury and its repair.
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14
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Abstract
Cells respond to viral infections through sensors that detect non-self-molecules, and through effectors, which can have direct antiviral activities or adapt cell physiology to limit viral infection and propagation. Eukaryotic translation initiation factor 2 alpha kinase 2, better known as PKR, acts as both a sensor and an effector in the response to viral infections. After sensing double-stranded RNA molecules in infected cells, PKR self-activates and majorly exerts its antiviral function by blocking the translation machinery and inducing apoptosis. The antiviral potency of PKR is emphasized by the number of strategies developed by viruses to antagonize the PKR pathway. In this review, we present an update on the diversity of such strategies, which range from preventing double-stranded RNA recognition upstream from PKR activation, to activating eIF2B downstream from PKR targets.
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Affiliation(s)
- Teresa Cesaro
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | - Thomas Michiels
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
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15
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Abstract
Fibrosis is not a unidirectional, linear process, but a dynamic one resulting from an interplay of fibrogenesis and fibrolysis depending on the extent and severity of a biologic insult, or lack thereof. Regression of fibrosis has been documented best in patients treated with phlebotomies for hemochromatosis, and after successful suppression and eradication of chronic hepatitis B and C infections. This evidence mandates a reconsideration of the term "cirrhosis," which implies an inevitable progression towards liver failure. Furthermore, it also necessitates a staging system that acknowledges the bidirectional nature of evolution of fibrosis, and has the ability to predict if the disease process is progressing or regressing. The Beijing classification attempts to fill this gap in contemporary practice. It is based on microscopic features termed "the hepatic repair complex," defined originally by Wanless and colleagues. The elements of the hepatic repair complex represent the 3 processes of fragmentation and regression of scar, vascular remodeling (resolution), and parenchymal regeneration. However, regression of fibrosis does not imply resolution of cirrhosis, which is more than just a stage of fibrosis. So far, there is little to no evidence to suggest that large regions of parenchymal extinction can be repopulated by regenerating hepatocytes. Similarly, the vascular lesions of cirrhosis persist, and there is no evidence of complete return to normal microcirculation in cirrhotic livers. In addition, the risk of hepatocellular carcinoma is higher compared with the general population and these patients need continued screening and surveillance.
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16
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Virus-Induced Tumorigenesis and IFN System. BIOLOGY 2021; 10:biology10100994. [PMID: 34681093 PMCID: PMC8533565 DOI: 10.3390/biology10100994] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 09/01/2021] [Accepted: 09/27/2021] [Indexed: 01/11/2023]
Abstract
Oncogenic viruses favor the development of tumors in mammals by persistent infection and specific cellular pathways modifications by deregulating cell proliferation and inhibiting apoptosis. They counteract the cellular antiviral defense through viral proteins as well as specific cellular effectors involved in virus-induced tumorigenesis. Type I interferons (IFNs) are a family of cytokines critical not only for viral interference but also for their broad range of properties that go beyond the antiviral action. In fact, they can inhibit cell proliferation and modulate differentiation, apoptosis, and migration. However, their principal role is to regulate the development and activity of most effector cells of the innate and adaptive immune responses. Various are the mechanisms by which IFNs exert their effects on immune cells. They can act directly, through IFN receptor triggering, or indirectly by the induction of chemokines, the secretion of further cytokines, or by the stimulation of cells useful for the activation of particular immune cells. All the properties of IFNs are crucial in the host defense against viruses and bacteria, as well as in the immune surveillance against tumors. IFNs may be affected by and, in turn, affect signaling pathways to mediate anti-proliferative and antiviral responses in virus-induced tumorigenic context. New data on cellular and viral microRNAs (miRNAs) machinery, as well as cellular communication and microenvironment modification via classical secretion mechanisms and extracellular vesicles-mediated delivery are reported. Recent research is reviewed on the tumorigenesis induced by specific viruses with RNA or DNA genome, belonging to different families (i.e., HPV, HTLV-1, MCPyV, JCPyV, Herpesviruses, HBV, HCV) and the IFN system involvement.
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17
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Rehman AU, Zhen G, Zhong B, Ni D, Li J, Nasir A, Gabr MT, Rafiq H, Wadood A, Lu S, Zhang J, Chen HF. Mechanism of zinc ejection by disulfiram in nonstructural protein 5A. Phys Chem Chem Phys 2021; 23:12204-12215. [PMID: 34008604 DOI: 10.1039/d0cp06360f] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.
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Affiliation(s)
- Ashfaq Ur Rehman
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China. and State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China and Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Guodong Zhen
- Department of VIP Clinic, Changhai Hospital, Navy Military Medical University, Shanghai, 200433, China
| | - Bozitao Zhong
- State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Duan Ni
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Jiayi Li
- State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Abdul Nasir
- Synthetic Protein Engineering Lab, Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea
| | - Moustafa T Gabr
- Department of Radiology, Stanford University, Stanford, California 94305, USA
| | - Humaira Rafiq
- Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Abdul Wadood
- Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Shaoyong Lu
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Jian Zhang
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Hai-Feng Chen
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China. and Shanghai Center for Bioinformation Technology, Shanghai, 200235, China
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18
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Neves WLL, Mariuba LAM, Alves KCS, Coelho KF, Tarragô AM, Costa AG, Chaves YO, Victoria FDS, Victoria MB, Malheiro A. Development of an immunoassay for the detection of human IgG against hepatitis C virus proteins using magnetic beads and flow cytometry. BIOTECHNOL BIOTEC EQ 2020. [DOI: 10.1080/13102818.2020.1839355] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Affiliation(s)
- Walter Luiz Lima Neves
- Post-graduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Post-graduate Program in Biotechnology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
| | - Luis André Morais Mariuba
- Post-graduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Post-graduate Program in Biotechnology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Leonidas & Maria Deane Research Institute, FIOCRUZ-Amazônia, Manaus, AM, Brazil
- Postgraduate Program Stricto sensu in Cellular and Molecular Biology of the Oswaldo Cruz Institute (PGBCM/IOC/Fiocruz), Rio de Janeiro, Brazil
| | - Késsia Caroline Souza Alves
- Post-graduate Program in Biotechnology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
| | - Kerolaine Fonseca Coelho
- Department of Education and Research, Amazonas Hospital Foundation of Hematology and Hemotherapy (HEMOAM), Manaus, AM, Brazil
- Post-graduate Program in Tropical Medicine, State University of Amazonas (UEA), Manaus, AM, Brazil
| | - Andrea Monteiro Tarragô
- Post-graduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Department of Education and Research, Amazonas Hospital Foundation of Hematology and Hemotherapy (HEMOAM), Manaus, AM, Brazil
- Post-graduate Program in Tropical Medicine, State University of Amazonas (UEA), Manaus, AM, Brazil
| | - Allyson Guimarães Costa
- Post-graduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Department of Education and Research, Amazonas Hospital Foundation of Hematology and Hemotherapy (HEMOAM), Manaus, AM, Brazil
- Post-graduate Program in Tropical Medicine, State University of Amazonas (UEA), Manaus, AM, Brazil
- Post-graduate Program in Hematology Sciences, State University of Amazonas (UEA), Manaus, AM, Brazil
- Carlos Borborema Clinical Research Institute, Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), Manaus, AM, Brazil
| | - Yury Oliveira Chaves
- Post-graduate Program in Hematology Sciences, State University of Amazonas (UEA), Manaus, AM, Brazil
| | - Flamir da Silva Victoria
- Post-graduate Program in Hematology Sciences, State University of Amazonas (UEA), Manaus, AM, Brazil
- Carlos Borborema Clinical Research Institute, Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), Manaus, AM, Brazil
| | - Marilu Barbieri Victoria
- Post-graduate Program in Hematology Sciences, State University of Amazonas (UEA), Manaus, AM, Brazil
- Carlos Borborema Clinical Research Institute, Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), Manaus, AM, Brazil
| | - Adriana Malheiro
- Post-graduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Post-graduate Program in Biotechnology, Institute of Biological Sciences, Federal University of Amazonas (UFAM), Manaus, AM, Brazil
- Post-graduate Program in Tropical Medicine, State University of Amazonas (UEA), Manaus, AM, Brazil
- Post-graduate Program in Hematology Sciences, State University of Amazonas (UEA), Manaus, AM, Brazil
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19
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Carpentier A, Sheldon J, Vondran FWR, Brown RJ, Pietschmann T. Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus. Gut 2020; 69:1659-1666. [PMID: 32114504 PMCID: PMC7456736 DOI: 10.1136/gutjnl-2019-319354] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Revised: 02/07/2020] [Accepted: 02/10/2020] [Indexed: 12/12/2022]
Abstract
OBJECTIVE AND DESIGN Human stem cell-derived hepatocyte-like cells (HLCs) have shown high potential as authentic model for dissection of the HCV life cycle and virus-induced pathogenesis. However, modest HCV replication, possibly due to robust innate immune responses, limits their broader use. To overcome these limitations and to dissect the mechanisms responsible for control of HCV, we analysed expression of key components of the interferon (IFN) system in HLCs, assessed permissiveness for different HCV strains and blocked innate immune signalling by pharmacological intervention. RESULTS Transcriptional profiling revealed that HLCs constitutively express messenger RNA of RLRs, and members of the IFN pathway. Moreover, HLCs upregulated IFNs and canonical interferon-regulated genes (IRGs) upon transfection with the double-stranded RNA mimic poly(I:C). Infection of HLCs with Jc1-HCVcc produced only limited viral progeny. In contrast, infection with p100, a Jc1-derived virus population with enhanced replication fitness and partial resistance to IFN, resulted in robust yet transient viraemia. Viral titres declined concomitant with a peak of IRG induction. Addition of ruxolitinib, a JAK/STAT inhibitor, permitted chronic infection and raised p100 infectious virus titres to 1×105 FFU/mL. IRGs expression profiling in infected HLCs revealed a landscape of HCV-dependent transcriptional changes similar to HCV-infected primary human hepatocytes, but distinct from Huh-7.5 cells. Withdrawal of ruxolitinib restored innate immune responses and resulted in HCV clearance. CONCLUSION This authentic human cell model is well suited to examine acute and chronic host-HCV interactions, particularly IFN-triggered antiviral effector functions and mechanisms of innate immune control of HCV infection.
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Affiliation(s)
- Arnaud Carpentier
- Institute of Experimental Virology, Twincore, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Julie Sheldon
- Institute of Experimental Virology, Twincore, Hannover, Germany
| | - Florian W R Vondran
- ReMediES, Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
| | - Richard Jp Brown
- Institute of Experimental Virology, Twincore, Hannover, Germany
- Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Thomas Pietschmann
- Institute of Experimental Virology, Twincore, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
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20
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Liu Y, Wang M, Cheng A, Yang Q, Wu Y, Jia R, Liu M, Zhu D, Chen S, Zhang S, Zhao XX, Huang J, Mao S, Ou X, Gao Q, Wang Y, Xu Z, Chen Z, Zhu L, Luo Q, Liu Y, Yu Y, Zhang L, Tian B, Pan L, Rehman MU, Chen X. The role of host eIF2α in viral infection. Virol J 2020; 17:112. [PMID: 32703221 PMCID: PMC7376328 DOI: 10.1186/s12985-020-01362-6] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Accepted: 06/23/2020] [Indexed: 12/24/2022] Open
Abstract
Background eIF2α is a regulatory node that controls protein synthesis initiation by its phosphorylation or dephosphorylation. General control nonderepressible-2 (GCN2), protein kinase R-like endoplasmic reticulum kinase (PERK), double-stranded RNA (dsRNA)-dependent protein kinase (PKR) and heme-regulated inhibitor (HRI) are four kinases that regulate eIF2α phosphorylation. Main body In the viral infection process, dsRNA or viral proteins produced by viral proliferation activate different eIF2α kinases, resulting in eIF2α phosphorylation, which hinders ternary tRNAMet-GTP-eIF2 complex formation and inhibits host or viral protein synthesis. The stalled messenger ribonucleoprotein (mRNP) complex aggregates under viral infection stress to form stress granules (SGs), which encapsulate viral RNA and transcription- and translation-related proteins, thereby limiting virus proliferation. However, many viruses have evolved a corresponding escape mechanism to synthesize their own proteins in the event of host protein synthesis shutdown and SG formation caused by eIF2α phosphorylation, and viruses can block the cell replication cycle through the PERK-eIF2α pathway, providing a favorable environment for their own replication. Subsequently, viruses can induce host cell autophagy or apoptosis through the eIF2α-ATF4-CHOP pathway. Conclusions This review summarizes the role of eIF2α in viral infection to provide a reference for studying the interactions between viruses and hosts.
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Affiliation(s)
- Yuanzhi Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China. .,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.
| | - Qiao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Ying Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Renyong Jia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Mafeng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Dekang Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Shaqiu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Xin-Xin Zhao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Juan Huang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Sai Mao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Xumin Ou
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Qun Gao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Yin Wang
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Zhiwen Xu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Zhengli Chen
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Ling Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Qihui Luo
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Yunya Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Yanling Yu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Ling Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Bin Tian
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Leichang Pan
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Mujeeb Ur Rehman
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
| | - Xiaoyue Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China
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21
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Colpitts CC, Ridewood S, Schneiderman B, Warne J, Tabata K, Ng CF, Bartenschlager R, Selwood DL, Towers GJ. Hepatitis C virus exploits cyclophilin A to evade PKR. eLife 2020; 9:e52237. [PMID: 32539931 PMCID: PMC7297535 DOI: 10.7554/elife.52237] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Accepted: 05/28/2020] [Indexed: 12/12/2022] Open
Abstract
Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.
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Affiliation(s)
- Che C Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s UniversityKingstonCanada
- Division of Infection and Immunity, University College LondonLondonUnited Kingdom
| | - Sophie Ridewood
- Division of Infection and Immunity, University College LondonLondonUnited Kingdom
| | - Bethany Schneiderman
- Division of Infection and Immunity, University College LondonLondonUnited Kingdom
| | - Justin Warne
- Wolfson Institute for Biomedical Research, UCLLondonUnited Kingdom
| | - Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg UniversityHeidelbergGermany
| | - Caitlin F Ng
- Division of Infection and Immunity, University College LondonLondonUnited Kingdom
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg UniversityHeidelbergGermany
- Division Virus-Associated Carcinogenesis, German Cancer Research CenterHeidelbergGermany
- German Center for Infection Research (DZIF), Heidelberg Partner SiteHeidelbergGermany
| | - David L Selwood
- Department of Medicine, Imperial College LondonLondonUnited Kingdom
| | - Greg J Towers
- Division of Infection and Immunity, University College LondonLondonUnited Kingdom
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22
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Goto K, Roca Suarez AA, Wrensch F, Baumert TF, Lupberger J. Hepatitis C Virus and Hepatocellular Carcinoma: When the Host Loses Its Grip. Int J Mol Sci 2020; 21:ijms21093057. [PMID: 32357520 PMCID: PMC7246584 DOI: 10.3390/ijms21093057] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 04/20/2020] [Accepted: 04/24/2020] [Indexed: 02/06/2023] Open
Abstract
Chronic infection with hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC). Novel treatments with direct-acting antivirals achieve high rates of sustained virologic response; however, the HCC risk remains elevated in cured patients, especially those with advanced liver disease. Long-term HCV infection causes a persistent and accumulating damage of the liver due to a combination of direct and indirect pro-oncogenic mechanisms. This review describes the processes involved in virus-induced disease progression by viral proteins, derailed signaling, immunity, and persistent epigenetic deregulation, which may be instrumental to develop urgently needed prognostic biomarkers and as targets for novel chemopreventive therapies.
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Affiliation(s)
- Kaku Goto
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Armando Andres Roca Suarez
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Florian Wrensch
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Thomas F. Baumert
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
- Pôle Hépato-digestif, Institut Hopitalo-Universitaire, F-67000 Strasbourg, France
- Institut Universitaire de France, F-75231 Paris, France
- Correspondence: (T.F.B.); (J.L.); Tel.: +33-3-68-85-37-03 (T.F.B. & J.L.); Fax: +33-3-68-85-37-24 (T.F.B. & J.L.)
| | - Joachim Lupberger
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
- Correspondence: (T.F.B.); (J.L.); Tel.: +33-3-68-85-37-03 (T.F.B. & J.L.); Fax: +33-3-68-85-37-24 (T.F.B. & J.L.)
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23
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Ferreira AR, Ramos B, Nunes A, Ribeiro D. Hepatitis C Virus: Evading the Intracellular Innate Immunity. J Clin Med 2020; 9:jcm9030790. [PMID: 32183176 PMCID: PMC7141330 DOI: 10.3390/jcm9030790] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2020] [Revised: 03/02/2020] [Accepted: 03/04/2020] [Indexed: 12/25/2022] Open
Abstract
Hepatitis C virus (HCV) infections constitute a major public health problem and are the main cause of chronic hepatitis and liver disease worldwide. The existing drugs, while effective, are expensive and associated with undesirable secondary effects. There is, hence, an urgent need to develop novel therapeutics, as well as an effective vaccine to prevent HCV infection. Understanding the interplay between HCV and the host cells will certainly contribute to better comprehend disease progression and may unravel possible new cellular targets for the development of novel antiviral therapeutics. Here, we review and discuss the interplay between HCV and the host cell innate immunity. We focus on the different cellular pathways that respond to, and counteract, HCV infection and highlight the evasion strategies developed by the virus to escape this intracellular response.
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Affiliation(s)
| | | | | | - Daniela Ribeiro
- Correspondence: ; Tel.: +351-234-247-014; Fax: +351-234-372-587
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24
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Wyles D, Mangia A, Cheng W, Shafran S, Schwabe C, Ouyang W, Hedskog C, McNally J, Brainard DM, Doehle BP, Svarovskaia E, Miller MD, Mo H, Dvory-Sobol H. Long-term persistence of HCV NS5A resistance-associated substitutions after treatment with the HCV NS5A inhibitor, ledipasvir, without sofosbuvir. Antivir Ther 2019. [PMID: 28650844 DOI: 10.3851/imp3181] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Data on persistence of NS5A resistance-associated substitutions (RASs) may have implications for resistance testing approaches and selection of initial and retreatment strategies. METHODS Long-term persistence of NS5A RASs in HCV genotype (GT) 1 infected subjects (n=76) who did not achieve sustained virological response after receiving ledipasvir (LDV) without sofosbuvir (SOF) and were subsequently enrolled in an ongoing 3-year follow-up registry study was investigated by population or deep sequencing. RESULTS Of the 76 subjects enrolled, 67 and 9 subjects had GT1a and GT1b infection, respectively. At pretreatment, NS5A RASs were detected in 14% of subjects (11/76) by population sequencing, with three subjects having >1 RAS. All RASs that were detected at pretreatment persisted and were observed at the 96 week visit in the follow-up study (FU96). For the remaining subjects with no detectable RASs at pretreatment, RASs were detected in 98% (63/64) of subjects at virological failure in the parent study and persisted at detectable levels through FU96 in 86% of subjects by deep sequencing (1% cutoff). However, a decline in the quasispecies frequency of most RASs and the number of RASs per subject was observed over time. Phenotypic analysis demonstrated that the majority of NS5A RASs confer similar levels of resistance to LDV and daclatasvir. CONCLUSIONS The majority of NS5A RASs can persist at detectable levels for >96 weeks post-treatment in subjects who failed treatment with regimens containing an NS5A inhibitor without SOF, suggesting relatively high fitness of NS5A RASs even in the absence of drug pressure.
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Affiliation(s)
- David Wyles
- Division of Infectious Diseases, Denver Health and Hospital Authority, Denver, CO, USA
| | - Alessandra Mangia
- Liver Unit, Department of Medical Sciences IRCCS 'Casa Sollievo della Sofferenza', San Giovanni Rotondo, Italy
| | - Wendy Cheng
- Gastroenterology and Hepatology, Royal Perth Hospital, Perth, Western Australia, Australia
| | - Stephen Shafran
- Department of Medicine, University of Alberta, Edmonton, AB, Canada
| | | | - Wen Ouyang
- Gilead Sciences, Inc., Foster City, CA, USA
| | | | | | | | | | | | | | - Hongmei Mo
- Gilead Sciences, Inc., Foster City, CA, USA
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25
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The Leader Protein of Theiler's Virus Prevents the Activation of PKR. J Virol 2019; 93:JVI.01010-19. [PMID: 31292248 DOI: 10.1128/jvi.01010-19] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2019] [Accepted: 07/02/2019] [Indexed: 11/20/2022] Open
Abstract
Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler's murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA.IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler's virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.
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26
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Amador-Cañizares Y, Bernier A, Wilson JA, Sagan SM. miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5' end from the cellular pyrophosphatases, DOM3Z and DUSP11. Nucleic Acids Res 2019; 46:5139-5158. [PMID: 29672716 PMCID: PMC6007490 DOI: 10.1093/nar/gky273] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Accepted: 04/05/2018] [Indexed: 12/21/2022] Open
Abstract
Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to the 5′ end of its genome. This interaction promotes viral RNA accumulation, but the precise mechanism(s) remain incompletely understood. Previous studies suggest that miR-122 is able to protect the HCV genome from 5′ exonucleases (Xrn1/2), but this protection is not sufficient to account for the effect of miR-122 on HCV RNA accumulation. Thus, we investigated whether miR-122 was also able to protect the viral genome from innate sensors of RNA or cellular pyrophosphatases. We found that miR-122 does not play a protective role against recognition by PKR, RIG-I-like receptors, or IFITs 1 and 5. However, we found that knockdown of both the cellular pyrophosphatases, DOM3Z and DUSP11, was able to rescue viral RNA accumulation of subgenomic replicons in the absence of miR-122. Nevertheless, pyrophosphatase knockdown increased but did not restore viral RNA accumulation of full-length HCV RNA in miR-122 knockout cells, suggesting that miR-122 likely plays an additional role(s) in the HCV life cycle, beyond 5′ end protection. Overall, our results support a model in which miR-122 stabilizes the HCV genome by shielding its 5′ terminus from cellular pyrophosphatase activity and subsequent turnover by exonucleases (Xrn1/2).
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Affiliation(s)
| | - Annie Bernier
- Department of Microbiology & Immunology, McGill University, Montréal, QC, Canada
| | - Joyce A Wilson
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK, Canada
| | - Selena M Sagan
- Department of Microbiology & Immunology, McGill University, Montréal, QC, Canada.,Department of Biochemistry, McGill University, Montréal, QC, Canada
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27
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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.
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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
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28
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Bou-Nader C, Gordon JM, Henderson FE, Zhang J. The search for a PKR code-differential regulation of protein kinase R activity by diverse RNA and protein regulators. RNA (NEW YORK, N.Y.) 2019; 25:539-556. [PMID: 30770398 PMCID: PMC6467004 DOI: 10.1261/rna.070169.118] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/06/2023]
Abstract
The interferon-inducible protein kinase R (PKR) is a key component of host innate immunity that restricts viral replication and propagation. As one of the four eIF2α kinases that sense diverse stresses and direct the integrated stress response (ISR) crucial for cell survival and proliferation, PKR's versatile roles extend well beyond antiviral defense. Targeted by numerous host and viral regulators made of RNA and proteins, PKR is subject to multiple layers of endogenous control and external manipulation, driving its rapid evolution. These versatile regulators include not only the canonical double-stranded RNA (dsRNA) that activates the kinase activity of PKR, but also highly structured viral, host, and artificial RNAs that exert a full spectrum of effects. In this review, we discuss our deepening understanding of the allosteric mechanism that connects the regulatory and effector domains of PKR, with an emphasis on diverse structured RNA regulators in comparison to their protein counterparts. Through this analysis, we conclude that much of the mechanistic details that underlie this RNA-regulated kinase await structural and functional elucidation, upon which we can then describe a "PKR code," a set of structural and chemical features of RNA that are both descriptive and predictive for their effects on PKR.
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Affiliation(s)
- Charles Bou-Nader
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA
| | - Jackson M Gordon
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA
| | - Frances E Henderson
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA
| | - Jinwei Zhang
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA
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29
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He B, Tran JT, Sanchez DJ. Manipulation of Type I Interferon Signaling by HIV and AIDS-Associated Viruses. J Immunol Res 2019; 2019:8685312. [PMID: 31089479 PMCID: PMC6476103 DOI: 10.1155/2019/8685312] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Accepted: 02/14/2019] [Indexed: 12/20/2022] Open
Abstract
Type I Interferons were first described for their profound antiviral abilities in cell culture and animal models, and later, they were translated into potent antiviral therapeutics. However, as additional studies into the function of Type I Interferons progressed, it was also seen that pathogenic viruses have coevolved to encode potent mechanisms allowing them to evade or suppress the impact of Type I Interferons on their replication. For chronic viral infections, such as HIV and many of the AIDS-associated viruses, including HTLV, HCV, KSHV, and EBV, the clinical efficacy of Type I Interferons is limited by these mechanisms. Here, we review some of the ways that HIV and AIDS-associated viruses thrive in Type I Interferon-rich environments via mechanisms that block the function of this important antiviral cytokine. Overall, a better understanding of these mechanisms creates avenues to better understand the innate immune response to these viruses as well as plan the development of antivirals that would allow the natural antiviral effect of Type I Interferons to manifest during these infections.
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Affiliation(s)
- Buyuan He
- Pharmaceutical Sciences Department, College of Pharmacy, Western University of Health Sciences, Pomona 91766, California, USA
| | - James T. Tran
- Pharmaceutical Sciences Department, College of Pharmacy, Western University of Health Sciences, Pomona 91766, California, USA
| | - David Jesse Sanchez
- Pharmaceutical Sciences Department, College of Pharmacy, Western University of Health Sciences, Pomona 91766, California, USA
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30
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Jassey A, Liu CH, Changou CA, Richardson CD, Hsu HY, Lin LT. Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy. Cells 2019; 8:cells8040290. [PMID: 30934919 PMCID: PMC6523690 DOI: 10.3390/cells8040290] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2018] [Revised: 03/12/2019] [Accepted: 03/26/2019] [Indexed: 12/13/2022] Open
Abstract
Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.
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Affiliation(s)
- Alagie Jassey
- International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
| | - Ching-Hsuan Liu
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
- Department of Microbiology & Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
| | - Chun A Changou
- Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
- Integrated Laboratory, Center of Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan.
- Core Facility, Taipei Medical University, Taipei 11031, Taiwan.
| | - Christopher D Richardson
- Department of Microbiology & Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
- Department of Pediatrics and Canadian Center for Vaccinology, Izaak Walton Killam Health Centre, Halifax, NS B3H 4R2, Canada.
| | - Hsue-Yin Hsu
- Department of Life Sciences, Tzu-Chi University, Hualien 970, Taiwan.
| | - Liang-Tzung Lin
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
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31
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Suzuki R, Matsuda M, Shimoike T, Watashi K, Aizaki H, Kato T, Suzuki T, Muramatsu M, Wakita T. Activation of protein kinase R by hepatitis C virus RNA-dependent RNA polymerase. Virology 2019; 529:226-233. [PMID: 30738360 DOI: 10.1016/j.virol.2019.01.024] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Revised: 01/28/2019] [Accepted: 01/29/2019] [Indexed: 12/12/2022]
Abstract
Hepatitis C virus (HCV) was shown to activate protein kinase R (PKR), which inhibits expression of interferon (IFN) and IFN-stimulated genes by controlling the translation of newly transcribed mRNAs. However, it is unknown exactly how HCV activates PKR. To address the molecular mechanism(s) of PKR activation mediated by HCV infection, we examined the effects of viral proteins on PKR activation. Here, we show that expression of HCV NS5B strongly induced PKR and eIF2α phosphorylation, and attenuated MHC class I expression. In contrast, expression of Japanese encephalitis virus RNA-dependent RNA polymerase did not induce phosphorylation of PKR. Co-immunoprecipitation analyses showed that HCV NS5B interacted with PKR. Furthermore, expression of NS5B with polymerase activity-deficient mutation failed to phosphorylate PKR, suggesting that RNA polymerase activity is required for PKR activation. These results suggest that HCV activates PKR by association with NS5B, resulting in translational suppression of MHC class I to establish chronic infection.
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Affiliation(s)
- Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama-shi, Tokyo 208-0011, Japan.
| | - Mami Matsuda
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
| | - Takashi Shimoike
- Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama-shi, Tokyo 208-0011, Japan.
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
| | - Tetsuro Suzuki
- Department of Infectious Diseases, Hamamatsu University School of Medicine, Shizuoka, Japan.
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
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Refolo G, Ciccosanti F, Di Rienzo M, Basulto Perdomo A, Romani M, Alonzi T, Tripodi M, Ippolito G, Piacentini M, Fimia GM. Negative Regulation of Mitochondrial Antiviral Signaling Protein-Mediated Antiviral Signaling by the Mitochondrial Protein LRPPRC During Hepatitis C Virus Infection. Hepatology 2019; 69:34-50. [PMID: 30070380 DOI: 10.1002/hep.30149] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 06/18/2018] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus (HCV) is highly efficient in establishing a chronic infection, having evolved multiple strategies to suppress the host antiviral responses. The HCV nonstructural 5A (NS5A) protein, in addition to its role in viral replication and assembly, has long been known to hamper the interferon (IFN) response. However, the mechanism of this inhibitory activity of NS5A remains partly characterized. In a functional proteomic screening carried out in HCV replicon cells, we identified the mitochondrial protein LRPPRC as an NS5A binding factor. Notably, we found that downregulation of LRPPRC expression results in a significant inhibition of HCV infection, which is associated with an increased activation of the IFN response. Moreover, we showed that LRPPRC acts as a negative regulator of the mitochondrial-mediated antiviral immunity, by interacting with mitochondrial antiviral signaling protein (MAVS) and inhibiting its association with TRAF3 and TRAF6. Finally, we demonstrated that NS5A is able to interfere with MAVS activity in a LRPPRC-dependent manner. Conclusion: Overall, our results indicate that NS5A contributes to the inhibition of innate immune pathways during HCV infection by exploiting the ability of LRPPRC to inhibit MAVS-regulated antiviral signaling.
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Affiliation(s)
- Giulia Refolo
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | - Fabiola Ciccosanti
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | - Martina Di Rienzo
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | | | - Marta Romani
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | - Tonino Alonzi
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | - Marco Tripodi
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy.,Department of Cellular Biotechnologies and Haematology, Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - Giuseppe Ippolito
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy
| | - Mauro Piacentini
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy.,Department of Biology, University of Rome 'Tor Vergata', Rome, Italy
| | - Gian Maria Fimia
- National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Rome, Italy.,Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, Italy
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Sharma G, Raheja H, Das S. Hepatitis C virus: Enslavement of host factors. IUBMB Life 2018; 70:41-49. [PMID: 29281185 DOI: 10.1002/iub.1702] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Revised: 11/20/2017] [Accepted: 11/28/2017] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus (HCV) has infected over 170 million people world-wide. This infection causes severe liver damage that can progress to hepatocellular carcinoma leading to death of the infected patients. Development of a cell culture model system for the study of HCV infection in the recent past has helped the researchers world-wide to understand the biology of this virus. Studies over the past decade have revealed the tricks played by the virus to sustain itself, for as long as 40 years, in the host setup without being eliminated by the immune system. Today we understand that the host organelles and different cellular proteins are affected during HCV infection. This cytoplasmic virus has all the cellular organelles at its disposal to successfully replicate, from ribosomes and intracellular membranous structures to the nucleus. It modulates these organelles at both the structural and the functional levels. The vast knowledge about the viral genome and viral proteins has also helped in the development of drugs against the virus. Despite the achieved success rate to cure the infected patients, we struggle to eliminate the cases of recurrence and the non-responders. Such cases might emerge owing to the property of the viral genome to accumulate mutations during its succeeding replication cycles which favours its survival. The current situation calls an urgent need for alternate therapeutic strategies to counter this major problem of human health. © 2017 IUBMB Life, 70(1):41-49, 2018.
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Affiliation(s)
- Geetika Sharma
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Harsha Raheja
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
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34
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Matz KM, Guzman RM, Goodman AG. The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2018; 345:35-136. [PMID: 30904196 PMCID: PMC6445394 DOI: 10.1016/bs.ircmb.2018.08.002] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Innate immunity, the first line of defense against invading pathogens, is an ancient form of host defense found in all animals, from sponges to humans. During infection, innate immune receptors recognize conserved molecular patterns, such as microbial surface molecules, metabolites produces during infection, or nucleic acids of the microbe's genome. When initiated, the innate immune response activates a host defense program that leads to the synthesis proteins capable of pathogen killing. In mammals, the induction of cytokines during the innate immune response leads to the recruitment of professional immune cells to the site of infection, leading to an adaptive immune response. While a fully functional innate immune response is crucial for a proper host response and curbing microbial infection, if the innate immune response is dysfunctional and is activated in the absence of infection, autoinflammation and autoimmune disorders can develop. Therefore, it follows that the innate immune response must be tightly controlled to avoid an autoimmune response from host-derived molecules, yet still unencumbered to respond to infection. In this review, we will focus on the innate immune response activated from cytosolic nucleic acids, derived from the microbe or host itself. We will depict how viruses and bacteria activate these nucleic acid sensing pathways and their mechanisms to inhibit the pathways. We will also describe the autoinflammatory and autoimmune disorders that develop when these pathways are hyperactive. Finally, we will discuss gaps in knowledge with regard to innate immune response failure and identify where further research is needed.
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Affiliation(s)
- Keesha M Matz
- School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, United States
| | - R Marena Guzman
- School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, United States
| | - Alan G Goodman
- School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, United States; Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA, United States.
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El-Dahshan D, Bahy D, Wahid A, Ahmed AE, Hanora A. Two novel SNPs in the promoter region of PKR gene in hepatitis C patients and their impact on disease outcome and response to treatment. Arab J Gastroenterol 2018; 19:106-115. [PMID: 30245117 DOI: 10.1016/j.ajg.2018.06.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2017] [Revised: 02/17/2018] [Accepted: 06/04/2018] [Indexed: 12/01/2022]
Abstract
BACKGROUND AND STUDY AIMS The double-stranded RNA dependent protein kinase (PKR) plays a vital role in the immune system. During HCV infection, PKR has antiviral effect by inhibition of protein synthesis of the HCV. The functional single nucleotide polymorphisms (SNPs) in PKR promoter region might have a relation to HCV disease outcome and response to treatment. The objective of the present work was threefold. First, it proposed an optimized protocol for PCR amplification of PKR promoter. Second, it screened the promoter region of PKR gene in HCV Egyptian patients to detect the possible SNPs' function. Third, to study the association between the detected SNPs and the response to treatment. PATIENTS AND METHODS The functional SNPs in PKR promoter region were detected using DNA sequencing in 40 HCV infected patients; 20 sustained virologic response (SVR) patients and 20 nonresponse (NR) patients after combined interferon/ribavirin therapy. Twenty healthy subjects were included as a control. RESULTS Two functional SNPs were detected: rs62133148T>G and rs12992188C>T within our target PKR promoter region. In rs62133148 polymorphism, there is a significant difference between patients and control subjects for TT and TG genotypes (p < 0.0001). In addition, the G allele is more predominant in HCV patients. In rs12992188 polymorphism, the CC genotype is significantly different between patients and healthy control subjects (OR/95% CI: 0.033/0.006-0.172, p < 0.0001). The presence of C allele was significantly associated with the NR patients (OR/95%CI: 0.25/0.097-0.643, p = 0.006). The TT genotype is significantly different between SVR and NR (OR/95%CI: 8.5/1.54-46.871, p = 0.014). CONCLUSION This study is a pioneer clinical study on these two functional SNPs (rs62133148T>G and rs12992188 C>T). The rs62133148 polymorphism does not show any association with response to treatment. The TT genotype in rs12992188 polymorphism shows association with response to treatment. Therefore, patients with TT genotypes were more likely to achieve SVR.
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Affiliation(s)
- Dina El-Dahshan
- Department of Clinical Pathology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
| | - Doaa Bahy
- Biotechnology Department, Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Beni-Suef, Egypt; Beni-Suef Health Insurance Hospital, Beni-Suef, Egypt.
| | - Ahmed Wahid
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
| | - Amr E Ahmed
- Biotechnology Department, Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Beni-Suef, Egypt
| | - Amro Hanora
- Microbiology and Immunology Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt
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Removal of the C6 Vaccinia Virus Interferon-β Inhibitor in the Hepatitis C Vaccine Candidate MVA-HCV Elicited in Mice High Immunogenicity in Spite of Reduced Host Gene Expression. Viruses 2018; 10:v10080414. [PMID: 30096846 PMCID: PMC6116028 DOI: 10.3390/v10080414] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2018] [Revised: 08/03/2018] [Accepted: 08/07/2018] [Indexed: 12/19/2022] Open
Abstract
Hepatitis C virus (HCV) represents a major global health problem for which a vaccine is not available. Modified vaccinia virus Ankara (MVA)-HCV is a unique HCV vaccine candidate based in the modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV genotype 1a that elicits CD8⁺ T-cell responses in mice. With the aim to improve the immune response of MVA-HCV and because of the importance of interferon (IFN) in HCV infection, we deleted in MVA-HCV the vaccinia virus (VACV) C6L gene, encoding an inhibitor of IFN-β that prevents activation of the interferon regulatory factors 3 and 7 (IRF3 and IRF7). The resulting vaccine candidate (MVA-HCV ΔC6L) expresses all HCV antigens and deletion of C6L had no effect on viral growth in permissive chicken cells. In human monocyte-derived dendritic cells, infection with MVA-HCV ΔC6L triggered severe down-regulation of IFN-β, IFN-β-induced genes, and cytokines in a manner similar to MVA-HCV, as defined by real-time polymerase chain reaction (PCR) and microarray analysis. In infected mice, both vectors had a similar profile of recruited immune cells and induced comparable levels of adaptive and memory HCV-specific CD8⁺ T-cells, mainly against p7 + NS2 and NS3 HCV proteins, with a T cell effector memory (TEM) phenotype. Furthermore, antibodies against E2 were also induced. Overall, our findings showed that while these vectors had a profound inhibitory effect on gene expression of the host, they strongly elicited CD8⁺ T cell and humoral responses against HCV antigens and to the virus vector. These observations add support to the consideration of these vectors as potential vaccine candidates against HCV.
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Suhail M, Sohrab SS, Qureshi A, Tarique M, Abdel-Hafiz H, Al-Ghamdi K, Qadri I. Association of HCV mutated proteins and host SNPs in the development of hepatocellular carcinoma. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2018; 60:160-172. [PMID: 29501636 DOI: 10.1016/j.meegid.2018.02.034] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/03/2017] [Revised: 02/14/2018] [Accepted: 02/28/2018] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus plays a significant role in the development of hepatocellular carcinoma (HCC) globally. The pathogenic mechanisms of hepatocellular carcinoma with HCV infection are generally linked with inflammation, cytokines, fibrosis, cellular signaling pathways, and liver cell proliferation modulating pathways. HCV encoded proteins (Core, NS3, NS4, NS5A) interact with a broad range of hepatocytes derived factors to modulate an array of activities such as cell signaling, DNA repair, transcription and translational regulation, cell propagation, apoptosis, membrane topology. These four viral proteins are also implicated to show a strong conversion potential in tissue culture. Furthermore, Core and NS5A also trigger the accretion of the β-catenin pathway as a common target to contribute viral induced transformation. There is a strong association between HCV variants within Core, NS4, and NS5A and host single nucleotide polymorphisms (SNPs) with the HCC pathogenesis. Identification of such viral mutants and host SNPs is very critical to determine the risk of HCC and response to antiviral therapy. In this review, we highlight the association of key variants, mutated proteins, and host SNPs in development of HCV induced HCC. How such viral mutants may modulate the interaction with cellular host machinery is also discussed.
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Affiliation(s)
- Mohd Suhail
- King Fahd Medical Research Center, King Abdulaziz University, PO Box 80216, Jeddah 21589, Saudi Arabia
| | - Sayed Sartaj Sohrab
- Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, PO Box 80216, Jeddah 21589, Saudi Arabia
| | - Abid Qureshi
- Biomedical Informatics Centre, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, J&K, India
| | - Mohd Tarique
- Department of Surgery, Sylvester Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136, United States
| | - Hany Abdel-Hafiz
- Dept of Medicine, University of Colorado Denver, Aurora, CO 80045, United States
| | - Khalid Al-Ghamdi
- Department of Biological Science, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Ishtiaq Qadri
- Department of Biological Science, King Abdulaziz University, Jeddah, Saudi Arabia.
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Chen S, Yang C, Zhang W, Mahalingam S, Wang M, Cheng A. Flaviviridae virus nonstructural proteins 5 and 5A mediate viral immune evasion and are promising targets in drug development. Pharmacol Ther 2018; 190:1-14. [PMID: 29742479 DOI: 10.1016/j.pharmthera.2018.05.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Infections with viruses in the Flaviviridae family have a vast global and economic impact because of the high morbidity and mortality. The pathogenesis of Flaviviridae infections is very complex and not fully understood because these viruses can inhibit multiple immune pathways including the complement system, NK cells, and IFN induction and signalling pathways. The non-structural (NS) 5 and 5A proteins of Flaviviridae viruses are highly conserved and play an important role in resisting host immunity through various evasion mechanisms. This review summarizes the strategies used by the NS5 and 5A proteins of Flaviviridae viruses for evading the innate immune response by inhibiting pattern recognition receptor (PRR) signalling pathways (TLR/MyD88, IRF7), suppressing interferon (IFN) signalling pathways (IFN-γRs, STAT1, STAT2), and impairing the function of IFN-stimulated genes (ISGs) (e.g. protein kinase R [PKR], oligoadenylate synthase [OAS]). All of these immune evasion mechanisms depend on the interaction of NS5 or NS5A with cellular proteins, such as MyD88 and IRF7, IFN-αRs, IFN-γRs, STAT1, STAT2, PKR and OAS. NS5 is the most attractive target for the discovery of broad spectrum compounds against Flaviviridae virus infection. The methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities of NS5 are the main therapeutic targets for antiviral drugs against Flaviviridae virus infection. Based on our site mapping, the sites involved in immune evasion provide some potential and promising targets for further novel antiviral therapeutics.
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Affiliation(s)
- Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan 611130, China.
| | - Chao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
| | - Wei Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
| | - Suresh Mahalingam
- Emerging Viruses and Inflammation Research Group, Institute for Glycomics, Griffith University, Gold Coast, Australia
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan 611130, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan 611130, China.
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Dzananovic E, McKenna SA, Patel TR. Viral proteins targeting host protein kinase R to evade an innate immune response: a mini review. Biotechnol Genet Eng Rev 2018; 34:33-59. [PMID: 29716441 DOI: 10.1080/02648725.2018.1467151] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The innate immune system offers a first line of defense by neutralizing foreign pathogens such as bacteria, fungi, and viruses. These pathogens express molecules (RNA and proteins) that have discrete structures, known as the pathogen-associated molecular patterns that are recognized by a highly specialized class of host proteins called pattern recognition receptors to facilitate the host's immune response against infection. The RNA-dependent Protein Kinase R (PKR) is one of the host's pattern recognition receptors that is a key component of an innate immune system. PKR recognizes imperfectly double-stranded non-coding viral RNA molecules via its N-terminal double-stranded RNA binding motifs, undergoes phosphorylation of the C-terminal kinase domain, ultimately resulting in inhibition of viral protein translation by inhibiting the guanine nucleotide exchange activity of eukaryotic initiation factor 2α. Not surprisingly, viruses have evolved mechanisms by which viral non-coding RNA or protein molecules inhibit PKR's activation and/or its downstream activity to allow viral replication. In this review, we will highlight the role of viral proteins in inhibiting PKR's activity and summarize currently known mechanisms by which viral proteins execute such inhibitory activity.
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Affiliation(s)
- Edis Dzananovic
- a Plant Pathology, Plant Protection and Molecular Biology , Agriculture and Agri-Food Canada , Saskatoon , Canada
| | - Sean A McKenna
- b Department of Chemistry, Manitoba Institute for Materials, Department of Biochemistry and Medical Genetics , University of Manitoba , Winnipeg , Canada
| | - Trushar R Patel
- c Department of Chemistry and Biochemistry , Alberta RNA Research and Training Institute, University of Lethbridge , Lethbridge , Canada.,d DiscoveryLab, Faculty of Medicine & Dentistry , University of Alberta , Edmonton , Canada.,e Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine , University of Calgary , Calgary , Canada
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Morozov VA, Lagaye S. Hepatitis C virus: Morphogenesis, infection and therapy. World J Hepatol 2018; 10:186-212. [PMID: 29527256 PMCID: PMC5838439 DOI: 10.4254/wjh.v10.i2.186] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2017] [Revised: 01/11/2018] [Accepted: 02/07/2018] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a major cause of liver diseases including liver cirrhosis and hepatocellular carcinoma. Approximately 3% of the world population is infected with HCV. Thus, HCV infection is considered a public healthy challenge. It is worth mentioning, that the HCV prevalence is dependent on the countries with infection rates around 20% in high endemic countries. The review summarizes recent data on HCV molecular biology, the physiopathology of infection (immune-mediated liver damage, liver fibrosis and lipid metabolism), virus diagnostic and treatment. In addition, currently available in vitro, ex vivo and animal models to study the virus life cycle, virus pathogenesis and therapy are described. Understanding of both host and viral factors may in the future lead to creation of new approaches in generation of an efficient therapeutic vaccine.
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Affiliation(s)
- Vladimir Alexei Morozov
- Center for HIV and Retrovirology, Department of Infectious Diseases, Robert Koch Institute, Berlin 13353, Germany
| | - Sylvie Lagaye
- Department of Immunology, Institut Pasteur, INSERM U1223, Paris 75015, France
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41
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Lampejo T, Agarwal K, Carey I. Interferon-free direct-acting antiviral therapy for acute hepatitis C virus infection in HIV-infected individuals: A literature review. Dig Liver Dis 2018; 50:113-123. [PMID: 29233687 DOI: 10.1016/j.dld.2017.11.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2017] [Revised: 10/29/2017] [Accepted: 11/15/2017] [Indexed: 02/07/2023]
Abstract
Dramatic rises in hepatitis C virus (HCV) coinfection rates in human immunodeficiency virus (HIV)-infected individuals have been observed recently, largely attributable to increasing recreational drug use combined with increased testing for HCV. In the era of direct-acting antiviral (DAA) therapy, treatment of acute HCV infection in HIV-infected individuals with short durations of these drugs may potentially reduce the disease and economic burden associated with HCV infection as well as reducing the likelihood of onward HCV transmission. We performed an extensive literature search of PubMed, Embase and Google Scholar up to 05 September 2017 for clinical trials of acute HCV infection in HIV-infected individuals. In the studies identified, rates of sustained virologic response at 12 weeks post-treatment (SVR12) ranged from 21% with 6 weeks of therapy up to 92% with 12 weeks of therapy with sofosbuvir and ribavirin. Ledipasvir/sofosbuvir for 6 weeks achieved an SVR of 77%. No HIV-related events occurred regardless of whether patients were receiving antiretroviral therapy (ART) and DAAs were well tolerated. Data is currently limited with regards to optimal regimens and durations of therapy, which need to be tailored based on potential interactions with concurrent ART and consideration for the fact that patients with higher baseline HCV RNA levels may require an extended duration of treatment.
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Affiliation(s)
- Temi Lampejo
- Institute of Liver Studies, King's College Hospital, London, United Kingdom.
| | - Kosh Agarwal
- Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Ivana Carey
- Institute of Liver Studies, King's College Hospital, London, United Kingdom
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Minami N, Abe T, Deng L, Matsui C, Fukuhara T, Matsuura Y, Shoji I. Unconjugated interferon-stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I. Microbiol Immunol 2017; 61:287-292. [PMID: 28543875 DOI: 10.1111/1348-0421.12493] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2017] [Revised: 05/22/2017] [Accepted: 05/23/2017] [Indexed: 11/28/2022]
Abstract
Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.
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Affiliation(s)
- Nanae Minami
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017
| | - Takayuki Abe
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017
| | - Lin Deng
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017
| | - Chieko Matsui
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Ikuo Shoji
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017
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Innate Immune Evasion Mediated by Flaviviridae Non-Structural Proteins. Viruses 2017; 9:v9100291. [PMID: 28991176 PMCID: PMC5691642 DOI: 10.3390/v9100291] [Citation(s) in RCA: 75] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 10/03/2017] [Accepted: 10/05/2017] [Indexed: 12/17/2022] Open
Abstract
Flaviviridae-caused diseases are a critical, emerging public health problem worldwide. Flaviviridae infections usually cause severe, acute or chronic diseases, such as liver damage and liver cancer resulting from a hepatitis C virus (HCV) infection and high fever and shock caused by yellow fever. Many researchers worldwide are investigating the mechanisms by which Flaviviridae cause severe diseases. Flaviviridae can interfere with the host’s innate immunity to achieve their purpose of proliferation. For instance, dengue virus (DENV) NS2A, NS2B3, NS4A, NS4B and NS5; HCV NS2, NS3, NS3/4A, NS4B and NS5A; and West Nile virus (WNV) NS1 and NS4B proteins are involved in immune evasion. This review discusses the interplay between viral non-structural Flaviviridae proteins and relevant host proteins, which leads to the suppression of the host’s innate antiviral immunity.
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Hepatitis C Virus NS5A Targets Nucleosome Assembly Protein NAP1L1 To Control the Innate Cellular Response. J Virol 2017; 91:JVI.00880-17. [PMID: 28659470 DOI: 10.1128/jvi.00880-17] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2017] [Accepted: 06/19/2017] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A.IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.
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Preclinical Pharmacokinetics and First-in-Human Pharmacokinetics, Safety, and Tolerability of Velpatasvir, a Pangenotypic Hepatitis C Virus NS5A Inhibitor, in Healthy Subjects. Antimicrob Agents Chemother 2017; 61:AAC.02084-16. [PMID: 28193657 DOI: 10.1128/aac.02084-16] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2016] [Accepted: 02/04/2017] [Indexed: 12/13/2022] Open
Abstract
Preclinical characterization of velpatasvir (VEL; GS-5816), an inhibitor of the hepatitis C virus (HCV) NS5A protein, demonstrated that it has favorable in vitro and in vivo properties, including potent antiviral activity against hepatitis C virus genotype 1 to 6 replicons, good metabolic stability, low systemic clearance, and adequate bioavailability and physicochemical properties, to warrant clinical evaluation. The phase 1 (first-in-human) study evaluated the safety, tolerability, and pharmacokinetics of VEL in healthy human subjects following administration of single and multiple (n = 7) once-daily ascending doses and of VEL in the presence and absence of food. Following administration of single and multiple doses, VEL was safe and well tolerated when administered at up to 450 mg and when administered with food. The pharmacokinetic behavior of VEL observed in humans was generally in agreement with that seen during preclinical characterization. Following administration of multiple doses, VEL trough concentrations were significantly greater than the protein-adjusted half-maximal (50%) effective concentration of VEL against HCV genotype 1 to 6 replicons at all evaluated doses greater than 5 mg. The pharmacokinetics of VEL were not significantly affected by administration with food. Collectively, the results of this study support the further clinical investigation of VEL administered once daily as part of a regimen with other pangenotypic direct-acting antivirals for the treatment of HCV infection.
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Chen M, Gan X, Yoshino KI, Kitakawa M, Shoji I, Deng L, Hotta H. Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain-containing 3 and induces activator protein 1 activation. Microbiol Immunol 2017; 60:407-17. [PMID: 27080060 DOI: 10.1111/1348-0421.12383] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2016] [Revised: 04/01/2016] [Accepted: 04/10/2016] [Indexed: 12/27/2022]
Abstract
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.
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Affiliation(s)
- Ming Chen
- Division of Microbiology.,Division of Infectious Disease Control, Kobe University Graduate School of Medicine
| | - Xiang Gan
- Division of Microbiology.,Institute of Biochemistry and Molecular Biology, Hubei University, Wuhan, China
| | | | | | - Ikuo Shoji
- Division of Infectious Disease Control, Kobe University Graduate School of Medicine
| | - Lin Deng
- Division of Microbiology.,Division of Infectious Disease Control, Kobe University Graduate School of Medicine
| | - Hak Hotta
- Division of Microbiology.,Department of Oral Vaccine and Drug Development, Kobe University Graduate School of Health Sciences, Kobe, Japan
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Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein. Proc Natl Acad Sci U S A 2017; 114:2018-2023. [PMID: 28159892 DOI: 10.1073/pnas.1614623114] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to down-regulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.
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Ivanenkov YA, Aladinskiy VA, Bushkov NA, Ayginin AA, Majouga AG, Ivachtchenko AV. Small-molecule inhibitors of hepatitis C virus (HCV) non-structural protein 5A (NS5A): a patent review (2010-2015). Expert Opin Ther Pat 2017; 27:401-414. [PMID: 27967269 DOI: 10.1080/13543776.2017.1272573] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
INTRODUCTION Non-structural 5A (NS5A) protein has achieved a considerable attention as an attractive target for the treatment of hepatitis C (HCV). A number of novel NS5A inhibitors have been reported to date. Several drugs having favorable ADME properties and mild side effects were launched into the pharmaceutical market. For instance, daclatasvir was launched in 2014, elbasvir is currently undergoing registration, ledipasvir was launched in 2014 as a fixed-dose combination with sofosbuvir (NS5B inhibitor). Areas covered: Thomson integrity database and SciFinder database were used as a valuable source to collect the patents on small-molecule NS5A inhibitors. All the structures were ranked by the date of priority. Patent holder and antiviral activity for each scaffold claimed were summarized and presented in a convenient manner. A particular focus was placed on the best-in-class bis-pyrrolidine-containing NS5A inhibitors. Expert opinion: Several first generation NS5A inhibitors have recently progressed into advanced clinical trials and showed superior efficacy in reducing viral load in infected subjects. Therapy schemes of using these agents in combination with other established antiviral drugs with complementary mechanisms of action can address the emergence of resistance and poor therapeutic outcome frequently attributed to antiviral drugs.
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Affiliation(s)
- Yan A Ivanenkov
- a Department of Biological and Medical Physics , Moscow Institute of Physics and Technology (State University) , Dolgoprudny City , Moscow Region , Russia.,b Department of Computational and Medicinal Chemistry , ChemDiv , San Diego , CA , USA.,c Chemistry Department , Moscow State University , Moscow , Russia
| | - Vladimir A Aladinskiy
- a Department of Biological and Medical Physics , Moscow Institute of Physics and Technology (State University) , Dolgoprudny City , Moscow Region , Russia
| | - Nikolay A Bushkov
- a Department of Biological and Medical Physics , Moscow Institute of Physics and Technology (State University) , Dolgoprudny City , Moscow Region , Russia
| | - Andrey A Ayginin
- a Department of Biological and Medical Physics , Moscow Institute of Physics and Technology (State University) , Dolgoprudny City , Moscow Region , Russia
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Mogalian E, German P, Kearney BP, Yang CY, Brainard D, McNally J, Moorehead L, Mathias A. Use of Multiple Probes to Assess Transporter- and Cytochrome P450-Mediated Drug-Drug Interaction Potential of the Pangenotypic HCV NS5A Inhibitor Velpatasvir. Clin Pharmacokinet 2016; 55:605-13. [PMID: 26519191 DOI: 10.1007/s40262-015-0334-7] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
BACKGROUND AND OBJECTIVES Velpatasvir (VEL; GS-5816) is a potent, pangenotypic hepatitis C virus (HCV), non-structural protein 5A inhibitor in clinical development for the treatment of chronic HCV infection. In vitro studies indicate that VEL may inhibit several drug transporters and be a substrate for enzyme/drug transport systems in vivo. The purpose of this study was to evaluate the potential of VEL as a perpetrator or victim of metabolic- and transporter-based drug-drug interactions using complementary probe drugs. METHODS This Phase 1 study was a randomized, cross-over, open-label, single- and multiple-dose, five-cohort study. Serial blood samples were collected following oral administration of reference and test treatments. The primary pharmacokinetic parameters of each analyte were compared when administered alone or in combination. The 90% confidence intervals (CI) for the ratio of the geometric least-squares means of the test and reference treatments was calculated for each analyte and parameter of interest. RESULTS This study demonstrated that VEL is a weak (P-gp, OATP) to moderate (breast cancer resistance protein) transport inhibitor. As a victim of interactions, VEL is moderately affected by potent inhibitors and to a greater extent, potent inducers of enzyme/drug transporter systems. CONCLUSIONS The impact of specific transporters and overall contribution of drug transport vs. metabolizing enzymes on the disposition of VEL was characterized through the use of complementary probes, despite the lack of phenotypic specificity, and informs a broad range of drug-drug interaction recommendations.
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Affiliation(s)
- Erik Mogalian
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA.
| | - Polina German
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - Brian P Kearney
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - Cheng Yong Yang
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - Diana Brainard
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - John McNally
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - Lisa Moorehead
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
| | - Anita Mathias
- Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA, 94404, USA
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50
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Hayes CN, Chayama K. Interferon stimulated genes and innate immune activation following infection with hepatitis B and C viruses. J Med Virol 2016; 89:388-396. [DOI: 10.1002/jmv.24659] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/08/2016] [Indexed: 12/28/2022]
Affiliation(s)
- C. Nelson Hayes
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences; Hiroshima University; Hiroshima Japan
- Liver Research Project Center; Hiroshima University; Hiroshima Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences; Hiroshima University; Hiroshima Japan
- Liver Research Project Center; Hiroshima University; Hiroshima Japan
- Laboratory for Digestive Diseases; Center for Genomic Medicine, RIKEN; Hiroshima Japan
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