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Pant A, Yao X, Lavedrine A, Viret C, Dockterman J, Chauhan S, Chong-Shan Shi, Manjithaya R, Cadwell K, Kufer TA, Kehrl JH, Coers J, Sibley LD, Faure M, Taylor GA, Chauhan S. Interactions of Autophagy and the Immune System in Health and Diseases. AUTOPHAGY REPORTS 2022; 1:438-515. [PMID: 37425656 PMCID: PMC10327624 DOI: 10.1080/27694127.2022.2119743] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/07/2023]
Abstract
Autophagy is a highly conserved process that utilizes lysosomes to selectively degrade a variety of intracellular cargo, thus providing quality control over cellular components and maintaining cellular regulatory functions. Autophagy is triggered by multiple stimuli ranging from nutrient starvation to microbial infection. Autophagy extensively shapes and modulates the inflammatory response, the concerted action of immune cells, and secreted mediators aimed to eradicate a microbial infection or to heal sterile tissue damage. Here, we first review how autophagy affects innate immune signaling, cell-autonomous immune defense, and adaptive immunity. Then, we discuss the role of non-canonical autophagy in microbial infections and inflammation. Finally, we review how crosstalk between autophagy and inflammation influences infectious, metabolic, and autoimmune disorders.
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Affiliation(s)
- Aarti Pant
- Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India
| | - Xiaomin Yao
- Kimmel Center for Biology and Medicine at the Skirball Institute, New York University Grossman School of Medicine, New York, New York, United States of America
- Department of Microbiology, New York University Grossman School of Medicine, New York, New York, United States of America
| | - Aude Lavedrine
- CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
- Equipe Labellisée par la Fondation pour la Recherche Médicale, FRM
| | - Christophe Viret
- CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
- Equipe Labellisée par la Fondation pour la Recherche Médicale, FRM
| | - Jake Dockterman
- Department of Immunology, Duke University, Medical Center, Durham, North Carolina, USA
| | - Swati Chauhan
- Cell biology and Infectious diseases, Institute of Life Sciences, Bhubaneswar, India
| | - Chong-Shan Shi
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States
| | - Ravi Manjithaya
- Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India
- Neuroscience Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India
| | - Ken Cadwell
- Kimmel Center for Biology and Medicine at the Skirball Institute, New York University Grossman School of Medicine, New York, New York, United States of America
- Department of Microbiology, New York University Grossman School of Medicine, New York, New York, United States of America
- Division of Gastroenterology and Hepatology, Department of Medicine, New York University Grossman School of Medicine, New York, New York, United States of America
| | - Thomas A. Kufer
- Department of Immunology, Institute of Nutritional Medicine, University of Hohenheim, Stuttgart, Germany
| | - John H. Kehrl
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States
| | - Jörn Coers
- Department of Immunology, Duke University, Medical Center, Durham, North Carolina, USA
- Department of Molecular Genetics and Microbiology, Duke University, Medical Center, Durham, North Carolina, USA
| | - L. David Sibley
- Department of Molecular Microbiology, Washington University Sch. Med., St Louis, MO, 63110, USA
| | - Mathias Faure
- CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
- Equipe Labellisée par la Fondation pour la Recherche Médicale, FRM
| | - Gregory A Taylor
- Department of Immunology, Duke University, Medical Center, Durham, North Carolina, USA
- Department of Molecular Genetics and Microbiology, Duke University, Medical Center, Durham, North Carolina, USA
- Department of Molecular Microbiology, Washington University Sch. Med., St Louis, MO, 63110, USA
- Geriatric Research, Education, and Clinical Center, VA Health Care Center, Durham, North Carolina, USA
- Departments of Medicine, Division of Geriatrics, and Center for the Study of Aging and Human Development, Duke University, Medical Center, Durham, North Carolina, USA
| | - Santosh Chauhan
- Cell biology and Infectious diseases, Institute of Life Sciences, Bhubaneswar, India
- CSIR–Centre For Cellular And Molecular Biology (CCMB), Hyderabad, Telangana
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Miller SR, McGrath ME, Zorn KM, Ekins S, Wright SH, Cherrington NJ. Response to Comments on "Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites". Mol Pharmacol 2022; 101:121-122. [PMID: 35105679 PMCID: PMC11037455 DOI: 10.1124/molpharm.121.000448] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Accepted: 11/11/2021] [Indexed: 11/22/2022] Open
Affiliation(s)
- Siennah R Miller
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.)
| | - Meghan E McGrath
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.)
| | - Kimberley M Zorn
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.)
| | - Sean Ekins
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.)
| | - Stephen H Wright
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.)
| | - Nathan J Cherrington
- College of Pharmacy, Department of Pharmacology & Toxicology (S.R.M., M.E.M., N.J.C.) and Department of Physiology (S.H.W.), University of Arizona, Tucson, Arizona; and Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina (K.M.Z., S.E.) cherrington@pharmacy
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Shi SL, Fukuda H, Chujo T, Kouwaki T, Oshiumi H, Tomizawa K, Wei FY. Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication. RNA Biol 2021; 18:478-495. [PMID: 34382915 PMCID: PMC8677048 DOI: 10.1080/15476286.2021.1960689] [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: 03/30/2021] [Revised: 07/14/2021] [Accepted: 07/23/2021] [Indexed: 11/25/2022] Open
Abstract
RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.
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Affiliation(s)
- Sheng-Lan Shi
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Hiroyuki Fukuda
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takeshi Chujo
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takahisa Kouwaki
- Department of Immunology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Hiroyuki Oshiumi
- Department of Immunology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kazuhito Tomizawa
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Fan-Yan Wei
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
- Department of Modomics Biology and Medicine, Institute of Development, Aging and Cancer, Tohoku University, Miyagi, Japan
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Szekerczés T, Gógl A, Illyés I, Mandl J, Borka K, Kiss A, Schaff Z, Lendvai G, Werling K. Autophagy, Mitophagy and MicroRNA Expression in Chronic Hepatitis C and Autoimmune Hepatitis. Pathol Oncol Res 2020; 26:2143-2151. [PMID: 32124227 PMCID: PMC7471137 DOI: 10.1007/s12253-020-00799-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Accepted: 02/11/2020] [Indexed: 12/13/2022]
Abstract
Although the role of autophagy has been implicated in several forms of chronic hepatitis, it is still not fully understood. Active autophagy eliminates damaged molecules and organelles (such as mitochondria) by lysosomal degradation. In the present study, we aimed to examine and compare autophagy activity in chronic hepatitis C (CHC) and autoimmune hepatitis (AIH) by detecting the expression of autophagy (LC3 and p62) and mitochondrium-related (TOMM20) proteins, as well as the levels of selected microRNAs (miR-101, -155, -204 and - 224) known to be involved in the regulation of autophagy. In addition, the expression levels were related to pathohistological parameters. Liver biopsy samples, including 45 CHC and 18 AIH cases, were immunohistochemically stained for LC3, p62 and TOMM20 and the expression of miRNAs was determined using real-time PCR. We found elevated LC3 and p62 in AIH samples as compared with CHC ones, indicating an activated autophagy that is impaired in AIH as no degradation of p62 seemed to occur. Moreover, p62 showed strong correlation with necroinflammatory grades in the AIH group. The observed elevated levels of TOMM20 and p62 suggest a less efficient elimination of damaged mitochondria in AIH as opposed to CHC, in which autophagy seems to have a more active function. The level of miR-101 was increased in case of CHC as compared with AIH, however, miR-155, -204 and 224 resulted in no expressional. Furthermore, miR-224 level correlated with steatosis and miR-155 expression with fibrosis stage in CHC. In conclusion, dissimilar autophagic activity was observed in CHC and AIH, suggesting a close association between impaired autophagy and severity of necroinflammation. This impairment may not be regulated by the analyzed miRNAs. Nevertheless, miR-224 and - 155 seem to be associated with CHC progression.
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MESH Headings
- Adolescent
- Adult
- Aged
- Autophagy
- Biomarkers, Tumor/genetics
- Disease Progression
- Female
- Follow-Up Studies
- Gene Expression Regulation, Neoplastic
- Hepatitis C, Chronic/genetics
- Hepatitis C, Chronic/metabolism
- Hepatitis C, Chronic/pathology
- Hepatitis C, Chronic/surgery
- Hepatitis, Autoimmune/genetics
- Hepatitis, Autoimmune/metabolism
- Hepatitis, Autoimmune/pathology
- Hepatitis, Autoimmune/surgery
- Humans
- Male
- MicroRNAs/genetics
- Middle Aged
- Mitophagy
- Prognosis
- Retrospective Studies
- Survival Rate
- Young Adult
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Affiliation(s)
- Tímea Szekerczés
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - Alíz Gógl
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - Ildikó Illyés
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - József Mandl
- Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 1094, Budapest, Hungary
| | - Katalin Borka
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - András Kiss
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - Zsuzsa Schaff
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary
| | - Gábor Lendvai
- 2nd Department of Pathology, Semmelweis University, Üllői 93, 1091, Budapest, Hungary.
| | - Klára Werling
- 2nd Department of Internal Medicine, Semmelweis University, 1088, Budapest, Hungary
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Dash S, Aydin Y, Wu T. Integrated stress response in hepatitis C promotes Nrf2-related chaperone-mediated autophagy: A novel mechanism for host-microbe survival and HCC development in liver cirrhosis. Semin Cell Dev Biol 2020; 101:20-35. [PMID: 31386899 PMCID: PMC7007355 DOI: 10.1016/j.semcdb.2019.07.015] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 06/26/2019] [Accepted: 07/30/2019] [Indexed: 02/06/2023]
Abstract
The molecular mechanism(s) how liver damage during the chronic hepatitis C virus (HCV) infection evolve into cirrhosis and hepatocellular carcinoma (HCC) is unclear. HCV infects hepatocyte, the major cell types in the liver. During infection, large amounts of viral proteins and RNA replication intermediates accumulate in the endoplasmic reticulum (ER) of the infected hepatocyte, which creates a substantial amount of stress response. Infected hepatocyte activates a different type of stress adaptive mechanisms such as unfolded protein response (UPR), antioxidant response (AR), and the integrated stress response (ISR) to promote virus-host cell survival. The hepatic stress is also amplified by another layer of innate and inflammatory response associated with cellular sensing of virus infection through the production of interferon (IFN) and inflammatory cytokines. The interplay between various types of cellular stress signal leads to different forms of cell death such as apoptosis, necrosis, and autophagy depending on the intensity of the stress and nature of the adaptive cellular response. How do the adaptive cellular responses decode such death programs that promote host-microbe survival leading to the establishment of chronic liver disease? In this review, we discuss how the adaptive cellular response through the Nrf2 pathway that promotes virus and cell survival. Furthermore, we provide a glimpse of novel stress-induced Nrf2 mediated compensatory autophagy mechanisms in virus-cell survival that degrade tumor suppressor gene and activation of oncogenic signaling during HCV infection. Based on these facts, we hypothesize that the balance between hepatic stress, inflammation and different types of cell death determines liver disease progression outcomes. We propose that a more nuanced understanding of virus-host interactions under excessive cellular stress may provide an answer to the fundamental questions why some individuals with chronic HCV infection remain at risk of developing cirrhosis, cancer and some do not.
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Affiliation(s)
- Srikanta Dash
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, 70112, USA.
| | - Yucel Aydin
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, 70112, USA
| | - Tong Wu
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, 70112, USA
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Liao W, Liu X, Yang Q, Liu H, Liang B, Jiang J, Huang J, Ning C, Zang N, Zhou B, Liao Y, Chen J, Tian L, Ho W, Abdullah AS, Kong L, Liang H, Chen H, Ye L. Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells. Antiviral Res 2020; 174:104704. [PMID: 31917237 DOI: 10.1016/j.antiviral.2020.104704] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Revised: 12/15/2019] [Accepted: 01/02/2020] [Indexed: 02/06/2023]
Abstract
AIMS Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. METHODS HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. RESULTS Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. CONCLUSIONS Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.
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Affiliation(s)
- Weibo Liao
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Xin Liu
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Quanlue Yang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Huifang Liu
- Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Bingyu Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Junjun Jiang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Jiegang Huang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Chuanyi Ning
- Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Ning Zang
- Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Bo Zhou
- Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Yanyan Liao
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Jingzhao Chen
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Li Tian
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Wenzhe Ho
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, 19140, USA
| | - Abu S Abdullah
- Boston University School of Medicine, Boston Medical Center, Boston, MA, 02118, USA
| | - Lingbao Kong
- Institute of Pathogenic Microorganism, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, 330045, Jiangxi, China
| | - Hao Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China.
| | - Hui Chen
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China.
| | - Li Ye
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China.
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Dash S, Aydin Y, Moroz K. Chaperone-Mediated Autophagy in the Liver: Good or Bad? Cells 2019; 8:E1308. [PMID: 31652893 PMCID: PMC6912708 DOI: 10.3390/cells8111308] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2019] [Revised: 10/17/2019] [Accepted: 10/22/2019] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection triggers autophagy processes, which help clear out the dysfunctional viral and cellular components that would otherwise inhibit the virus replication. Increased cellular autophagy may kill the infected cell and terminate the infection without proper regulation. The mechanism of autophagy regulation during liver disease progression in HCV infection is unclear. The autophagy research has gained a lot of attention recently since autophagy impairment is associated with the development of hepatocellular carcinoma (HCC). Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) are three autophagy processes involved in the lysosomal degradation and extracellular release of cytosolic cargoes under excessive stress. Autophagy processes compensate for each other during extreme endoplasmic reticulum (ER) stress to promote host and microbe survival as well as HCC development in the highly stressed microenvironment of the cirrhotic liver. This review describes the molecular details of how excessive cellular stress generated during HCV infection activates CMA to improve cell survival. The pathological implications of stress-related CMA activation resulting in the loss of hepatic innate immunity and tumor suppressors, which are most often observed among cirrhotic patients with HCC, are discussed. The oncogenic cell programming through autophagy regulation initiated by a cytoplasmic virus may facilitate our understanding of HCC mechanisms related to non-viral etiologies and metabolic conditions such as uncontrolled type II diabetes. We propose that a better understanding of how excessive cellular stress leads to cancer through autophagy modulation may allow therapeutic development and early detection of HCC.
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Affiliation(s)
- Srikanta Dash
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
- Southeast Louisiana Veterans Health Care System, 2400 Canal Street, New Orleans, LA 70119, USA.
| | - Yucel Aydin
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
| | - Krzysztof Moroz
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
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Sempere RN, Arias A. Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides. Viruses 2019; 11:E560. [PMID: 31212939 PMCID: PMC6630443 DOI: 10.3390/v11060560] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2019] [Revised: 06/02/2019] [Accepted: 06/15/2019] [Indexed: 12/30/2022] Open
Abstract
Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5' end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.
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Affiliation(s)
- Raquel Navarro Sempere
- Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
- Abiopep Sociedad Limitada, Parque Científico de Murcia, 30100 Murcia, Spain.
| | - Armando Arias
- Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
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9
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Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model. Sci Rep 2018. [PMID: 29545599 DOI: 10.1038/s41598–018–22620–2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Ribavirin (RBV) is a broad-spectrum antiviral active against a wide range of RNA viruses. Despite having been used for decades in the treatment of chronic hepatitis C virus (HCV) infection, the precise mechanism of action of RBV is unknown. In other viruses, it inhibits propagation by increasing the rate of G-to-A and C-to-U transitions. Here, we utilized the J6/JFH1 HCV cell-culture system to investigate whether RBV inhibits HCV through the same mechanism. Infected Huh7.5 cells were treated with increasing concentrations of RBV or its phosphorylated forms. A fragment of the HCV NS5B-polymerase gene was amplified, cloned, and sequenced to estimate genetic distances. We confirm that the antiviral effect of all three RBV-drug forms on HCV relies on induction of specific transitions (G-to-A and C-to-U). These mutations lead to generation of non-infectious virions, reflected by decreased spread of HCV in cell culture despite relatively limited effect on virus genome titers. Moreover, treatment experiments conducted on a novel Huh7.5 cell line stably overexpressing adenosine kinase, a key enzyme for RBV activation, yielded comparable results. This study indicates that RBV action on HCV in hepatoma cell-culture is exerted through increase in mutagenesis, mediated by RBV triphosphate, and leading to production of non-infectious viruses.
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10
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Galli A, Mens H, Gottwein JM, Gerstoft J, Bukh J. Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model. Sci Rep 2018; 8:4619. [PMID: 29545599 PMCID: PMC5854589 DOI: 10.1038/s41598-018-22620-2] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2017] [Accepted: 02/26/2018] [Indexed: 01/18/2023] Open
Abstract
Ribavirin (RBV) is a broad-spectrum antiviral active against a wide range of RNA viruses. Despite having been used for decades in the treatment of chronic hepatitis C virus (HCV) infection, the precise mechanism of action of RBV is unknown. In other viruses, it inhibits propagation by increasing the rate of G-to-A and C-to-U transitions. Here, we utilized the J6/JFH1 HCV cell-culture system to investigate whether RBV inhibits HCV through the same mechanism. Infected Huh7.5 cells were treated with increasing concentrations of RBV or its phosphorylated forms. A fragment of the HCV NS5B-polymerase gene was amplified, cloned, and sequenced to estimate genetic distances. We confirm that the antiviral effect of all three RBV-drug forms on HCV relies on induction of specific transitions (G-to-A and C-to-U). These mutations lead to generation of non-infectious virions, reflected by decreased spread of HCV in cell culture despite relatively limited effect on virus genome titers. Moreover, treatment experiments conducted on a novel Huh7.5 cell line stably overexpressing adenosine kinase, a key enzyme for RBV activation, yielded comparable results. This study indicates that RBV action on HCV in hepatoma cell-culture is exerted through increase in mutagenesis, mediated by RBV triphosphate, and leading to production of non-infectious viruses.
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Affiliation(s)
- Andrea Galli
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Helene Mens
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
| | - Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jan Gerstoft
- Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
- Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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11
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Aydin Y, Chatterjee A, Chandra PK, Chava S, Chen W, Tandon A, Dash A, Chedid M, Moehlen MW, Regenstein F, Balart LA, Cohen A, Lu H, Wu T, Dash S. Interferon-alpha-induced hepatitis C virus clearance restores p53 tumor suppressor more than direct-acting antivirals. Hepatol Commun 2017; 1:256-269. [PMID: 29404458 PMCID: PMC5721446 DOI: 10.1002/hep4.1025] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Revised: 02/07/2017] [Accepted: 02/11/2017] [Indexed: 12/15/2022] Open
Abstract
The mechanism why hepatitis C virus (HCV) clearance by direct-acting antivirals (DAAs) does not eliminate the risk of hepatocellular carcinoma (HCC) among patients with advanced cirrhosis is unclear. Many viral and bacterial infections degrade p53 in favor of cell survival to adapt an endoplasmic reticulum (ER)-stress response. In this study, we examined whether HCV clearance by interferon-alpha or DAAs normalizes the ER stress and restores the expression of p53 tumor suppressor in cell culture. We found that HCV infection induces chronic ER stress and unfolded protein response in untransformed primary human hepatocytes. The unfolded protein response induces chaperone-mediated autophagy (CMA) in infected primary human hepatocytes and Huh-7.5 cells that results in degradation of p53 and induced expression of mouse double minute 2 (Mdm2). Inhibition of p53/Mdm2 interactions by small molecule (nutlin-3) or silencing Mdm2 did not rescue the p53 degradation, indicating that HCV infection induces degradation of p53 independent of the Mdm2 pathway. Interestingly, we found that HCV infection degrades p53 in a lysosome-dependent mechanism because lysosome-associated membrane protein 2A silencing restored p53 degradation. Our results show that HCV clearance induced by interferon-alpha-based antiviral therapies normalizes the ER-stress response and restores p53, whereas HCV clearance by DAAs does neither. We show that decreased expression of p53 in HCV-infected cirrhotic liver is associated with expression of chaperones associated with ER stress and the CMA response. Conclusion: HCV-induced ER stress and CMA promote p53 degradation in advanced liver cirrhosis. HCV clearance by DAAs does not restore p53, which provides a potential explanation for why a viral cure by DAAs does not eliminate the HCC risk among patients with advanced liver disease. We propose that resolving the ER-stress response is an alternative approach to reducing HCC risk among patients with cirrhosis after viral cure. (Hepatology Communications 2017;1:256-269).
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Affiliation(s)
- Yucel Aydin
- Department of Medicine, Division of Gastroenterology and Hepatology Tulane University Health Sciences Center New Orleans LA
| | - Animesh Chatterjee
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Partha K Chandra
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Srinivas Chava
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Weina Chen
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Anamika Tandon
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Asha Dash
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Milad Chedid
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Martin W Moehlen
- Department of Medicine, Division of Gastroenterology and Hepatology Tulane University Health Sciences Center New Orleans LA
| | - Frederic Regenstein
- Department of Medicine, Division of Gastroenterology and Hepatology Tulane University Health Sciences Center New Orleans LA
| | - Luis A Balart
- Department of Medicine, Division of Gastroenterology and Hepatology Tulane University Health Sciences Center New Orleans LA
| | - Ari Cohen
- Liver Transplant Surgery Section Ochsner Medical Center New Orleans LA
| | - Hua Lu
- Department of Biochemistry Tulane University Health Sciences Center New Orleans LA
| | - Tong Wu
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
| | - Srikanta Dash
- Department of Medicine, Division of Gastroenterology and Hepatology Tulane University Health Sciences Center New Orleans LA.,Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center New Orleans LA
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12
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Fang S, Su J, Liang B, Li X, Li Y, Jiang J, Huang J, Zhou B, Ning C, Li J, Ho W, Li Y, Chen H, Liang H, Ye L. Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells. Sci Rep 2017; 7:44039. [PMID: 28276509 PMCID: PMC5343675 DOI: 10.1038/srep44039] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Accepted: 02/02/2017] [Indexed: 12/20/2022] Open
Abstract
Previous studies have shown that mycophenolic acid (MPA) has an anti-HCV activity. However, the mechanism of MPA-mediated inhibition of HCV replication remains to be determined. This study investigated whether MPA has an effect on autophagy, a cellular machinery required for HCV replication, thereby, inhibits HCV replication in Huh7 cells. MPA treatment of Huh7 cells could suppress autophagy, evidenced by decreased LC3B-II level and conversion of LC3B-I to LC3B-II, decreased autophagosome formation, and increased p62 level compared to MPA-untreated cells. Tunicamycin treatment or HCV infection could induce cellular autophagy, however, MPA also exhibited its inhibitory effect on tunicamycin- or HCV infection-induced autophagy. The expression of three autophagy-related genes, Atg3, Atg5, and Atg7 were identified to be inhibited by MPA treatment. Over-expression of these genes could partly recover HCV replication inhibited by MPA; however, silencing their expression by siRNAs could enhance the inhibitory effect of MPA on HCV. Collectively, these results reveal that suppression of autophagy by MPA plays a role in its anti-HCV activity. Down-regulating the expression of three autophagy-related genes by MPA involves in its antiviral mechanism.
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Affiliation(s)
- Shoucai Fang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jinming Su
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Division of HIV/AIDS Control and Prevention, Guangxi Center for Disease Control and Prevention, Nanning 530021, Guangxi, China
| | - Bingyu Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Xu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Yu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Junjun Jiang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jiegang Huang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Bo Zhou
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Chuanyi Ning
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jieliang Li
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA
| | - Wenzhe Ho
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA
| | - Yiping Li
- Institute of Human Virology and Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
| | - Hui Chen
- Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Hao Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Li Ye
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
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13
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Li X, Li Y, Fang S, Su J, Jiang J, Liang B, Huang J, Zhou B, Zang N, Ho W, Li J, Li Y, Chen H, Ye L, Liang H. Downregulation of autophagy-related gene ATG5 and GABARAP expression by IFN-λ1 contributes to its anti-HCV activity in human hepatoma cells. Antiviral Res 2017; 140:83-94. [PMID: 28131804 DOI: 10.1016/j.antiviral.2017.01.016] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2016] [Accepted: 01/23/2017] [Indexed: 02/07/2023]
Abstract
Type-III interferon (IFN-λ), the most recently discovered family of IFNs, shares common features with type I IFNs, but also has many distinctive activities. It is not clear that whether IFN-λ has additional antiviral mechanisms. In this study, we investigated the effects of IFN-λ on autophagy, a cellular process closely related to hepatitis C virus (HCV) infection in human hepatoma Huh7 cells. Our results showed that IFN-λ1 treatment inhibit autophagic activity in Huh7 cells, as evidenced by the decreased expression of microtubule-associated protein 1 light chain 3B (LC3B)-II and conversion of LC3B-I to LC3B-II, decreased formation of GFP-LC3 puncta and accumulation of autophagosomes. IFN-λ1 could also inhibit HCV-induced or tunicamycin (a known inducer of autophagy with similar mechanism to HCV infection) -induced LC3B-II expression and autophagosome formation. Through PCR array, real time RT PCR, and western blot, two autophagy-related genes, ATG5 and GABARAP, were identified and verified to be down-regulated by IFN-λ1 treatment, either in HCV-uninfected Huh7 cells or in HCV JFH-1-infected cells. Overexpression of ATG5 and/or GABARAP could partly recover the IFN-λ1-inhibited HCV replication. Mechanism research demonstrated that IFN-λ1 could induce the expression of miR-181a and miR-214 (targeting ATG5 and GABARAP respectively), by which down-regulates ATG5 and GABARAP expression. Taken together, our results indicate that suppression of the autophagy response by IFN-λ1 contributes to IFN-λ1 anti-HCV activity. The results also provide a theoretical basis for improving the effectiveness of IFN treatment of HCV infection through inhibition of the HCV-induced autophagy response.
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Affiliation(s)
- Xu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Yu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Medical Insurance Department, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, 530021, Guangxi, China
| | - Shoucai Fang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Jinming Su
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Division of HIV/AIDS Control and Prevention, Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention, Nanning, 530021, Guangxi, China
| | - Junjun Jiang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Bingyu Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Jiegang Huang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Bo Zhou
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Ning Zang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
| | - Wenzhe Ho
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, 19140, USA
| | - Jieliang Li
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, 19140, USA
| | - Yiping Li
- Institute of Human Virology and Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China
| | - Hui Chen
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of GuangXi Medical University, Nanning, 530021, Guangxi, China
| | - Li Ye
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China.
| | - Hao Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China; Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China.
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14
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Baloch K, Chen L, Memon AA, Dexter L, Irving W, Ilyas M, Thomson BJ. Equilibrative nucleoside transporter 1 expression in primary human hepatocytes is highly variable and determines uptake of ribavirin. Antivir Chem Chemother 2017; 25:2-10. [PMID: 28417642 DOI: 10.1177/2040206616686894] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Aims Ribavirin is a nucleoside analogue and remains a necessary component of both interferon-based and directly acting anti-viral regimens for the treatment of hepatitis C virus infection. The achievable concentration of ribavirin within hepatocytes is likely to be an important determinant of therapeutic outcome. In vitro expression levels of equilibrative nucleoside transporter 1 (ENT1) has been shown to be a predictor of treatment response in patients receiving nucleoside-based chemotherapeutic agents. We therefore investigated whether a similar relationship existed between ENT1 expression and ribavirin uptake in freshly isolated primary hepatocytes. Methods Primary hepatocytes were cultured on collagen-coated plates and exposed to ribavirin. Parallel samples were taken for high-performance liquid chromatography to assess ribavirin uptake and for quantitative polymerase chain reaction to evaluate ENT1 expression. Similar assays were performed on the human hepatoma cell line (Huh7). ENT1 gene sequence was analysed by cloning of polymerase chain reaction amplified complementary DNA followed by direct sequencing. Results There was a strong direct correlation between expression of ENT1 in primary hepatocytes and ribavirin uptake at 24 hr. Huh7 cells expressed ENT1 at similar levels to the majority of primary hepatocytes, but did not take up ribavirin. Sequencing revealed that ENT1 in Huh7 cells is wild type. Conclusions In this study, we clearly demonstrate that ribavirin uptake in primary human hepatocytes is variable and correlates with ENT1 expression. This variation in ENT1 expression may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy.
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Affiliation(s)
- Kanwal Baloch
- 1 School of Medicine, University of Nottingham, Nottingham, UK.,2 Department of Pathology, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan
| | - Liqiong Chen
- 3 School of Pharmacy, University of Nottingham, Nottingham, UK.,4 AEM iMed, AstraZeneca, Shanghai, China
| | - Ameer A Memon
- 2 Department of Pathology, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan
| | - Laura Dexter
- 3 School of Pharmacy, University of Nottingham, Nottingham, UK.,5 Wales Specialist Virology Centre, University Hospital of Wales, Heath Park, Cardiff, UK
| | - William Irving
- 6 Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, UK.,7 Nottingham Digestive Diseases Centre Biomedical Research Unit, Nottingham University Hospitals, Nottingham, UK
| | - Mohammad Ilyas
- 1 School of Medicine, University of Nottingham, Nottingham, UK
| | - Brian J Thomson
- 1 School of Medicine, University of Nottingham, Nottingham, UK.,7 Nottingham Digestive Diseases Centre Biomedical Research Unit, Nottingham University Hospitals, Nottingham, UK
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15
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Hepatitis C Virus Infection Induces Autophagy as a Prosurvival Mechanism to Alleviate Hepatic ER-Stress Response. Viruses 2016; 8:v8050150. [PMID: 27223299 PMCID: PMC4885105 DOI: 10.3390/v8050150] [Citation(s) in RCA: 58] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2016] [Revised: 05/04/2016] [Accepted: 05/18/2016] [Indexed: 12/17/2022] Open
Abstract
Hepatitis C virus (HCV) infection frequently leads to chronic liver disease, liver cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms by which HCV infection leads to chronic liver disease and HCC are not well understood. The infection cycle of HCV is initiated by the attachment and entry of virus particles into a hepatocyte. Replication of the HCV genome inside hepatocytes leads to accumulation of large amounts of viral proteins and RNA replication intermediates in the endoplasmic reticulum (ER), resulting in production of thousands of new virus particles. HCV-infected hepatocytes mount a substantial stress response. How the infected hepatocyte integrates the viral-induced stress response with chronic infection is unknown. The unfolded protein response (UPR), an ER-associated cellular transcriptional response, is activated in HCV infected hepatocytes. Over the past several years, research performed by a number of laboratories, including ours, has shown that HCV induced UPR robustly activates autophagy to sustain viral replication in the infected hepatocyte. Induction of the cellular autophagy response is required to improve survival of infected cells by inhibition of cellular apoptosis. The autophagy response also inhibits the cellular innate antiviral program that usually inhibits HCV replication. In this review, we discuss the physiological implications of the HCV-induced chronic ER-stress response in the liver disease progression.
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16
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Ferraris P, Chandra PK, Panigrahi R, Aboulnasr F, Chava S, Kurt R, Pawlotsky JM, Wilkens L, Osterlund P, Hartmann R, Balart LA, Wu T, Dash S. Cellular Mechanism for Impaired Hepatitis C Virus Clearance by Interferon Associated with IFNL3 Gene Polymorphisms Relates to Intrahepatic Interferon-λ Expression. THE AMERICAN JOURNAL OF PATHOLOGY 2016; 186:938-51. [PMID: 26896692 PMCID: PMC5807932 DOI: 10.1016/j.ajpath.2015.11.027] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2015] [Revised: 11/13/2015] [Accepted: 11/24/2015] [Indexed: 12/12/2022]
Abstract
The single nucleotide polymorphism located within the IFNL3 (also known as IL28B) promoter is one of the host factors associated with hepatitis C virus (HCV) clearance by interferon (IFN)-α therapy; however the mechanism remains unknown. We investigated how IL28B gene polymorphism influences HCV clearance with infected primary human hepatocytes, liver biopsies, and hepatoma cell lines. Our study confirms that the rs12979860-T/T genotype has a strong correlation with ss469415590-ΔG/ΔG single nucleotide polymorphism that produces IFN-λ4 protein. We found that IFN-α and IFN-λ1 antiviral activity against HCV was impaired in IL28B T/T infected hepatocytes compared with C/C genotype. Western blot analysis showed that IL28B TT genotype hepatocytes expressed higher levels of IFN-λ proteins (IL28B, IL-29), preactivated IFN-stimulated gene (ISG) expression, and impaired Stat phosphorylation when stimulated with either IFN-α or IFN-λ1. Furthermore, we showed that silencing IFN-λ1 in T/T cell line reduced basal ISG expression and improved antiviral activity. Likewise, overexpression of IFN-λ (1 to 4) in C/C cells induced basal ISG expression and prevented IFN-α antiviral activity. We showed that IFN-λ4, produced at low level only in T/T cells induced expression of IL28B and IL-29 and prevented IFN-α antiviral activity in HCV cell culture. Our results suggest that IFN-λ4 protein expression associated with the IL28B-T/T variant preactivates the Janus kinase-Stat signaling, leading to impaired HCV clearance by both IFN-α and IFN-λ.
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Affiliation(s)
- Pauline Ferraris
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Partha K Chandra
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Rajesh Panigrahi
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Fatma Aboulnasr
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Srinivas Chava
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Ramazan Kurt
- Department of Medicine, Gastroenterology, and Hepatology, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Jean-Michel Pawlotsky
- Department of Molecular Virology and Immunology, Institut Mondor de la Recherche, Creteil, France
| | - Ludwig Wilkens
- Institute of Pathology, University of Bern, Bern, Switzerland
| | - Pamela Osterlund
- Department of Vaccination and Immune Protection Viral Infections, National Institute for Health and Welfare, Helsinki, Finland
| | - Rune Hartmann
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
| | - Luis A Balart
- Department of Medicine, Gastroenterology, and Hepatology, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Tong Wu
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana
| | - Srikanta Dash
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana; Department of Medicine, Gastroenterology, and Hepatology, Tulane University Health Sciences Center, New Orleans, Louisiana.
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Aboulnasr F, Hazari S, Nayak S, Chandra PK, Panigrahi R, Ferraris P, Chava S, Kurt R, Song K, Dash A, Balart LA, Garry RF, Wu T, Dash S. IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System. PLoS One 2015; 10:e0141655. [PMID: 26657215 PMCID: PMC4686105 DOI: 10.1371/journal.pone.0141655] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Accepted: 10/12/2015] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND HCV replication in persistently infected cell culture remains resistant to IFN-α/RBV combination treatment, whereas IFN-λ1 induces viral clearance. The antiviral mechanisms by which IFN-λ1 induces sustained HCV clearance have not been determined. AIM To investigate the mechanisms by which IFN-λ clears HCV replication in an HCV cell culture model. METHODS IFN-α sensitive (S3-GFP) and resistant (R4-GFP) cells were treated with equivalent concentrations of either IFN-α or IFN-λ. The relative antiviral effects of IFN-α and IFN-λ1 were compared by measuring the HCV replication, quantification of HCV-GFP expression by flow cytometry, and viral RNA levels by real time RT-PCR. Activation of Jak-Stat signaling, interferon stimulated gene (ISG) expression, and miRNA-122 transcription in S3-GFP and R4-GFP cells were examined. RESULTS We have shown that IFN-λ1 induces HCV clearance in IFN-α resistant and sensitive replicon cell lines in a dose dependent manner through Jak-Stat signaling, and induces STAT 1 and STAT 2 activation, ISRE-luciferase promoter activation and ISG expression. Stat 3 activation is also involved in IFN-λ1 induced antiviral activity in HCV cell culture. IFN-λ1 induced Stat 3 phosphorylation reduces the expression of hepatocyte nuclear factor 4 alpha (HNF4α) through miR-24 in R4-GFP cells. Reduced expression of HNF4α is associated with decreased expression of miR-122 resulting in an anti-HCV effect. Northern blot analysis confirms that IFN-λ1 reduces miR-122 levels in R4-GFP cells. Our results indicate that IFN-λ1 activates the Stat 3-HNF4α feedback inflammatory loop to inhibit miR-122 transcription in HCV cell culture. CONCLUSIONS In addition to the classical Jak-Stat antiviral signaling pathway, IFN-λ1 inhibits HCV replication through the suppression of miRNA-122 transcription via an inflammatory Stat 3-HNF4α feedback loop. Inflammatory feedback circuits activated by IFNs during chronic inflammation expose non-responders to the risk of hepatocellular carcinoma.
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Affiliation(s)
- Fatma Aboulnasr
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Sidhartha Hazari
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Satyam Nayak
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Partha K. Chandra
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Rajesh Panigrahi
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Pauline Ferraris
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Srinivas Chava
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Ramazan Kurt
- Department of Medicine, Division of Gastroenterology and Hepatology
| | - Kyongsub Song
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Asha Dash
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Luis A. Balart
- Department of Medicine, Division of Gastroenterology and Hepatology
| | - Robert F. Garry
- Microbiology and Immunology Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Tong Wu
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
| | - Srikanta Dash
- Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA-70112, United States of America
- Department of Medicine, Division of Gastroenterology and Hepatology
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18
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Panigrahi R, Dash S. ENT1 and treatment of viral diseases. Oncotarget 2015; 6:32281-2. [PMID: 26431496 PMCID: PMC4741678 DOI: 10.18632/oncotarget.5859] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2015] [Accepted: 09/28/2015] [Indexed: 11/25/2022] Open
Affiliation(s)
- Rajesh Panigrahi
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA
| | - Srikanta Dash
- Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA
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19
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Ploen D, Hildt E. Hepatitis C virus comes for dinner: How the hepatitis C virus interferes with autophagy. World J Gastroenterol 2015; 21:8492-8507. [PMID: 26229393 PMCID: PMC4515832 DOI: 10.3748/wjg.v21.i28.8492] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Revised: 05/10/2015] [Accepted: 06/16/2015] [Indexed: 02/06/2023] Open
Abstract
Autophagy is a highly-regulated, conserved cellular process for the degradation of intracellular components in lysosomes to maintain the energetic balance of the cell. It is a pro-survival mechanism that plays an important role during development, differentiation, apoptosis, ageing and innate and adaptive immune response. Besides, autophagy has been described to be involved in the development of various human diseases, e.g., chronic liver diseases and the development of hepatocellular carcinoma. The hepatitis C virus (HCV) is a major cause of chronic liver diseases. It has recently been described that HCV, like other RNA viruses, hijacks the autophagic machinery to improve its replication. However, the mechanisms underlying its activation are conflicting. HCV replication and assembly occurs at the so-called membranous web that consists of lipid droplets and rearranged endoplasmic reticulum-derived membranes including single-, double- and multi-membrane vesicles. The double-membrane vesicles have been identified to contain NS3, NS5A, viral RNA and the autophagosomal marker microtubule-associated protein 1 light chain 3, corroborating the involvement of the autophagic pathway in the HCV life-cycle. In this review, we will highlight the crosstalk of the autophagosomal compartment with different steps of the HCV life-cycle and address its implications on favoring the survival of infected hepatocytes.
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Rothan HA, Bahrani H, Mohamed Z, Teoh TC, Shankar EM, Rahman NA, Yusof R. A combination of doxycycline and ribavirin alleviated chikungunya infection. PLoS One 2015; 10:e0126360. [PMID: 25970853 PMCID: PMC4430285 DOI: 10.1371/journal.pone.0126360] [Citation(s) in RCA: 83] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2014] [Accepted: 04/01/2015] [Indexed: 01/18/2023] Open
Abstract
Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.
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Affiliation(s)
- Hussin A. Rothan
- Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
- * E-mail:
| | - Hirbod Bahrani
- Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
| | - Zulqarnain Mohamed
- Institute of biological sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
| | - Teow Chong Teoh
- Institute of biological sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
| | - Esaki M. Shankar
- Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
| | - Noorsaadah A. Rahman
- Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
| | - Rohana Yusof
- Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
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21
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Kurt R, Chandra PK, Aboulnasr F, Panigrahi R, Ferraris P, Aydin Y, Reiss K, Wu T, Balart LA, Dash S. Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture. PLoS One 2015; 10:e0125962. [PMID: 25961570 PMCID: PMC4427131 DOI: 10.1371/journal.pone.0125962] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 03/27/2015] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Hepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired. AIM To investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment. METHOD HCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated. RESULTS FFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA). CONCLUSION Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.
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Affiliation(s)
- Ramazan Kurt
- Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Partha K. Chandra
- Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Fatma Aboulnasr
- Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Rajesh Panigrahi
- Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Pauline Ferraris
- Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Yucel Aydin
- Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Krzysztof Reiss
- Neurological Cancer Research, Stanley S Scott Cancer Center, LSU Health Science Center, New Orleans, Louisiana, United States of America
| | - Tong Wu
- Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Luis A. Balart
- Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Srikanta Dash
- Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
- Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
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