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Zhong Q, Qi J, Su N, Li Z, Wang C, Zeng H, Liu R, Li Y, Yang Q. In vivo investigation of PEDV transmission via nasal infection: mechanisms of CD4 + T-cell-mediated intestinal infection. J Virol 2025:e0176124. [PMID: 40094365 DOI: 10.1128/jvi.01761-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2024] [Accepted: 02/03/2025] [Indexed: 03/19/2025] Open
Abstract
The porcine epidemic diarrhea virus (PEDV), a highly pathogenic coronavirus, poses significant challenges to global swine agriculture with severe economic consequences. Our research reveals that in addition to known transmission routes, PEDV can be airborne, initially invading the nasal mucosa and subsequently being transported by dendritic cells and peripheral blood T cells, ultimately leading to intestinal disease in piglets. This study elucidates the cellular mechanisms behind the process, demonstrating how PEDV is internalized by CD4+ T cells after being transferred by dendritic cells, where it establishes a latent infection. Crucially, PEDV induces the upregulation of the integrin α4β7 homing receptor, facilitating the migration of these infected CD4+ T cells to the small intestine. Furthermore, our findings reveal that the activation of the α4β7-Rho-GTPases-Cofilin signaling pathway by PEDV reorganizes the actin cytoskeleton, enabling CD4+ T-cell transmigration through high endothelial venules into the intestinal mucosa, resulting in the infection of intestinal epithelial cells. These insights not only illuminate the molecular mechanisms PEDV employs to hijack CD4+ T cells for transmission from the respiratory tract to the intestine but also identify novel targets for therapeutic intervention, providing new perspectives for effectively preventing and managing PEDV infection with broader implications for controlling similar pathogens in diverse hosts.IMPORTANCEPorcine epidemic diarrhea virus (PEDV), characterized by rapid transmission and widespread prevalence, poses a significant long-term threat to the global pig farming industry. Our previous research revealed that, in addition to the classic fecal-oral infection route, PEDV can invade through the nasal mucosa, leading to intestinal infection. This study further investigated the molecular mechanisms by which the virus is transported by T lymphocytes from the respiratory tract to the intestines. We found that PEDV establishes a latent infection in CD4+ T cells and promotes their intestinal homing by upregulating the homing receptor integrin α4β7. Additionally, we elucidated the activation of the integrin α4β7-mediated Rho-GTPase-Cofilin signaling axis by PEDV, which regulates pseudopod formation and facilitates CD4+ T-cell migration to the intestinal mucosal lamina propria post-homing. This study elucidates the mechanism underlying the lymphocyte-dependent dissemination of PEDV following nasal infection, providing new insights into strategies for preventing PEDV invasion.
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Affiliation(s)
- Qiu Zhong
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Jiaxin Qi
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Na Su
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Zi Li
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, Jilin, China
| | - Chengcheng Wang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Hui Zeng
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Ruiling Liu
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Yuchen Li
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
| | - Qian Yang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China
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2
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Ke PY, Yeh CT. Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy. Pathogens 2024; 13:980. [PMID: 39599533 PMCID: PMC11597459 DOI: 10.3390/pathogens13110980] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2024] [Revised: 10/30/2024] [Accepted: 11/07/2024] [Indexed: 11/29/2024] Open
Abstract
Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host-virus interactions.
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Affiliation(s)
- Po-Yuan Ke
- Department of Biochemistry and Molecular Biology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
| | - Chau-Ting Yeh
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
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3
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Van Campenhout R, Vinken M. Hepatic cell junctions: Pulling a double-duty. Liver Int 2024; 44:2873-2889. [PMID: 39115254 DOI: 10.1111/liv.16045] [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: 02/29/2024] [Revised: 06/26/2024] [Accepted: 07/09/2024] [Indexed: 10/25/2024]
Abstract
Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in the structural and functional organization of multicellular tissues, including in liver. Specifically, hepatic cell junctions mediate intercellular adhesion and communication between liver cells. The establishment of the hepatic cell junction network is a prerequisite for normal liver functioning. Hepatic cell junctions indeed support liver-specific features and control essential aspects of the hepatic life cycle. This review paper summarizes the role of cell junctions and their components in relation to liver physiology, thereby also discussing their involvement in hepatic dysfunctionality, including liver disease and toxicity.
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Affiliation(s)
- Raf Van Campenhout
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mathieu Vinken
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
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Ma Y, Xu J, Hao L, Wang G, Huang W, Liu Z. MiR-181a Negatively Regulates Claudin-3 to Facilitate Lateolabrax maculatus Iridovirus Replication in Lateolabrax maculatus Astroglia Cells. Viruses 2024; 16:1589. [PMID: 39459923 PMCID: PMC11512373 DOI: 10.3390/v16101589] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 09/22/2024] [Accepted: 10/01/2024] [Indexed: 10/28/2024] Open
Abstract
Lateolabrax maculatus iridovirus (LMIV) is a variant strain of red sea bream iridovirus (RSIV), causing serious economic losses in aquaculture. Claudins (CLDNs) are major components of tight junctions (TJs) forming an important line of defense against pathogens. Our pilot miRNA-mRNA joint analysis indicated the degradation of CLDN3, as well as its interaction with miR-181a during LMIV infection. To elucidate the miR-181a/CLDN3/LMIV interactions, in vitro assays were carried out on LMB-L cells. We first confirmed that LMIV infection could decrease the expression of CLDN3, accompanied by the enhancement of permeability, suggesting the dysfunction of TJs. Contrary to the inhibition of CLDN3, the activation of miR-181a was proved, presenting a negative correlation between miR-181a and CLDN3 (Pearson r = -0.773 and p < 0.01). In addition, the influence of CLDN3 on LMIV replication was analyzed by knockdown and over-expression of CLDN3. When CLDN3 was silenced in LMB-L cells with siCLDN3-623 at 9 days post transfection (dpt), LMIV copies and titers were significantly up-regulated by 1.59-fold and 13.87-fold, respectively. By contrast, LMIV replication in LMB-L cells was reduced by 60% and 71%, post transfection with pcDNA3.1-CLDN3 over-expressed plasmid at 6 dpt and 9 dpt, respectively. Ultimately, the regulatory relationship between miR-181a and CLDN3 was further validated by dual luciferase reporter assays. Taking into account the above-described results, we proposed a "miR-181a/CLDN3/LMIV" regulatory relationship. This study provides a new insight for understanding the mechanism of LMIV replication.
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Affiliation(s)
- Yanping Ma
- Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; (Y.M.); (J.X.); (L.H.); (G.W.)
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
| | - Jingjing Xu
- Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; (Y.M.); (J.X.); (L.H.); (G.W.)
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
| | - Le Hao
- Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; (Y.M.); (J.X.); (L.H.); (G.W.)
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
| | - Gang Wang
- Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; (Y.M.); (J.X.); (L.H.); (G.W.)
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
| | - Wen Huang
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
| | - Zhenxing Liu
- Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; (Y.M.); (J.X.); (L.H.); (G.W.)
- Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
- Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
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Du F, Xie Y, Wu S, Ji M, Dong B, Zhu C. Expression and Targeted Application of Claudins Family in Hepatobiliary and Pancreatic Diseases. J Hepatocell Carcinoma 2024; 11:1801-1821. [PMID: 39345937 PMCID: PMC11439345 DOI: 10.2147/jhc.s483861] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2024] [Accepted: 09/12/2024] [Indexed: 10/01/2024] Open
Abstract
Hepatobiliary and pancreatic diseases are becoming increasingly common worldwide and associated cancers are prone to recurrence and metastasis. For a more accurate treatment, new therapeutic strategies are urgently needed. The claudins (CLDN) family comprises a class of membrane proteins that are the main components of tight junctions, and are essential for forming intercellular barriers and maintaining cellular polarity. In mammals, the claudin family contains at least 27 transmembrane proteins and plays a major role in mediating cell adhesion and paracellular permeability. Multiple claudin proteins are altered in various cancers, including gastric cancer (GC), esophageal cancer (EC), hepatocellular carcinoma (HCC), pancreatic cancer (PC), colorectal cancer (CRC) and breast cancer (BC). An increasing number of studies have shown that claudins are closely associated with the occurrence and development of hepatobiliary and pancreatic diseases. Interestingly, claudin proteins exhibit different effects on cancer progression in different tumor tissues, including tumor suppression and promotion. In addition, various claudin proteins are currently being studied as potential diagnostic and therapeutic targets, including claudin-3, claudin-4, claudin-18.2, etc. In this article, the functional phenotype, molecular mechanism, and targeted application of the claudin family in hepatobiliary and pancreatic diseases are reviewed, with an emphasis on claudin-1, claudin-4, claudin-7 and claudin-18.2, and the current situation and future prospects are proposed.
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Affiliation(s)
- Fangqian Du
- Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China
| | - Yuwei Xie
- Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China
| | - Shengze Wu
- Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China
| | - Mengling Ji
- Department of Surgery, Institute of Biomedical Sciences, Tokushima University, Tokushima, Japan
| | - Bingzi Dong
- Department of Endocrinology and Metabolism, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China
| | - Chengzhan Zhu
- Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China
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Saviano A, Roehlen N, Baumert TF. Tight Junction Proteins as Therapeutic Targets to Treat Liver Fibrosis and Hepatocellular Carcinoma. Semin Liver Dis 2024; 44:180-190. [PMID: 38648796 DOI: 10.1055/s-0044-1785646] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 04/25/2024]
Abstract
In the last decade tight junction proteins exposed at the surface of liver or cancer cells have been uncovered as mediators of liver disease biology: Claudin-1 and Occludin are host factors for hepatitis C virus entry and Claudin-1 has been identified as a driver for liver fibrosis and hepatocellular carcinoma (HCC). Moreover, Claudins have emerged as therapeutic targets for liver disease and HCC. CLDN1 expression is upregulated in liver fibrosis and HCC. Monoclonal antibodies (mAbs) targeting Claudin-1 have completed preclinical proof-of-concept studies for treatment of liver fibrosis and HCC and are currently in clinical development for advanced liver fibrosis. Claudin-6 overexpression is associated with an HCC aggressive phenotype and treatment resistance. Claudin-6 mAbs or chimeric antigen receptor-T cells therapies are currently being clinically investigated for Claudin-6 overexpressing tumors. In conclusion, targeting Claudin proteins offers a novel clinical opportunity for the treatment of patients with advanced liver fibrosis and HCC.
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Affiliation(s)
- Antonio Saviano
- Inserm, U1110, Institute of Translational Medicine and Liver Disease, Strasbourg, France
- University of Strasbourg, Strasbourg, France
- Service d'hépato-gastroentérologie, Pôle Hépato-digestif, Institut-Hospitalo-Universitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - Natascha Roehlen
- Department of Medicine II, Gastroenterology, Hepatology, Endocrinology and Infectious Diseases, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Berta-Ottenstein-Programme, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Thomas F Baumert
- Inserm, U1110, Institute of Translational Medicine and Liver Disease, Strasbourg, France
- University of Strasbourg, Strasbourg, France
- Service d'hépato-gastroentérologie, Pôle Hépato-digestif, Institut-Hospitalo-Universitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
- Institut Universitaire de France, Paris, France
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7
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Vincenzi M, Mercurio FA, Leone M. EPHA2 Receptor as a Possible Therapeutic Target in Viral Infections. Curr Med Chem 2024; 31:5670-5701. [PMID: 37828671 DOI: 10.2174/0109298673256638231003111234] [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: 04/06/2023] [Revised: 08/02/2023] [Accepted: 08/24/2023] [Indexed: 10/14/2023]
Abstract
BACKGROUND The receptor tyrosine kinase EphA2 plays a role in many diseases, like cancer, cataracts, and osteoporosis. Interestingly, it has also been linked to viral infections. OBJECTIVE Herein, current literature has been reviewed to clarify EphA2 functions in viral infections and explore its potential role as a target in antiviral drug discovery strategies. METHODS Research and review articles along with preprints connecting EphA2 to different viruses have been searched through PubMed and the web. Structures of complexes between EphA2 domains and viral proteins have been retrieved from the PDB database. RESULTS EphA2 assumes a key role in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein Barr virus (EBV) infections by directly binding, through its ligand binding domain, viral glycoproteins. For human cytomegalovirus (HCMV), the role of EphA2 in maintaining virus latency state, through cooperation with specific viral proteins, has also been speculated. In certain cells, with high EphA2 expression levels, following ligand stimulation, receptor activation might contribute to severe symptoms accompanying a few viral infections, including lung injuries often related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). CONCLUSION Since EphA2 works as a host receptor for certain viruses, it might be worth more deeply investigating known compounds targeting its extracellular ligand binding domain as antiviral therapeutics. Due to EphA2's function in inflammation, its possible correlation with SARS-CoV-2 cannot be excluded, but more experimental studies are needed in this case to undoubtedly attribute the role of this receptor in viral infections.
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Affiliation(s)
- Marian Vincenzi
- Institute of Biostructures and Bioimaging, National Research Council of Italy (CNR-IBB), Naples, Italy
| | - Flavia Anna Mercurio
- Institute of Biostructures and Bioimaging, National Research Council of Italy (CNR-IBB), Naples, Italy
| | - Marilisa Leone
- Institute of Biostructures and Bioimaging, National Research Council of Italy (CNR-IBB), Naples, Italy
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Carriquí-Madroñal B, Lasswitz L, von Hahn T, Gerold G. Genetic and pharmacological perturbation of hepatitis-C virus entry. Curr Opin Virol 2023; 62:101362. [PMID: 37678113 DOI: 10.1016/j.coviro.2023.101362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Revised: 06/30/2023] [Accepted: 08/08/2023] [Indexed: 09/09/2023]
Abstract
Hepatitis-C virus (HCV) chronically infects 58 million individuals worldwide with variable disease outcome. While a subfraction of individuals exposed to the virus clear the infection, the majority develop chronic infection if untreated. Another subfraction of chronically ill proceeds to severe liver disease. The underlying causes of this interindividual variability include genetic polymorphisms in interferon genes. Here, we review available data on the influence of genetic or pharmacological perturbation of HCV host dependency factors on the clinically observed interindividual differences in disease outcome. We focus on host factors mediating virus entry into human liver cells. We assess available data on genetic variants of the major entry factors scavenger receptor class-B type I, CD81, claudin-1, and occludin as well as pharmacological perturbation of these entry factors. We review cell culture experimental and clinical cohort study data and conclude that entry factor perturbation may contribute to disease outcome of hepatitis C.
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Affiliation(s)
- Belén Carriquí-Madroñal
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany
| | - Lisa Lasswitz
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany
| | - Thomas von Hahn
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany; Department of Gastroenterology, Hepatology and Interventional Endoscopy, Asklepios Hospital Barmbek, Semmelweis University, Campus Hamburg, 22307 Hamburg, Germany
| | - Gisa Gerold
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umeå, Sweden; Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden.
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Sabei FY, Y Safhi A, Almoshari Y, Salawi A, H Sultan M, Ali Bakkari M, Alsalhi A, A Madkhali O, M Jali A, Ahsan W. Structure-based virtual screening of natural compounds as inhibitors of HCV using molecular docking and molecular dynamics simulation studies. J Biomol Struct Dyn 2023; 42:11574-11585. [PMID: 37776007 DOI: 10.1080/07391102.2023.2263588] [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: 04/03/2023] [Accepted: 08/28/2023] [Indexed: 10/01/2023]
Abstract
The hepatitis C virus (HCV), which causes hepatitis C, is a viral infection that damages the liver and causes inflammation in the liver. New potentially effective antiviral drugs are required for its treatment owing to various issues associated with the existing medications, including moderate to severe adverse effects, higher costs, and the emergence of drug-resistant strains. The objective of the current study was to utilize computational techniques to assess the anti-HCV efficacy of certain phytochemicals against tetraspanin (CD81) and claudin 1 (CLDN1) entry proteins. A 200-nanosecond molecular dynamics (MD) simulation was employed to examine the stability of the lead-protein complexes. Free binding energy and molecular docking calculations were conducted utilizing MM/GBSA method, and the selectivity of hit compounds for CD81 and CLDN1 was determined. Five significant CD81 and CLDN1 inhibitors were identified: Petasiphenone, Silibinin, Tanshinone IIA, Taxifolin, and Topaquinone. The MM/GBSA analysis of the compounds revealed high free binding energies. All the identified compounds were stable within the CD81 and CLDN1 binding pockets. This study indicated the promising inhibitory potential of the identified compounds against CD81 and CLDN1 receptors and might develop into potential viral entry inhibitors. However, to validate the chemotherapeutic capabilities of the discovered leads extensive preclinical research is required.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Fahad Y Sabei
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Awaji Y Safhi
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Yosif Almoshari
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Ahmad Salawi
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Muhammad H Sultan
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Mohammed Ali Bakkari
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Abdullah Alsalhi
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Osama A Madkhali
- Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Abdulmajeed M Jali
- Department of Pharmacology and Toxicology, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
| | - Waquar Ahsan
- Department of Pharmaceutical Chemistry and Pharmacognosy, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
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Toon K, Kalemera MD, Palor M, Rose NJ, Takeuchi Y, Grove J, Mattiuzzo G. GB Virus B and Hepatitis C Virus, Distantly Related Hepaciviruses, Share an Entry Factor, Claudin-1. J Virol 2023; 97:e0046923. [PMID: 37310242 PMCID: PMC10373534 DOI: 10.1128/jvi.00469-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Accepted: 05/10/2023] [Indexed: 06/14/2023] Open
Abstract
Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest that they have undergone specific adaptation and have evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus B (GBV-B), the first hepacivirus described in an animal after hepatitis C virus (HCV). GBV-B-pseudotyped viral particles (GBVBpp) were shown to be uniquely sensitive to the sera of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of human hepatoma cell lines that were CRISPR/Cas9 engineered to ablate the expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating the GBV-B and HCV share an entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on a C-terminal region containing the second extracellular loop. The observation that claudin-1 is an entry factor shared between these two hepaciviruses suggests that the tight junction protein is of fundamental mechanistic importance during cell entry. IMPORTANCE Hepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection and are at risk of developing cirrhosis and liver cancer. To achieve the World Health Organization's target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters cells can inform the design of new vaccines and treatments targeting the first stage of infection. However, the HCV cell entry mechanism is complex and has been sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as membrane fusion, and inform structure-guided HCV vaccine design; in this work, we have identified a protein, claudin-1, that facilitates the entry of an HCV-related hepacivirus but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil a commonality of entry factors and, possibly, new mechanisms.
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Affiliation(s)
- Kamilla Toon
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Mphatso D. Kalemera
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Machaela Palor
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Nicola J. Rose
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
| | - Yasuhiro Takeuchi
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Joe Grove
- Division of Infection and Immunity, University College London, London, United Kingdom
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Giada Mattiuzzo
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
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11
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Deng L, Liang P, Cui H. Pseudotyped lentiviral vectors: Ready for translation into targeted cancer gene therapy? Genes Dis 2022. [PMID: 37492721 PMCID: PMC10363566 DOI: 10.1016/j.gendis.2022.03.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Gene therapy holds great promise for curing cancer by editing the deleterious genes of tumor cells, but the lack of vector systems for efficient delivery of genetic material into specific tumor sites in vivo has limited its full therapeutic potential in cancer gene therapy. Over the past two decades, increasing studies have shown that lentiviral vectors (LVs) modified with different glycoproteins from a donating virus, a process referred to as pseudotyping, have altered tropism and display cell-type specificity in transduction, leading to selective tumor cell killing. This feature of LVs together with their ability to enable high efficient gene delivery in dividing and non-dividing mammalian cells in vivo make them to be attractive tools in future cancer gene therapy. This review is intended to summarize the status quo of some typical pseudotypings of LVs and their applications in basic anti-cancer studies across many malignancies. The opportunities of translating pseudotyped LVs into clinic use in cancer therapy have also been discussed.
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12
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Tight junction protein claudin-2 promotes cell entry of Bombyx mori cypovirus. Appl Microbiol Biotechnol 2021; 105:6019-6031. [PMID: 34324010 DOI: 10.1007/s00253-021-11456-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 06/24/2021] [Accepted: 07/03/2021] [Indexed: 10/20/2022]
Abstract
Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: •Claudin-2 could promote BmCPV infection of cells. •Claudin-2 interacted with BmCPV during BmCPV infection. •Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.
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13
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Beyenbach KW, Schöne F, Breitsprecher LF, Tiburcy F, Furuse M, Izumi Y, Meyer H, Jonusaite S, Rodan AR, Paululat A. The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster. Am J Physiol Cell Physiol 2020; 318:C1107-C1122. [PMID: 32267718 DOI: 10.1152/ajpcell.00061.2020] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.
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Affiliation(s)
- Klaus W Beyenbach
- Department of Zoology/Developmental Biology, University of Osnabrück, Osnabrück, Germany.,Department of Animal Physiology, University of Osnabrück, Osnabrück, Germany
| | - Frederike Schöne
- Department of Zoology/Developmental Biology, University of Osnabrück, Osnabrück, Germany
| | | | - Felix Tiburcy
- Department of Animal Physiology, University of Osnabrück, Osnabrück, Germany
| | - Mikio Furuse
- Division of Cell Structure, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan.,Department of Physiological Sciences, School of Life Science, Sokendai, The Graduate University for Advanced Studies, Okazaki, Japan
| | - Yasushi Izumi
- Division of Cell Structure, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan.,Department of Physiological Sciences, School of Life Science, Sokendai, The Graduate University for Advanced Studies, Okazaki, Japan
| | - Heiko Meyer
- Department of Zoology/Developmental Biology, University of Osnabrück, Osnabrück, Germany
| | - Sima Jonusaite
- Division of Nephrology and Hypertension, Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, Utah.,Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada
| | - Aylin R Rodan
- Division of Nephrology and Hypertension, Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, Utah
| | - Achim Paululat
- Department of Zoology/Developmental Biology, University of Osnabrück, Osnabrück, Germany
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14
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Mailly L, Baumert TF. Hepatitis C virus infection and tight junction proteins: The ties that bind. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2020; 1862:183296. [PMID: 32268133 DOI: 10.1016/j.bbamem.2020.183296] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/13/2020] [Revised: 03/25/2020] [Accepted: 03/28/2020] [Indexed: 02/07/2023]
Abstract
The hepatitis C virus (HCV) is a major cause of liver diseases ranging from liver inflammation to advanced liver diseases like cirrhosis and hepatocellular carcinoma (HCC). HCV infection is restricted to the liver, and more specifically to hepatocytes, which represent around 80% of liver cells. The mechanism of HCV entry in human hepatocytes has been extensively investigated since the discovery of the virus 30 years ago. The entry mechanism is a multi-step process relying on several host factors including heparan sulfate proteoglycan (HSPG), low density lipoprotein receptor (LDLR), tetraspanin CD81, Scavenger Receptor class B type I (SR-BI), Epidermal Growth Factor Receptor (EGFR) and Niemann-Pick C1-like 1 (NPC1L1). Moreover, in order to establish a persistent infection, HCV entry is dependent on the presence of tight junction (TJ) proteins Claudin-1 (CLDN1) and Occludin (OCLN). In the liver, tight junction proteins play a role in architecture and homeostasis including sealing the apical pole of adjacent cells to form bile canaliculi and separating the basolateral domain drained by sinusoidal blood flow. In this review, we will highlight the role of liver tight junction proteins in HCV infection, and we will discuss the potential targeted therapeutic approaches to improve virus eradication.
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Affiliation(s)
- Laurent Mailly
- Université de Strasbourg, INSERM, UMR-S1110, Institut de Recherche sur les Maladies Virales et Hépatiques, F-67000 Strasbourg, France.
| | - Thomas F Baumert
- Université de Strasbourg, INSERM, UMR-S1110, Institut de Recherche sur les Maladies Virales et Hépatiques, F-67000 Strasbourg, France; Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg, France; Institut Universitaire de France, F-75231 Paris, France.
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15
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Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process. Int J Mol Sci 2020; 21:ijms21062091. [PMID: 32197477 PMCID: PMC7140000 DOI: 10.3390/ijms21062091] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 03/15/2020] [Accepted: 03/16/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. Its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. This review provides an overview of the viral and host factors involved in HCV entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors.
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16
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Clément CM, Deffieu MS, Dorobantu CM, Baumert TF, Ayala‐Nunez NV, Mély Y, Ronde P, Gaudin R. Characterisation of endogenous Claudin‐1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock‐in cells. Biol Cell 2020; 112:140-151. [DOI: 10.1111/boc.201900085] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Accepted: 01/30/2020] [Indexed: 01/06/2023]
Affiliation(s)
- Camille M.H. Clément
- CNRSIRIM Institut de Recherche en infectiologie de Montpellier Montpellier 34293 France
- Université de Montpellier Montpellier 34090 France
- Inserm U1110Institut de Recherche sur les Maladies Virales et Hépatiques (IVH) Strasbourg 67000 France
- Université de Strasbourg Strasbourg 67000 France
| | - Maika S. Deffieu
- CNRSIRIM Institut de Recherche en infectiologie de Montpellier Montpellier 34293 France
- Université de Montpellier Montpellier 34090 France
| | - Cristina M. Dorobantu
- Inserm U1110Institut de Recherche sur les Maladies Virales et Hépatiques (IVH) Strasbourg 67000 France
- Université de Strasbourg Strasbourg 67000 France
| | - Thomas F. Baumert
- Inserm U1110Institut de Recherche sur les Maladies Virales et Hépatiques (IVH) Strasbourg 67000 France
- Université de Strasbourg Strasbourg 67000 France
- Pole Hépato‐digestifHôpitaux Universitaires de StrasbourgInstitut Hospitalo‐universitaire Strasbourg 67000 France
| | - Nilda Vanesa Ayala‐Nunez
- CNRSIRIM Institut de Recherche en infectiologie de Montpellier Montpellier 34293 France
- Université de Montpellier Montpellier 34090 France
- Inserm U1110Institut de Recherche sur les Maladies Virales et Hépatiques (IVH) Strasbourg 67000 France
- Université de Strasbourg Strasbourg 67000 France
| | - Yves Mély
- Université de Strasbourg Strasbourg 67000 France
- CNRS UMR 7021Laboratoire de Bioimagerie et PathologiesFaculté de pharmacie Illkirch 67401 France
| | - Philippe Ronde
- Université de Strasbourg Strasbourg 67000 France
- CNRS UMR 7021Laboratoire de Bioimagerie et PathologiesFaculté de pharmacie Illkirch 67401 France
| | - Raphael Gaudin
- CNRSIRIM Institut de Recherche en infectiologie de Montpellier Montpellier 34293 France
- Université de Montpellier Montpellier 34090 France
- Inserm U1110Institut de Recherche sur les Maladies Virales et Hépatiques (IVH) Strasbourg 67000 France
- Université de Strasbourg Strasbourg 67000 France
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17
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Dong D, Xie W, Liu M. Alteration of cell junctions during viral infection. Thorac Cancer 2020; 11:519-525. [PMID: 32017415 PMCID: PMC7049484 DOI: 10.1111/1759-7714.13344] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Revised: 01/13/2020] [Accepted: 01/14/2020] [Indexed: 12/21/2022] Open
Abstract
Cell junctions serve as a protective barrier for cells and provide an important channel for information transmission between cells and the surrounding environment. Viruses are parasites that invade and commandeer components of host cells in order to survive and replicate, and they have evolved various mechanisms to alter cell junctions to facilitate viral infection. In this review, we examined the current state of knowledge on the action of viruses on host cell junctions. The existing evidence suggests that targeting the molecules involved in the virus-cell junction interaction can prevent the spread of viral diseases.
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Affiliation(s)
- Dan Dong
- Institute of Biomedical Sciences, Shandong Provincial Key Laboratory of Animal Resistance Biology, Collaborative Innovation Center of Cell Biology in Universities of Shandong, College of Life Sciences, Shandong Normal University, Jinan, China
| | - Wei Xie
- Institute of Biomedical Sciences, Shandong Provincial Key Laboratory of Animal Resistance Biology, Collaborative Innovation Center of Cell Biology in Universities of Shandong, College of Life Sciences, Shandong Normal University, Jinan, China
| | - Min Liu
- Institute of Biomedical Sciences, Shandong Provincial Key Laboratory of Animal Resistance Biology, Collaborative Innovation Center of Cell Biology in Universities of Shandong, College of Life Sciences, Shandong Normal University, Jinan, China
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18
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Gerold G, Moeller R, Pietschmann T. Hepatitis C Virus Entry: Protein Interactions and Fusion Determinants Governing Productive Hepatocyte Invasion. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a036830. [PMID: 31427285 DOI: 10.1101/cshperspect.a036830] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) entry is among the best-studied uptake processes for human pathogenic viruses. Uptake follows a spatially and temporally tightly controlled program. Numerous host factors including proteins, lipids, and glycans promote productive uptake of HCV particles into human liver cells. The virus initially attaches to surface proteoglycans, lipid receptors such as the scavenger receptor BI (SR-BI), and to the tetraspanin CD81. After lateral translocation of virions to tight junctions, claudin-1 (CLDN1) and occludin (OCLN) are essential for entry. Clathrin-mediated endocytosis engulfs HCV particles, which fuse with endosomal membranes after pH drop. Uncoating of the viral RNA genome in the cytoplasm completes the entry process. Here we systematically review and classify HCV entry factors by their mechanistic role, relevance, and level of evidence. Finally, we report on more recent knowledge on determinants of membrane fusion and close with an outlook on future implications of HCV entry research.
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Affiliation(s)
- Gisa Gerold
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany.,Department of Clinical Microbiology, Virology & Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, 901 85 Umeå, Sweden
| | - Rebecca Moeller
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany
| | - Thomas Pietschmann
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany
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19
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Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells. Int J Mol Sci 2019; 20:ijms20194774. [PMID: 31561440 PMCID: PMC6801472 DOI: 10.3390/ijms20194774] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2019] [Revised: 09/11/2019] [Accepted: 09/22/2019] [Indexed: 12/15/2022] Open
Abstract
Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.
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20
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Lavie M, Linna L, Moustafa RI, Belouzard S, Fukasawa M, Dubuisson J. Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection. Traffic 2019; 20:753-773. [PMID: 31328852 DOI: 10.1111/tra.12680] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 07/17/2019] [Accepted: 07/17/2019] [Indexed: 12/12/2022]
Abstract
The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.
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Affiliation(s)
- Muriel Lavie
- Universite de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, Lille, France
| | - Lydia Linna
- Universite de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, Lille, France
| | - Rehab I Moustafa
- Universite de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, Lille, France.,Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt
| | - Sandrine Belouzard
- Universite de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, Lille, France
| | - Masayoshi Fukasawa
- Department of Biochemistry & Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Jean Dubuisson
- Universite de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, Lille, France
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21
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Pol S, Lagaye S. The remarkable history of the hepatitis C virus. Microbes Infect 2019; 21:263-270. [PMID: 31295571 DOI: 10.1016/j.micinf.2019.06.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 03/25/2019] [Accepted: 04/01/2019] [Indexed: 12/23/2022]
Abstract
The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations which are mainly reversible. The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8-12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016.
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Affiliation(s)
- Stanislas Pol
- Université Paris Descartes, Paris, France; Département d'Hépatologie, Hôpital Cochin, APHP, Paris, France; INSERM UMS-20, Institut Pasteur, Paris, France; Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France; INSERM U1223, Institut Pasteur, Paris, France.
| | - Sylvie Lagaye
- Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France; INSERM U1223, Institut Pasteur, Paris, France.
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22
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Pol S, Lagaye S. The remarkable history of the hepatitis C virus. Genes Immun 2019; 20:436-446. [PMID: 31019253 DOI: 10.1038/s41435-019-0066-z] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 03/25/2019] [Accepted: 04/01/2019] [Indexed: 02/06/2023]
Abstract
The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease, which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection, which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations, which are mainly reversible. The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8-12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016.
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Affiliation(s)
- Stanislas Pol
- Université Paris Descartes, Paris, France. .,Département d'Hépatologie, Hôpital Cochin, APHP, Paris, France. .,INSERM UMS-20, Institut Pasteur, Paris, France. .,Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France. .,INSERM U1223, Institut Pasteur, Paris, France.
| | - Sylvie Lagaye
- Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France. .,INSERM U1223, Institut Pasteur, Paris, France.
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23
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Zeisel MB, Dhawan P, Baumert TF. Tight junction proteins in gastrointestinal and liver disease. Gut 2019; 68:547-561. [PMID: 30297438 PMCID: PMC6453741 DOI: 10.1136/gutjnl-2018-316906] [Citation(s) in RCA: 202] [Impact Index Per Article: 33.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 08/16/2018] [Accepted: 08/19/2018] [Indexed: 12/11/2022]
Abstract
Over the past two decades a growing body of evidence has demonstrated an important role of tight junction (TJ) proteins in the physiology and disease biology of GI and liver disease. On one side, TJ proteins exert their functional role as integral proteins of TJs in forming barriers in the gut and the liver. Furthermore, TJ proteins can also be expressed outside TJs where they play important functional roles in signalling, trafficking and regulation of gene expression. A hallmark of TJ proteins in disease biology is their functional role in epithelial-to-mesenchymal transition. A causative role of TJ proteins has been established in the pathogenesis of colorectal cancer and gastric cancer. Among the best characterised roles of TJ proteins in liver disease biology is their function as cell entry receptors for HCV-one of the most common causes of hepatocellular carcinoma. At the same time TJ proteins are emerging as targets for novel therapeutic approaches for GI and liver disease. Here we review our current knowledge of the role of TJ proteins in the pathogenesis of GI and liver disease biology and discuss their potential as therapeutic targets.
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Affiliation(s)
- Mirjam B. Zeisel
- Inserm U1052, CNRS UMR 5286, Cancer Research Center of Lyon (CRCL), Université de Lyon (UCBL), Lyon, France
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
| | - Punita Dhawan
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE
- Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE
- VA Nebraska-Western Iowa Health Care System, Omaha, NE
| | - Thomas F. Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
- Institut Hospitalo-Universitaire, Pôle hépato-digestif, Nouvel Hôpital Civil, Strasbourg, France
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Ke PY. The Multifaceted Roles of Autophagy in Flavivirus-Host Interactions. Int J Mol Sci 2018; 19:ijms19123940. [PMID: 30544615 PMCID: PMC6321027 DOI: 10.3390/ijms19123940] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 12/05/2018] [Accepted: 12/05/2018] [Indexed: 02/06/2023] Open
Abstract
Autophagy is an evolutionarily conserved cellular process in which intracellular components are eliminated via lysosomal degradation to supply nutrients for organelle biogenesis and metabolic homeostasis. Flavivirus infections underlie multiple human diseases and thus exert an immense burden on public health worldwide. Mounting evidence indicates that host autophagy is subverted to modulate the life cycles of flaviviruses, such as hepatitis C virus, dengue virus, Japanese encephalitis virus, West Nile virus and Zika virus. The diverse interplay between autophagy and flavivirus infection not only regulates viral growth in host cells but also counteracts host stress responses induced by viral infection. In this review, we summarize the current knowledge on the role of autophagy in the flavivirus life cycle. We also discuss the impacts of virus-induced autophagy on the pathogeneses of flavivirus-associated diseases and the potential use of autophagy as a therapeutic target for curing flavivirus infections and related human diseases.
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Affiliation(s)
- Po-Yuan Ke
- Department of Biochemistry & Molecular Biology and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan.
- Division of Allergy, Immunology and Rheumatology, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan.
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25
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Baktash Y, Madhav A, Coller KE, Randall G. Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process. Cell Host Microbe 2018; 23:382-394.e5. [PMID: 29544098 DOI: 10.1016/j.chom.2018.02.005] [Citation(s) in RCA: 78] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2017] [Revised: 01/30/2018] [Accepted: 02/16/2018] [Indexed: 12/12/2022]
Abstract
Hepatitis C virus (HCV) enters hepatocytes via various entry factors, including scavenger receptor BI (SR-B1), cluster of differentiation 81 (CD81), epidermal growth factor receptor (EGFR), claudin-1 (CLDN1), and occludin (OCLN). As CLDN1 and OCLN are not readily accessible due to their tight junctional localization, HCV likely accesses them by either disrupting cellular polarity or migrating to the tight junction. In this study, we image HCV entry into a three-dimensional polarized hepatoma system and reveal that the virus sequentially engages these entry factors through actin-dependent mechanisms. HCV initially localizes with the early entry factors SR-B1, CD81, and EGFR at the basolateral membrane and then accumulates at the tight junction in an actin-dependent manner. HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR. Thus, HCV uses a dynamic and multi-step process to engage and enter host cells.
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Affiliation(s)
- Yasmine Baktash
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Anisha Madhav
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Kelly E Coller
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Glenn Randall
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
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26
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Tarr AW, Backx M, Hamed MR, Urbanowicz RA, McClure CP, Brown RJP, Ball JK. Immunization with a synthetic consensus hepatitis C virus E2 glycoprotein ectodomain elicits virus-neutralizing antibodies. Antiviral Res 2018; 160:25-37. [PMID: 30217650 DOI: 10.1016/j.antiviral.2018.09.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 09/06/2018] [Accepted: 09/10/2018] [Indexed: 01/06/2023]
Abstract
Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly-neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing of vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies.
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Affiliation(s)
- Alexander W Tarr
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK
| | - Matthijs Backx
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK
| | - Mohamed R Hamed
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK; Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Egypt
| | - Richard A Urbanowicz
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK
| | - C Patrick McClure
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK
| | - Richard J P Brown
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK
| | - Jonathan K Ball
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, UK; School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK.
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27
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Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB. PLoS Pathog 2018; 14:e1007111. [PMID: 30024968 PMCID: PMC6053247 DOI: 10.1371/journal.ppat.1007111] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Accepted: 05/18/2018] [Indexed: 12/24/2022] Open
Abstract
Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells. CD81 is a cell membrane protein, which functions as entry factor for hepatitis C virus (HCV) and malaria sporozoites in the human liver. Currently, it remains enigmatic how CD81 guides the entry process of both pathogens and whether it functions in a similar way during liver cell invasion of HCV and malaria parasites. Here, we use high resolution quantitative proteomics to identify CD81 associated host proteins in liver cells. We found that at least 33 proteins form a complex with CD81, 23 of which were not reported as interaction partners before. We further determined that at least five CD81 interactors are HCV host factors, among them calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB). All tested HCV genotypes require CAPN5 and CBLB for full infection, but neither malaria parasites nor other tested enveloped virus rely on CAPN5 or CBLB. Our study maps the liver cell interactome of CD81 and provides new insight into the distinct cell invasion mechanisms of HCV and malaria parasites.
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Florin L, Lang T. Tetraspanin Assemblies in Virus Infection. Front Immunol 2018; 9:1140. [PMID: 29887866 PMCID: PMC5981178 DOI: 10.3389/fimmu.2018.01140] [Citation(s) in RCA: 80] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 05/07/2018] [Indexed: 12/23/2022] Open
Abstract
Tetraspanins (Tspans) are a family of four-span transmembrane proteins, known as plasma membrane “master organizers.” They form Tspan-enriched microdomains (TEMs or TERMs) through lateral association with one another and other membrane proteins. If multiple microdomains associate with each other, larger platforms can form. For infection, viruses interact with multiple cell surface components, including receptors, activating proteases, and signaling molecules. It appears that Tspans, such as CD151, CD82, CD81, CD63, CD9, Tspan9, and Tspan7, coordinate these associations by concentrating the interacting partners into Tspan platforms. In addition to mediating viral attachment and entry, these platforms may also be involved in intracellular trafficking of internalized viruses and assist in defining virus assembly and exit sites. In conclusion, Tspans play a role in viral infection at different stages of the virus replication cycle. The present review highlights recently published data on this topic, with a focus on events at the plasma membrane. In light of these findings, we propose a model for how Tspan interactions may organize cofactors for viral infection into distinct molecular platforms.
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Affiliation(s)
- Luise Florin
- Department of Medical Microbiology and Hygiene, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Thorsten Lang
- Department of Membrane Biochemistry, Life & Medical Sciences Institute, University of Bonn, Bonn, Germany
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Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry. J Virol 2018; 92:JVI.01982-17. [PMID: 29491159 DOI: 10.1128/jvi.01982-17] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2017] [Accepted: 02/20/2018] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry.IMPORTANCE The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.
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CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells. Viruses 2018; 10:v10040207. [PMID: 29677132 PMCID: PMC5923501 DOI: 10.3390/v10040207] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2018] [Revised: 04/14/2018] [Accepted: 04/19/2018] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.
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Riad SE, Elhelw DS, Shawer H, El-Ekiaby N, Salah A, Zekri A, Esmat G, Amleh A, Abdelaziz AI. Disruption of Claudin-1 Expression by miRNA-182 Alters the Susceptibility to Viral Infectivity in HCV Cell Models. Front Genet 2018; 9:93. [PMID: 29616082 PMCID: PMC5869927 DOI: 10.3389/fgene.2018.00093] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2017] [Accepted: 03/05/2018] [Indexed: 01/01/2023] Open
Abstract
HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.
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Affiliation(s)
- Sarah E Riad
- Pharmacology and Toxicology Department, German University in Cairo, New Cairo, Egypt
| | - Dalia S Elhelw
- Pharmaceutical Chemistry Department, German University in Cairo, New Cairo, Egypt
| | - Heba Shawer
- Biology Department, School of Science and Engineering, American University in Cairo, New Cairo, Egypt
| | - Nada El-Ekiaby
- Pharmacology and Toxicology Department, German University in Cairo, New Cairo, Egypt.,School of Medicine, NewGiza University, Cairo, Egypt
| | - Ayman Salah
- Department of Surgery, Cairo University, Cairo, Egypt
| | - Abdelrahman Zekri
- Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt
| | - Gamal Esmat
- Department of Endemic Medicine and Hepatology, Cairo University, Cairo, Egypt
| | - Asma Amleh
- Biology Department, School of Science and Engineering, American University in Cairo, New Cairo, Egypt
| | - Ahmed I Abdelaziz
- Pharmacology and Toxicology Department, German University in Cairo, New Cairo, Egypt.,School of Medicine, NewGiza University, Cairo, Egypt
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32
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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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33
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King B, Tarr AW. How have retrovirus pseudotypes contributed to our understanding of viral entry? Future Virol 2017. [DOI: 10.2217/fvl-2017-0062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Study of virus entry into host cells is important for understanding viral tropism and pathogenesis. Studying the entry of in vitro cultured viruses is not always practicable. Study of highly pathogenic viruses, viruses that do not grow in culture, and viruses that rapidly change phenotype in vitro can all benefit from alternative models of entry. Retrovirus particles can be engineered to display the envelope proteins of heterologous enveloped viruses. This approach, broadly termed ‘pseudotyping’, is an important technique for interrogating virus entry. In this perspective we consider how retrovirus pseudotypes have addressed these challenges and improved our understanding of the entry pathways of diverse virus species, including Ebolavirus, human immunodeficiency virus and hepatitis C virus.
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Affiliation(s)
- Barnabas King
- National Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust & the University of Nottingham, Nottingham, UK
- School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham, UK
| | - Alexander W Tarr
- National Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust & the University of Nottingham, Nottingham, UK
- School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham, UK
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Grove J, Hu K, Farquhar MJ, Goodall M, Walker L, Jamshad M, Drummer HE, Bill RM, Balfe P, McKeating JA. A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81. Wellcome Open Res 2017; 2:82. [PMID: 29090272 PMCID: PMC5657224 DOI: 10.12688/wellcomeopenres.12058.1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/16/2017] [Indexed: 12/26/2022] Open
Abstract
Background: Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies. Methods: We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy. Results: The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains. Conclusions: These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general.
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Affiliation(s)
- Joe Grove
- Institute of Immunity and Transplantation, Division of Infection and Immunity, , University College London, London, NW3 2PF, UK
| | - Ke Hu
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK
| | - Michelle J. Farquhar
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK
| | - Margaret Goodall
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK
| | - Lucas Walker
- Institute of Immunity and Transplantation, Division of Infection and Immunity, , University College London, London, NW3 2PF, UK
| | - Mohammed Jamshad
- Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Heidi E. Drummer
- Centre for Biomedical Resear, Burnet Institute, Melbourne, VIC, 3004, Australia
| | - Roslyn M. Bill
- School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK
| | - Peter Balfe
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK
| | - Jane A. McKeating
- Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, UK
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Attachment and Postattachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission. J Virol 2017; 91:JVI.00280-17. [PMID: 28404852 DOI: 10.1128/jvi.00280-17] [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] [Received: 02/17/2017] [Accepted: 04/10/2017] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission.IMPORTANCE The importance and underlying molecular mechanisms of cell surface receptors in HCV cell-free and cell-to-cell transmission are poorly understood. The role of some of the HCV attachment and postattachment receptors in HCV infection and cell-to-cell spread remains controversial. Using CRISPR-Cas9-mediated knockouts of specific cellular genes, we demonstrate that both SDC-1 and SDC-2, but not SDC-3 or SDC-4, are bona fide HCV attachment receptors. We also used a newly developed luciferase-based reporter system to quantitatively determine the importance of attachment and postattachment receptors in HCV cell-to-cell transmission. SDC-1, SDC-2, TIM-1, and SR-BI were found to modestly promote HCV cell-to-cell spread. CD81, CLDN1, OCLN, and LDLR play more important roles in HCV cell-to-cell transmission. Likewise, apolipoprotein E (apoE) is critically important for HCV cell-to-cell spread, unlike VLDL-containing mouse serum, which did not affect HCV cell-to-cell spread. These findings suggest that the mechanism(s) of HCV cell-to-cell spread differs from that of cell-free infection.
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Regulated Entry of Hepatitis C Virus into Hepatocytes. Viruses 2017; 9:v9050100. [PMID: 28486435 PMCID: PMC5454413 DOI: 10.3390/v9050100] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Revised: 04/24/2017] [Accepted: 05/02/2017] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is a model for the study of virus–host interaction and host cell responses to infection. Virus entry into hepatocytes is the first step in the HCV life cycle, and this process requires multiple receptors working together. The scavenger receptor class B type I (SR-BI) and claudin-1 (CLDN1), together with human cluster of differentiation (CD) 81 and occludin (OCLN), constitute the minimal set of HCV entry receptors. Nevertheless, HCV entry is a complex process involving multiple host signaling pathways that form a systematic regulatory network; this network is centrally controlled by upstream regulators epidermal growth factor receptor (EGFR) and transforming growth factor β receptor (TGFβ-R). Further feedback regulation and cell-to-cell spread of the virus contribute to the chronic maintenance of HCV infection. A comprehensive and accurate disclosure of this critical process should provide insights into the viral entry mechanism, and offer new strategies for treatment regimens and targets for HCV therapeutics.
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Tetraspanins in infections by human cytomegalo- and papillomaviruses. Biochem Soc Trans 2017; 45:489-497. [PMID: 28408489 DOI: 10.1042/bst20160295] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Revised: 02/15/2017] [Accepted: 02/16/2017] [Indexed: 12/30/2022]
Abstract
Members of the tetraspanin family have been identified as essential cellular membrane proteins in infectious diseases by nearly all types of pathogens. The present review highlights recently published data on the role of tetraspanin CD151, CD81, and CD63 and their interaction partners in host cell entry by human cytomegalo- and human papillomaviruses. Moreover, we discuss a model for tetraspanin assembly into trafficking platforms at the plasma membrane. These platforms might persist during intracellular viral trafficking.
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Abstract
PURPOSE OF REVIEW Direct-acting antiviral agents (DAAs) have markedly improved the prognosis of hepatitis C virus (HCV)-genotype 3 (GT3), a highly prevalent infection worldwide. However, in patients with hepatic fibrosis, cirrhosis, or hepatocellular carcinoma (HCC), GT3 infection presents a treatment challenge compared with other genotypes. The dependence of the HCV life cycle on host lipid metabolism suggests the possible utility of targeting host cellular factors for combination anti-HCV therapy. We discuss current and emergent DAA regimens for HCV-GT3 treatment. We then summarize recent research findings on the reliance of HCV entry, replication, and virion assembly on host lipid metabolism. RECENT FINDINGS Current HCV treatment guidelines recommend the use of daclatasvir plus sofosbuvir (DCV/SOF) or sofosbuvir plus velpatasvir (SOF/VEL) for the management of GT3 based upon clinical efficacy [≥88% overall sustained virological response (SVR)] and tolerability. Potential future DAA options, such as SOF/VEL co-formulated with GS-9857, also look promising in treating cirrhotic GT3 patients. However, HCV resistance to DAAs will likely continue to impact the therapeutic efficacy of interferon-free treatment regimens. Disruption of HCV entry by targeting required host cellular receptors shows potential in minimizing HCV resistance and broadening therapeutic options for certain subpopulations of GT3 patients. The use of cholesterol biosynthesis and transport inhibitors may also improve health outcomes for GT3 patients when used synergistically with DAAs. Due to the morbidity and mortality associated with HCV-GT3 infection compared to other genotypes, efforts should be made to address current limitations in the therapeutic prevention and management of HCV-GT3 infection.
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Colpitts CC, Baumert TF. Claudins in viral infection: from entry to spread. Pflugers Arch 2016; 469:27-34. [PMID: 27885488 DOI: 10.1007/s00424-016-1908-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Revised: 11/14/2016] [Accepted: 11/16/2016] [Indexed: 12/20/2022]
Abstract
Tight junctions are critically important for many physiological functions, including the maintenance of cell polarity, regulation of paracellular permeability, and involvement in signal transduction pathways to regulate integral cellular processes. Furthermore, tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection. Viruses, in turn, have evolved mechanisms to exploit tight junction proteins to gain access to cells or spread through tissues in an infected host. Claudin family proteins are integral components of tight junctions and are thought to play crucial roles in regulating their permeability. Claudins have been implicated in the infection process of several medically important human pathogens, including hepatitis C virus, dengue virus, West Nile virus, and human immunodeficiency virus, among others. In this review, we summarize the role of claudins in viral infections and discuss their potential as novel antiviral targets. A better understanding of claudins during viral infection may provide insight into physiological roles of claudins and uncover novel therapeutic antiviral strategies.
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Affiliation(s)
- Che C Colpitts
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 3 Rue Koeberlé, 67000, Strasbourg, France.
- Université de Strasbourg, 67000, Strasbourg, France.
| | - Thomas F Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 3 Rue Koeberlé, 67000, Strasbourg, France.
- Université de Strasbourg, 67000, Strasbourg, France.
- Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hopitaux Universitaires de Strasbourg, 67000, Strasbourg, France.
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Florian PE, Rouillé Y, Ruta S, Nichita N, Roseanu A. Recent advances in human viruses imaging studies. J Basic Microbiol 2016; 56:591-607. [PMID: 27059598 DOI: 10.1002/jobm.201500575] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Accepted: 02/27/2016] [Indexed: 12/13/2022]
Affiliation(s)
- Paula Ecaterina Florian
- Department of ; Ligand-Receptor Interactions; Institute of Biochemistry of the Romanian Academy; Bucharest Romania
| | - Yves Rouillé
- Center for Infection and Immunity of Lille (CIIL); Inserm U1019; CNRS UMR8204; Institut Pasteur de Lille; Université Lille Nord de France; Lille France
| | - Simona Ruta
- Department of Emergent Diseases; Stefan S. Nicolau Institute of Virology; Bucharest 030304 Romania
| | - Norica Nichita
- Department of Viral Glycoproteins; Institute of Biochemistry of the Romanian Academy; Bucharest Romania
| | - Anca Roseanu
- Department of ; Ligand-Receptor Interactions; Institute of Biochemistry of the Romanian Academy; Bucharest Romania
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Liu F, Koval M, Ranganathan S, Fanayan S, Hancock WS, Lundberg EK, Beavis RC, Lane L, Duek P, McQuade L, Kelleher NL, Baker MS. Systems Proteomics View of the Endogenous Human Claudin Protein Family. J Proteome Res 2016; 15:339-59. [PMID: 26680015 DOI: 10.1021/acs.jproteome.5b00769] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation.
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Affiliation(s)
| | - Michael Koval
- Department of Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, and Department of Cell Biology, Emory University School of Medicine , 205 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, Georgia 30322, United States
| | | | | | - William S Hancock
- Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States
| | - Emma K Lundberg
- SciLifeLab, School of Biotechnology, Royal Institute of Technology (KTH) , SE-171 21 Solna, Stockholm, Sweden
| | - Ronald C Beavis
- Department of Biochemistry and Medical Genetics, University of Manitoba , 744 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada
| | - Lydie Lane
- SIB-Swiss Institute of Bioinformatics , CMU - Rue Michel-Servet 1, 1211 Geneva, Switzerland
| | - Paula Duek
- SIB-Swiss Institute of Bioinformatics , CMU - Rue Michel-Servet 1, 1211 Geneva, Switzerland
| | | | - Neil L Kelleher
- Department of Chemistry, Department of Molecular Biosciences, and Proteomics Center of Excellence, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States
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Qian XJ, Zhu YZ, Zhao P, Qi ZT. Entry inhibitors: New advances in HCV treatment. Emerg Microbes Infect 2016; 5:e3. [PMID: 26733381 PMCID: PMC4735057 DOI: 10.1038/emi.2016.3] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Revised: 10/28/2015] [Accepted: 11/02/2015] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus (HCV) infection affects approximately 3% of the world's population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry.
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Affiliation(s)
- Xi-Jing Qian
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Yong-Zhe Zhu
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Ping Zhao
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Zhong-Tian Qi
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
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44
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Fénéant L, Ghosn J, Fouquet B, Helle F, Belouzard S, Vausselin T, Séron K, Delfraissy JF, Dubuisson J, Misrahi M, Cocquerel L. Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro. PLoS One 2015; 10:e0142539. [PMID: 26561856 PMCID: PMC4643007 DOI: 10.1371/journal.pone.0142539] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 10/22/2015] [Indexed: 12/12/2022] Open
Abstract
The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.
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Affiliation(s)
- Lucie Fénéant
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Jade Ghosn
- Assistance Publique—Hôpitaux de Paris, Unité Fonctionnelle de Thérapeutique en Immuno-Infectiologie, Hôpital Universitaire Hôtel Dieu, Paris, France
- Université Paris Descartes, EA 7327, Faculté de Médecine site Necker, Paris, France
| | - Baptiste Fouquet
- Univ Paris Sud, Faculté de Médecine, Hôpitaux Universitaires Paris Sud, Le Kremlin-Bicêtre and Inserm-U1193, Hôpital Paul Brousse, F-94800 Villejuif, France
| | - François Helle
- Virology Department, Amiens University Hospital, Amiens, France
| | - Sandrine Belouzard
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Thibaut Vausselin
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Karin Séron
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Jean-François Delfraissy
- Assistance Publique—Hôpitaux de Paris, Service de Médecine Interne et Maladies Infectieuses, Centre Hospitalier Universitaire de Bicêtre, Le Kremlin-Bicêtre, France
| | - Jean Dubuisson
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Micheline Misrahi
- Univ Paris Sud, Faculté de Médecine, Hôpitaux Universitaires Paris Sud, Le Kremlin-Bicêtre and Inserm-U1193, Hôpital Paul Brousse, F-94800 Villejuif, France
| | - Laurence Cocquerel
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
- * E-mail:
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45
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Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus. World J Hepatol 2015. [PMID: 26464752 DOI: 10.4254/wjh.v7.i22.2369.] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Hepatitis C virus (HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies (mAb) of mice are predominantly used for the immunodiagnosis of several viral, bacterial, and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mAb are used in the immunotherapy of several blood malignancies, such as lymphoma and leukemia, as well as for autoimmune diseases. In this review article, we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mAb in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV, which is a major health problem throughout the world, particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mAb. Finally, we will discuss available and new trends to produce antibodies, such as egg yolk-based antibodies (IgY), production in transgenic plants, and the synthetic antibody mimics approach.
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46
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Tabll A, Abbas AT, El-Kafrawy S, Wahid A. Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus. World J Hepatol 2015; 7:2369-2383. [PMID: 26464752 PMCID: PMC4598607 DOI: 10.4254/wjh.v7.i22.2369] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2015] [Revised: 08/05/2015] [Accepted: 09/08/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies (mAb) of mice are predominantly used for the immunodiagnosis of several viral, bacterial, and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mAb are used in the immunotherapy of several blood malignancies, such as lymphoma and leukemia, as well as for autoimmune diseases. In this review article, we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mAb in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV, which is a major health problem throughout the world, particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mAb. Finally, we will discuss available and new trends to produce antibodies, such as egg yolk-based antibodies (IgY), production in transgenic plants, and the synthetic antibody mimics approach.
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47
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Colpitts CC, Zeisel MB, Baumert TF. When one receptor closes, another opens: claudins and the hepatitis C virus E1 glycoprotein. Hepatology 2015; 62:991-3. [PMID: 25930717 DOI: 10.1002/hep.27876] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 04/25/2015] [Indexed: 01/21/2023]
Affiliation(s)
- Che C Colpitts
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Mirjam B Zeisel
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Thomas F Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.,Université de Strasbourg, Strasbourg, France.,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
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Aizawa Y, Seki N, Nagano T, Abe H. Chronic hepatitis C virus infection and lipoprotein metabolism. World J Gastroenterol 2015; 21:10299-10313. [PMID: 26420957 PMCID: PMC4579877 DOI: 10.3748/wjg.v21.i36.10299] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2015] [Revised: 07/11/2015] [Accepted: 08/31/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a hepatotrophic virus and a major cause of chronic liver disease, including hepatocellular carcinoma, worldwide. The life cycle of HCV is closely associated with the metabolism of lipids and lipoproteins. The main function of lipoproteins is transporting lipids throughout the body. Triglycerides, free cholesterol, cholesteryl esters, and phospholipids are the major components of the transported lipids. The pathway of HCV assembly and secretion is closely linked to lipoprotein production and secretion, and the infectivity of HCV particles largely depends on the interaction of lipoproteins. Moreover, HCV entry into hepatocytes is strongly influenced by lipoproteins. The key lipoprotein molecules mediating these interactions are apolipoproteins. Apolipoproteins are amphipathic proteins on the surface of a lipoprotein particle, which help stabilize lipoprotein structure. They perform a key role in lipoprotein metabolism by serving as receptor ligands, enzyme co-factors, and lipid transport carriers. Understanding the association between the life cycle of HCV and lipoprotein metabolism is important because each step of the life cycle of HCV that is associated with lipoprotein metabolism is a potential target for anti-HCV therapy. In this article, we first concisely review the nature of lipoprotein and its metabolism to better understand the complicated interaction of HCV with lipoprotein. Then, we review the outline of the processes of HCV assembly, secretion, and entry into hepatocytes, focusing on the association with lipoproteins. Finally, we discuss the clinical aspects of disturbed lipid/lipoprotein metabolism and the significance of dyslipoproteinemia in chronic HCV infection with regard to abnormal apolipoproteins.
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Tawar RG, Colpitts CC, Timm J, Fehm T, Roggendorf M, Meisel H, Meyer N, Habersetzer F, Cosset FL, Berg T, Zeisel MB, Baumert TF. Acute hepatitis C virus infection induces anti-host cell receptor antibodies with virus-neutralizing properties. Hepatology 2015; 62:726-36. [PMID: 26010076 DOI: 10.1002/hep.27906] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2014] [Accepted: 05/19/2015] [Indexed: 12/17/2022]
Abstract
UNLABELLED Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies (Abs) against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study, we aimed to investigate whether anti-receptor Abs are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an enzyme-linked immunosorbant assay using a recombinant CD81-claudin-1 (CLDN1) fusion protein to detect and quantify Abs directed against extracellular epitopes of the HCV CD81-CLDN1 coreceptor complex. The presence of anti-receptor Abs was studied in serum of patients from a well-defined cohort of a single-source HCV outbreak of pregnant women and several control groups, including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection, and healthy individuals. Virus-neutralizing activity of Abs was determined using recombinant cell culture-derived HCV (HCVcc). Our results demonstrate that HCV-infected patients have statistically significantly higher anti-CD81/CLDN1 Ab titers during the early phase of infection than controls. The titers were significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV-neutralizing anti-receptor Abs are induced in the early phase of HCV infection, but not in control groups. CONCLUSION The virus-neutralizing properties of these Abs suggest a role for control of viral infection in conjunction with antiviral responses. Characterization of these anti-receptor Abs opens new avenues to prevent and treat HCV infection.
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Affiliation(s)
- Rajiv G Tawar
- Inserm, U1110, Institut des Maladies Virales et Hépatiques, Strasbourg, France.,University of Strasbourg, Strasbourg, France
| | - Che C Colpitts
- Inserm, U1110, Institut des Maladies Virales et Hépatiques, Strasbourg, France.,University of Strasbourg, Strasbourg, France
| | - Jörg Timm
- Institute for Virology, University Hospital Düsseldorf, Heinrich-Heine-University, Düsseldorf, Germany
| | - Tanja Fehm
- Department of Gynecology and Obstetrics, University Hospital Düsseldorf, Heinrich-Heine-University, Düsseldorf, Germany
| | | | - Helga Meisel
- Institute of Medical Virology, Helmut-Ruska-Haus, Charité University Medicine, Berlin, Germany
| | - Nicolas Meyer
- Pôle de Santé Publique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - François Habersetzer
- Inserm, U1110, Institut des Maladies Virales et Hépatiques, Strasbourg, France.,University of Strasbourg, Strasbourg, France.,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - François-Loïc Cosset
- CIRI-International Center for Infectiology Research, Lyon, France; Inserm, U1111, Lyon France; Ecole Normale Supérieure; CNRS UMR 5308, Lyon, France; LabEx Ecofect, University of Lyon, Lyon, France
| | - Thomas Berg
- Department of Internal Medicine, Neurology and Dermatology, Gastroenterology and Rheumatology Clinic, Section of Hepatology, University of Leipzig, Leipzig, Germany
| | - Mirjam B Zeisel
- Inserm, U1110, Institut des Maladies Virales et Hépatiques, Strasbourg, France.,University of Strasbourg, Strasbourg, France
| | - Thomas F Baumert
- Inserm, U1110, Institut des Maladies Virales et Hépatiques, Strasbourg, France.,University of Strasbourg, Strasbourg, France.,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
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50
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Futamura K, Sekino M, Hata A, Ikebuchi R, Nakanishi Y, Egawa G, Kabashima K, Watanabe T, Furuki M, Tomura M. Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement. Cytometry A 2015. [PMID: 26217952 PMCID: PMC5132038 DOI: 10.1002/cyto.a.22725] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use.
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Affiliation(s)
- Koji Futamura
- FCM Business Department, Life Science Business Division, Medical Business Unit, Sony Corporation, Minato-Ku, Tokyo, 108-0075, Japan
| | - Masashi Sekino
- Concept Development Department, Application Technology Development Division, System R&D Group, RDS Platform, Sony Corporation, Shinagawa-Ku, Tokyo, 141-0001, Japan
| | - Akihiro Hata
- Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan
| | - Ryoyo Ikebuchi
- Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan.,Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiorikita, Tondabayashi-City, Osaka Prefecture, 584-8540, Japan
| | - Yasutaka Nakanishi
- Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan
| | - Gyohei Egawa
- Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan
| | - Kenji Kabashima
- Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan
| | - Takeshi Watanabe
- Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan.,The Tazuke-Kofukai Medical Research Institute/Kitano Hospital, 2-4-20 Ohgimachi, Kita-Ku, Osaka, 530-8480, Japan
| | - Motohiro Furuki
- FCM Business Department, Life Science Business Division, Medical Business Unit, Sony Corporation, Minato-Ku, Tokyo, 108-0075, Japan
| | - Michio Tomura
- Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan.,Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiorikita, Tondabayashi-City, Osaka Prefecture, 584-8540, Japan
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