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Cosset FL, Denolly S. Lipoprotein receptors: A little grease for enveloped viruses to open the lock? J Biol Chem 2024; 300:107849. [PMID: 39357828 PMCID: PMC11550601 DOI: 10.1016/j.jbc.2024.107849] [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: 08/07/2024] [Revised: 09/20/2024] [Accepted: 09/22/2024] [Indexed: 10/04/2024] Open
Abstract
Several studies recently highlighted the role of lipoprotein receptors in viral entry. These receptors are evolutionarily ancient proteins, key for the transport of lipids as well as other signaling molecules across the plasma membrane. Here, we discuss the different families of lipoprotein receptors and how they are hijacked by enveloped viruses to promote their entry into infected cells. While the usage of lipoprotein receptors was known for members of the Flaviviridae family and vesicular stomatitis virus, the last 4 years have seen the discovery that these receptors are used by many genetically unrelated viruses. We also emphasize how viral particles interact with these receptors and the possible targeting of these host factors as antiviral strategies.
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Affiliation(s)
- François-Loïc Cosset
- CIRI - Centre International de Recherche en Infectiologie, Université de Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France.
| | - Solène Denolly
- Centre de Recherche en Cancérologie de Lyon, Inserm U1052-CNRS UMR5286, Université de Lyon, Université Claude Bernard Lyon1, Centre Léon Bérard, Lyon, France.
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Matthaei A, Joecks S, Frauenstein A, Bruening J, Bankwitz D, Friesland M, Gerold G, Vieyres G, Kaderali L, Meissner F, Pietschmann T. Landscape of protein-protein interactions during hepatitis C virus assembly and release. Microbiol Spectr 2024; 12:e0256222. [PMID: 38230952 PMCID: PMC10846047 DOI: 10.1128/spectrum.02562-22] [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: 07/05/2022] [Accepted: 10/11/2023] [Indexed: 01/18/2024] Open
Abstract
Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.
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Affiliation(s)
- Alina Matthaei
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
| | - Sebastian Joecks
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
| | - Annika Frauenstein
- RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Planegg, Bavaria, Germany
| | - Janina Bruening
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
| | - Dorothea Bankwitz
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
| | - Martina Friesland
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
| | - Gisa Gerold
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
- Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden
- Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umeå, Sweden
| | - Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
- Junior Research Group “Cell Biology of RNA Viruses,” Leibniz Institute of Experimental Virology, Hamburg, Germany
| | - Lars Kaderali
- Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany
| | - Felix Meissner
- RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Planegg, Bavaria, Germany
- Systems Immunology and Proteomics, Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany
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Vieyres G, Pietschmann T. The role of human lipoproteins for hepatitis C virus persistence. Curr Opin Virol 2023; 60:101327. [PMID: 37031484 DOI: 10.1016/j.coviro.2023.101327] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2022] [Revised: 01/23/2023] [Accepted: 03/05/2023] [Indexed: 04/11/2023]
Abstract
Hepatitis C virus (HCV) is a hepatotropic virus that establishes a chronic infection in most individuals. Effective treatments are available; however, many patients are not aware of their infection. Consequently, they do not receive treatment and HCV transmission remains high, particularly among groups at high risk of exposure such as people who inject intravenous drugs. A prophylactic vaccine may reduce HCV transmission, but is currently not available. HCV has evolved immune evasion strategies, which facilitate persistence and complicate development of a protective vaccine. The peculiar association of HCV particles with human lipoproteins is thought to facilitate evasion from humoral immune response and viral homing to liver cells. A better understanding of these aspects provides the basis for development of protective vaccination strategies. Here, we review key information about the composition of HCV particles, the mechanisms mediating lipoprotein incorporation, and the functional consequences of this interaction.
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Affiliation(s)
- Gabrielle Vieyres
- Leibniz Institute of Virology, Hamburg, Germany; Integrative Analysis of Pathogen-Induced Compartments, Leibniz ScienceCampus InterACt, Hamburg, Germany
| | - Thomas Pietschmann
- 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.
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Trifan A, Cuciureanu T, Nastasa R, Stratina E, Zenovia S, Muzica CM, Huiban L, Singeap AM, Chiriac S, Sfarti C, Cojocariu C, Girleanu I, Minea H, Stafie R, Rotaru A, Stanciu C. Changes in Components of Metabolic Syndrome after Antiviral Eradication in Hepatitis C Virus Infection. Life (Basel) 2023; 13:534. [PMID: 36836890 PMCID: PMC9959799 DOI: 10.3390/life13020534] [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: 01/11/2023] [Revised: 02/10/2023] [Accepted: 02/12/2023] [Indexed: 02/17/2023] Open
Abstract
Chronic hepatitis C infection is a systemic disease that affects over 71 million patients all over the world and it is to be considered nowadays as a new cardiometabolic risk factor. This study aimed to evaluate the weight and metabolic changes after viral eradication in patients with hepatitis C virus (HCV) infection. We conducted a prospective study between October 2017 to December 2021, in a tertiary care center, in which we included 132 patients with HCV or cirrhosis. All patients received treatment with direct antivirals (DAAs) and achieved sustained viral response at 12 weeks (SVR12). During the study, clinical laboratory data and Fibroscan examinations were recorded in all patients. The study group was evaluated at the initiation of antiviral treatment, at SVR12, and within an average follow-up period of 6 months to 12 months after the previous evaluation. Evaluation at SVR12 and the data recorded in the post-SVR surveillance period show a further increase in BMI compared with baseline measurements with a statistically significant difference (27.11 ± 3.22 vs. 27.415 ± 3.03 vs. 28.04 ± 1.11 kg/m2, p = 0.012). The same observation was noticed for waist circumference (WC) at post-SVR evaluation (87.6 ± 13.1 vs. 88.4 ± 13.6 cm, p = 0.031). Moreover, the study population registered an increase in the average total cholesterol (TC) values at post-SVR evaluation (177.01 ± 42.2 mg/dL, p = 0.014) compared to baseline. In addition, the serum level of triglycerides had been modified after viral clearance, with a minimal decrease in the mean values of triglycerides (TGD) at SVR-12 assessment (133.48 ± 41.8 mg/dL, p = 0.78), followed by a significant increase to the mean value of 145.4 ± 47.2 mg/dL (p = 0.026) in the third evaluation. Our study highlights that HCV eradication does not improve the lipid profile in the short term, and these patients still have an additional cardiovascular risk factor due to high levels of TC, TGD, and weight gain.
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Affiliation(s)
- Anca Trifan
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Tudor Cuciureanu
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Robert Nastasa
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Ermina Stratina
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Sebastian Zenovia
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Cristina Maria Muzica
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Laura Huiban
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Ana-Maria Singeap
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Stefan Chiriac
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Catalin Sfarti
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Camelia Cojocariu
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Irina Girleanu
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Horia Minea
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Remus Stafie
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Adrian Rotaru
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
| | - Carol Stanciu
- Department of Gastroenterology, Grigore T. Popa University of Medicine and Pharmacy, 700115 Iasi, Romania
- Institute of Gastroenterology and Hepatology, “St. Spiridon” University Hospital, 700111 Iasi, Romania
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Apolipoprotein C3 facilitates internalization of cationic lipid nanoparticles into bone marrow-derived mouse mast cells. Sci Rep 2023; 13:431. [PMID: 36624108 PMCID: PMC9828384 DOI: 10.1038/s41598-022-25737-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Accepted: 12/05/2022] [Indexed: 01/11/2023] Open
Abstract
Mast cells (MCs), are hematopoetically-derived secretory immune cells that release preformed as well as de novo synthesized inflammatory mediators in response to activation by several stimuli. Based on their role in inflammatory responses, particularly in the lung and skin, MCs provide an effective target for anti-inflammatory therapeutic strategies. Drug-delivery of lipophilic payloads to MCs can be challenging due to their functionally distinct intracellular structures. In the present study, pH-sensitive cationic lipid-based nanoparticles (LNPs) composed of DODMA, DODAP or DOTAP lipids that encapsulated a GFP or eGFP plasmid were constructed using non-turbulent microfluidic mixing. This approach achieved up to 75-92% encapsulation efficiency. Dynamic light scattering revealed a uniformly sized and homogeneous dispersion of LNPs. To promote cellular internalization, LNPs were complexed with apolipoproteins, amphipathic proteins capable of binding lipids and facilitating their transport into cells. Cryo-TEM analysis showed that LNP structure was differentially modified when associated with different types of apolipoproteins. LNP preparations made up of DODMA or DODMA, DODAP and DOTAP lipids were coated with seven apolipoproteins (Apo A1, B, C3, D, E2, E4 and H). Differentiated bone-marrow derived mouse mast cells (BMMCs) were exposed to apolipoprotein-LNP and internalization was measured using flow cytometry. Out of all the apolipoproteins tested, ApoC3 most efficiently facilitated cellular internalization of the LNP into BMMCs as determined by GFP fluorescence using flow cytometry. These effects were confirmed in a less differentiated but also interleukin-3-dependent model of mouse mast cells, MC/9. ApoC3-LNP enhanced internalization by BMMC in a concentration-dependent manner and this was significantly increased when BMMC were pre-treated with inhibitors of actin polymerization, suggesting a dependence on intracellular shuttling. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) decreased ApoC3-LNP internalization and reduced the expression of apolipoprotein E receptor 2 (ApoER2), suggesting that ApoC3-LNP binding to ApoER2 may be responsible for its enhanced internalization. Furthermore, ApoC3 fails to facilitate internalization of LNPs in Lrp8-/- KO BMMC that do not express ApoER2 on their cell surface. Altogether, our studies reveal an important role of ApoC3 in facilitating internalization of cationic LNPs into MCs.
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Diaz O, Vidalain PO, Ramière C, Lotteau V, Perrin-Cocon L. What role for cellular metabolism in the control of hepatitis viruses? Front Immunol 2022; 13:1033314. [PMID: 36466918 PMCID: PMC9713817 DOI: 10.3389/fimmu.2022.1033314] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 11/02/2022] [Indexed: 11/26/2023] Open
Abstract
Hepatitis B, C and D viruses (HBV, HCV, HDV, respectively) specifically infect human hepatocytes and often establish chronic viral infections of the liver, thus escaping antiviral immunity for years. Like other viruses, hepatitis viruses rely on the cellular machinery to meet their energy and metabolite requirements for replication. Although this was initially considered passive parasitism, studies have shown that hepatitis viruses actively rewire cellular metabolism through molecular interactions with specific enzymes such as glucokinase, the first rate-limiting enzyme of glycolysis. As part of research efforts in the field of immunometabolism, it has also been shown that metabolic changes induced by viruses could have a direct impact on the innate antiviral response. Conversely, detection of viral components by innate immunity receptors not only triggers the activation of the antiviral defense but also induces in-depth metabolic reprogramming that is essential to support immunological functions. Altogether, these complex triangular interactions between viral components, innate immunity and hepatocyte metabolism may explain why chronic hepatitis infections progressively lead to liver inflammation and progression to cirrhosis, fibrosis and hepatocellular carcinoma (HCC). In this manuscript, we first present a global overview of known connections between the innate antiviral response and cellular metabolism. We then report known molecular mechanisms by which hepatitis viruses interfere with cellular metabolism in hepatocytes and discuss potential consequences on the innate immune response. Finally, we present evidence that drugs targeting hepatocyte metabolism could be used as an innovative strategy not only to deprive viruses of key metabolites, but also to restore the innate antiviral response that is necessary to clear infection.
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Affiliation(s)
- Olivier Diaz
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Pierre-Olivier Vidalain
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Christophe Ramière
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France
| | - Vincent Lotteau
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Laure Perrin-Cocon
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
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Tréguier Y, Bull-Maurer A, Roingeard P. Apolipoprotein E, a Crucial Cellular Protein in the Lifecycle of Hepatitis Viruses. Int J Mol Sci 2022; 23:ijms23073676. [PMID: 35409035 PMCID: PMC8998859 DOI: 10.3390/ijms23073676] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Revised: 03/19/2022] [Accepted: 03/25/2022] [Indexed: 02/01/2023] Open
Abstract
Apolipoprotein E (ApoE) is a multifunctional protein expressed in several tissues, including those of the liver. This lipoprotein component is responsible for maintaining lipid content homeostasis at the plasma and tissue levels by transporting lipids between the liver and peripheral tissues. The ability of ApoE to interact with host-cell surface receptors and its involvement in several cellular pathways raised questions about the hijacking of ApoE by hepatotropic viruses. Hepatitis C virus (HCV) was the first hepatitis virus reported to be dependent on ApoE for the completion of its lifecycle, with ApoE being part of the viral particle, mediating its entry into host cells and contributing to viral morphogenesis. Recent studies of the hepatitis B virus (HBV) lifecycle have revealed that this virus and its subviral envelope particles also incorporate ApoE. ApoE favors HBV entry and is crucial for the morphogenesis of infectious particles, through its interaction with HBV envelope glycoproteins. This review summarizes the data highlighting the crucial role of ApoE in the lifecycles of HBV and HCV and discusses its potential role in the lifecycle of other hepatotropic viruses.
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Affiliation(s)
- Yannick Tréguier
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
| | - Anne Bull-Maurer
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
| | - Philippe Roingeard
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
- Plateforme IBiSA des Microscopies, Université de Tours et CHU de Tours, 37032 Tours, France
- Correspondence: ; Tel.: +33-0247-366-232
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Shimotohno K. HCV Assembly and Egress via Modifications in Host Lipid Metabolic Systems. Cold Spring Harb Perspect Med 2021; 11:cshperspect.a036814. [PMID: 32122916 PMCID: PMC7778218 DOI: 10.1101/cshperspect.a036814] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Hepatitis C virus (HCV) proliferates by hijacking the host lipid machinery. In vitro replication systems revealed many aspects of the virus life cycle; in particular, viral utilization of host lipid metabolism during HCV proliferation. HCV interacts with lipid droplets (LDs) before starting the process of virus capsid formation at the lipid-rich endoplasmic reticulum (ER) membrane compartment. HCV buds into the ER via lipoprotein assembly and secretion. Exchangeable apolipoproteins, represented by apolipoprotein E (apoE), play pivotal roles in enhancing HCV-specific infectivity. HCV virions are likely to interact with other lipoproteins circulating in blood vessels and incorporate apolipoproteins as well as lipids. This review focuses on virus assembly and egress by briefly describing the recent advances in this area.
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9
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Cosset FL, Mialon C, Boson B, Granier C, Denolly S. HCV Interplay with Lipoproteins: Inside or Outside the Cells? Viruses 2020; 12:v12040434. [PMID: 32290553 PMCID: PMC7232430 DOI: 10.3390/v12040434] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 04/05/2020] [Accepted: 04/10/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a major public health issue leading to chronic liver diseases. HCV particles are unique owing to their particular lipid composition, namely the incorporation of neutral lipids and apolipoproteins. The mechanism of association between HCV virion components and these lipoproteins factors remains poorly understood as well as its impact in subsequent steps of the viral life cycle, such as entry into cells. It was proposed that the lipoprotein biogenesis pathway is involved in HCV morphogenesis; yet, recent evidence indicated that HCV particles can mature and evolve biochemically in the extracellular medium after egress. In addition, several viral, cellular and blood components have been shown to influence and regulate this specific association. Finally, this specific structure and composition of HCV particles was found to influence entry into cells as well as their stability and sensitivity to neutralizing antibodies. Due to its specific particle composition, studying the association of HCV particles with lipoproteins remains an important goal towards the rational design of a protective vaccine.
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Cai H, Yao W, Huang J, Xiao J, Chen W, Hu L, Mai R, Liang M, Chen D, Jiang N, Zhou L, Peng T. Apolipoprotein M, identified as a novel hepatitis C virus (HCV) particle associated protein, contributes to HCV assembly and interacts with E2 protein. Antiviral Res 2020; 177:104756. [PMID: 32119870 DOI: 10.1016/j.antiviral.2020.104756] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 01/18/2020] [Accepted: 02/25/2020] [Indexed: 02/08/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases such as steatosis, cirrhosis, and hepatocellular carcinoma. HCV particles have been found to associate with apolipoproteins, and apolipoproteins not only participate in the HCV life cycle, but also help HCV escape recognition by the host immune system, which pose challenges for the development of both HCV treatments and vaccines. However, no study has reported on the comprehensive identification of apolipoprotein associations with HCV particles. In the present study, we performed proteome analysis by affinity purification coupled with mass spectrometry (AP-MS) to comprehensively identify the apolipoprotein associations with HCV particles, and ApoM was first identified by AP-MS besides the previously reported ApoE, ApoB, ApoA-I and ApoC-I. Additionally, three assays further confirmed that ApoM was a novel virus particle associated protein. We also showed that ApoM was required for HCV production, especially for the assembly/release step of HCV life cycle. Furthermore, ApoM interacted with the HCV E2 protein. Finally, HCV infection reduced ApoM expression both in vitro and in vivo. Collectively, our study demonstrates that ApoM, identified as a novel HCV particle associated protein, contributes to HCV assembly/release and interacts with HCV E2 protein. It provides new insights on how HCV and the host apolipoproteins are reciprocally influenced and lays a basis for research in developing innovative antiviral strategies.
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Affiliation(s)
- Hua Cai
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Wenxia Yao
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China.
| | - Jingxian Huang
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Jing Xiao
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Wenli Chen
- Department of Infectious Diseases, Guangdong Provincial People's Hospital, Guangzhou, China
| | - Longbo Hu
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Runming Mai
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Mengdi Liang
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Di Chen
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Nan Jiang
- The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Li Zhou
- The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Tao Peng
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China.
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11
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Fuior EV, Gafencu AV. Apolipoprotein C1: Its Pleiotropic Effects in Lipid Metabolism and Beyond. Int J Mol Sci 2019; 20:ijms20235939. [PMID: 31779116 PMCID: PMC6928722 DOI: 10.3390/ijms20235939] [Citation(s) in RCA: 107] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Revised: 11/19/2019] [Accepted: 11/21/2019] [Indexed: 12/20/2022] Open
Abstract
Apolipoprotein C1 (apoC1), the smallest of all apolipoproteins, participates in lipid transport and metabolism. In humans, APOC1 gene is in linkage disequilibrium with APOE gene on chromosome 19, a proximity that spurred its investigation. Apolipoprotein C1 associates with triglyceride-rich lipoproteins and HDL and exchanges between lipoprotein classes. These interactions occur via amphipathic helix motifs, as demonstrated by biophysical studies on the wild-type polypeptide and representative mutants. Apolipoprotein C1 acts on lipoprotein receptors by inhibiting binding mediated by apolipoprotein E, and modulating the activities of several enzymes. Thus, apoC1 downregulates lipoprotein lipase, hepatic lipase, phospholipase A2, cholesterylester transfer protein, and activates lecithin-cholesterol acyl transferase. By controlling the plasma levels of lipids, apoC1 relates directly to cardiovascular physiology, but its activity extends beyond, to inflammation and immunity, sepsis, diabetes, cancer, viral infectivity, and-not last-to cognition. Such correlations were established based on studies using transgenic mice, associated in the recent years with GWAS, transcriptomic and proteomic analyses. The presence of a duplicate gene, pseudogene APOC1P, stimulated evolutionary studies and more recently, the regulatory properties of the corresponding non-coding RNA are steadily emerging. Nonetheless, this prototypical apolipoprotein is still underexplored and deserves further research for understanding its physiology and exploiting its therapeutic potential.
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Affiliation(s)
- Elena V. Fuior
- Institute of Cellular Biology and Pathology “N. Simionescu”, 050568 Bucharest, Romania;
| | - Anca V. Gafencu
- Institute of Cellular Biology and Pathology “N. Simionescu”, 050568 Bucharest, Romania;
- Correspondence:
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12
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Abouelasrar Salama S, Lavie M, De Buck M, Van Damme J, Struyf S. Cytokines and serum amyloid A in the pathogenesis of hepatitis C virus infection. Cytokine Growth Factor Rev 2019; 50:29-42. [PMID: 31718982 DOI: 10.1016/j.cytogfr.2019.10.006] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Revised: 10/17/2019] [Accepted: 10/21/2019] [Indexed: 02/07/2023]
Abstract
Expression of the acute phase protein serum amyloid A (SAA) is dependent on the release of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α during infection and inflammation. Hepatitis C virus (HCV) upregulates SAA-inducing cytokines. In line with this, a segment of chronically infected individuals display increased circulating levels of SAA. SAA has even been proposed to be a potential biomarker to evaluate treatment efficiency and the course of disease. SAA possesses antiviral activity against HCV via direct interaction with the viral particle, but might also divert infectivity through its function as an apolipoprotein. On the other hand, SAA shares inflammatory and angiogenic activity with chemotactic cytokines by activating the G protein-coupled receptor, formyl peptide receptor 2. These latter properties might promote chronic inflammation and hepatic injury. Indeed, up to 80 % of infected individuals develop chronic disease because they cannot completely clear the infection, due to diversion of the immune response. In this review, we summarize the interconnection between SAA and cytokines in the context of HCV infection and highlight the dual role SAA could play in this disease. Nevertheless, more research is needed to establish whether the balance between those opposing activities can be tilted in favor of the host defense.
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Affiliation(s)
- Sara Abouelasrar Salama
- Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, 3000, Belgium
| | - Muriel Lavie
- University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France
| | - Mieke De Buck
- Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, 3000, Belgium
| | - Jo Van Damme
- Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, 3000, Belgium
| | - Sofie Struyf
- Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, 3000, Belgium.
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13
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Plissonnier ML, Cottarel J, Piver E, Kullolli M, Centonze FG, Pitteri S, Farhan H, Meunier JC, Zoulim F, Parent R. LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity. Virus Res 2019; 271:197679. [PMID: 31398365 DOI: 10.1016/j.virusres.2019.197679] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Accepted: 07/27/2019] [Indexed: 12/23/2022]
Abstract
Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, as well as immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity.
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Affiliation(s)
- Marie-Laure Plissonnier
- Pathogenesis of Hepatitis B and C -DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, F-69008, Lyon, France
| | - Jessica Cottarel
- Pathogenesis of Hepatitis B and C -DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, F-69008, Lyon, France
| | - Eric Piver
- Morphogenesis and Antigenicity of HIV and Hepatitis Viruses, INSERM U966, Université de Tours, F-37000, Tours, France
| | - Majlinda Kullolli
- Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA, 94304, USA
| | | | - Sharon Pitteri
- Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA, 94304, USA
| | - Hesso Farhan
- Institute of Basic Medical Science, University of Oslo, N-0372, Olso, Norway
| | - Jean-Christophe Meunier
- Morphogenesis and Antigenicity of HIV and Hepatitis Viruses, INSERM U966, Université de Tours, F-37000, Tours, France
| | - Fabien Zoulim
- Pathogenesis of Hepatitis B and C -DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, F-69008, Lyon, France; Lyon University Hospital (Hospices civils de Lyon), Hepatogastroenterology Service, F-69001, Lyon, France
| | - Romain Parent
- Pathogenesis of Hepatitis B and C -DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, F-69008, Lyon, France.
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14
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Syntenin regulates hepatitis C virus sensitivity to neutralizing antibody by promoting E2 secretion through exosomes. J Hepatol 2019; 71:52-61. [PMID: 30880226 DOI: 10.1016/j.jhep.2019.03.006] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Revised: 02/22/2019] [Accepted: 03/05/2019] [Indexed: 02/08/2023]
Abstract
BACKGROUND & AIMS Assembly of infectious hepatitis C virus (HCV) particles is known to involve host lipoproteins, giving rise to unique lipo-viro-particles (LVPs), but proteome studies now suggest that additional cellular proteins are associated with HCV virions or other particles containing the viral envelope glycoprotein E2. Many of these host cell proteins are common markers of exosomes, most notably the intracellular adaptor protein syntenin, which is required for exosome biogenesis. We aimed to elucidate the role of syntenin/E2 in HCV infection. METHODS Using cell culture-derived HCV, we studied the biogenesis and function of E2-coated exosomes in both hepatoma cells and primary human hepatocytes (PHHs). RESULTS Knockout of syntenin had a negligible impact on HCV replication and virus production, whereas ectopic expression of syntenin at physiological levels reduced intracellular E2 abundance, while concomitantly increasing the secretion of E2-coated exosomes. Importantly, cells expressing syntenin and HCV structural proteins efficiently released exosomes containing E2 but lacking the core protein. Furthermore, infectivity of HCV released from syntenin-expressing hepatoma cells and PHHs was more resistant to neutralization by E2-specific antibodies and chronic-phase patient serum. We also found that high E2/syntenin levels in sera correlate with lower serum neutralization capability. CONCLUSIONS E2- and syntenin-containing exosomes are a major type of particle released from cells expressing high levels of syntenin. Efficient production of E2-coated exosomes renders HCV infectivity less susceptible to antibody neutralization in hepatoma cells and PHHs. LAY SUMMARY This study identifies a key role for syntenin in the regulation of E2 secretion via exosomes. Efficient production of E2-coated exosomes was shown to make hepatitis C virus less sensitive to antibody neutralization. These results may have implications for the development of a hepatitis C virus vaccine.
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15
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Vieyres G, Pietschmann T. HCV Pit Stop at the Lipid Droplet: Refuel Lipids and Put on a Lipoprotein Coat before Exit. Cells 2019; 8:cells8030233. [PMID: 30871009 PMCID: PMC6468556 DOI: 10.3390/cells8030233] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 02/28/2019] [Accepted: 03/04/2019] [Indexed: 02/07/2023] Open
Abstract
The replication cycle of the liver-tropic hepatitis C virus (HCV) is tightly connected to the host lipid metabolism, during the virus entry, replication, assembly and egress stages, but also while the virus circulates in the bloodstream. This interplay coins viral particle properties, governs viral cell tropism, and facilitates immune evasion. This review summarizes our knowledge of these interactions focusing on the late steps of the virus replication cycle. It builds on our understanding of the cell biology of lipid droplets and the biosynthesis of liver lipoproteins and attempts to explain how HCV hijacks these organelles and pathways to assemble its lipo-viro-particles. In particular, this review describes (i) the mechanisms of viral protein translocation to and from the lipid droplet surface and the orchestration of an interface between replication and assembly complexes, (ii) the importance of the triglyceride mobilization from the lipid droplets for HCV assembly, (iii) the interplay between HCV and the lipoprotein synthesis pathway including the role played by apolipoproteins in virion assembly, and finally (iv) the consequences of these complex virus–host interactions on the virion composition and its biophysical properties. The wealth of data accumulated in the past years on the role of the lipid metabolism in HCV assembly and its imprint on the virion properties will guide vaccine design efforts and reinforce our understanding of the hepatic lipid metabolism in health and disease.
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Affiliation(s)
- Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), 30625 Hannover, Germany.
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), 30625 Hannover, Germany.
- German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 38124 Braunschweig, Germany.
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16
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Abstract
Apolipoprotein E (apoE) plays dual functions in the HCV life cycle by promoting HCV infection and virion assembly and production. ApoE is a structural component on the HCV envelope. It mediates HCV cell attachment through specific interactions with the cell surface receptors such as syndecan-1 (SDC-1) and SDC-2 heparan sulfate proteoglycans (HSPGs). It also interacts with NS5A and E2, resulting in an enhancement of HCV morphogenesis. It can bind HCV extracellularly and promotes HCV infection. It is critical for HCV cell-to-cell transmission and may also play a role in HCV persistence by interfering with the action of HCV-neutralizing antibodies. Other apolipoproteins particularly apoB and apoC1 were also found on the HCV envelope, but their roles in the HCV life cycle remain unclear. In the last decade, a number of genomic, immunological, structural, and cell biology methodologies have been developed and used for determining the importance of apoE in the HCV life cycle. These methods and protocols will continue to be valuable to further understand the importance and the underlying molecular mechanism of various apolipoproteins in HCV infection and pathogenesis.
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Affiliation(s)
- Luhua Qiao
- Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA
| | - Guangxiang George Luo
- Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA.
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17
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Wang X, Yan Y, Gan T, Yang X, Li D, Zhou D, Sun Q, Huang Z, Zhong J. A trivalent HCV vaccine elicits broad and synergistic polyclonal antibody response in mice and rhesus monkey. Gut 2019; 68:140-149. [PMID: 29180585 DOI: 10.1136/gutjnl-2017-314870] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2017] [Revised: 10/26/2017] [Accepted: 11/11/2017] [Indexed: 12/26/2022]
Abstract
OBJECTIVE Despite the development of highly effective direct-acting antivirals, a prophylactic vaccine is needed for eradicating HCV. A major hurdle of HCV vaccine development is to induce immunity against HCV with high genome diversity. We previously demonstrated that a soluble E2 (sE2) expressed from insect cells induces broadly neutralising antibodies (NAbs) and prevents HCV infection. The objective of this study is to develop a multivalent HCV vaccine to increase the antigenic coverage. DESIGN We designed a trivalent vaccine containing sE2 from genotype 1a, 1b and 3a. Mice and rhesus macaques were immunised with monovalent or trivalent sE2 vaccine, and sera or purified immunoglobulin were assessed for neutralisation against a panel of cell culture-derived virion (HCVcc) of genotype 1-7 in cell culture. Splenocytes from the vaccinated macaques were assessed for HCV-specific T cell response. RESULTS We showed that the trivalent vaccine elicited pangenotypic NAbs in mice, which neutralised HCVcc of all the seven genotypes more potently than the monovalent vaccine. Further analyses demonstrated that each sE2 component of this trivalent vaccine elicited unique spectrum of NAbs which acted synergistically to inhibit HCV infection. Finally, the trivalent vaccine triggered stronger and more uniform multigenotypic neutralising antibody response than the monovalent vaccine in rhesus macaques. CONCLUSIONS In summary, we developed a trivalent HCV vaccine that induces broad and synergistic-acting neutralising antibodies in mice and non-human primates.
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Affiliation(s)
- Xuesong Wang
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,Unit of Vaccinology and Antiviral Strategies, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Yu Yan
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Tianyu Gan
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Xi Yang
- University of Chinese Academy of Sciences, Beijing, China.,Vaccine Research Center, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - Dapeng Li
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,Unit of Vaccinology and Antiviral Strategies, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Dongming Zhou
- University of Chinese Academy of Sciences, Beijing, China.,Vaccine Research Center, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - Qiang Sun
- University of Chinese Academy of Sciences, Beijing, China.,Suzhou Non-human Primate Facility, Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
| | - Zhong Huang
- Unit of Vaccinology and Antiviral Strategies, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Jin Zhong
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
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18
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Similarities and Differences Between HCV Pseudoparticle (HCVpp) and Cell Culture HCV (HCVcc) in the Study of HCV. Methods Mol Biol 2019; 1911:33-45. [PMID: 30593616 DOI: 10.1007/978-1-4939-8976-8_2] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
For a long time, the study of the HCV infectious cycle has been a major challenge for researchers because of the difficulties in generating an efficient cell culture system leading to a productive viral infection. The development of HCVpp and later on HCVcc model allowing for functional studies of HCV in cell culture completely revolutionized HCV research. The aim of this review is to provide the reader with a brief overview of the development of these two models. We describe the advantages of each model as well as their limitations in the study of the HCV life cycle, with a particular emphasis on virus entry. A comparison between these two models is presented in terms of virion composition and their use as tools for the characterization of entry factors, envelope glycoprotein functions, and antibody neutralization. We also compare the production and biosafety level of these two types of viral particles. Globally, this review provides a general description of the most adequate applications for HCVpp and HCVcc in HCV research.
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19
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McPherson A, Larson SB. The structure of human apolipoprotein C-1 in four different crystal forms. J Lipid Res 2018; 60:400-411. [PMID: 30559175 PMCID: PMC6358290 DOI: 10.1194/jlr.m089441] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2018] [Revised: 11/28/2018] [Indexed: 02/02/2023] Open
Abstract
Human apolipoprotein C1 (APOC1) is a 57 amino acid long polypeptide that, through its potent inhibition of cholesteryl ester transferase protein, helps regulate the transfer of lipids between lipid particles. We have now determined the structure of APOC1 in four crystal forms by X-ray diffraction. A molecule of APOC1 is a single, slightly bent, α-helix having 13–14 turns and a length of about 80 Å. APOC1 exists as a dimer, but the dimers are not the same in the four crystals. In two monoclinic crystals, two helices closely engage one another in an antiparallel fashion. The interactions between monomers are almost entirely hydrophobic with sparse electrostatic complements. In the third monoclinic crystal, the two monomers spread at one end of the dimer, like a scissor opening, and, by translation along the crystallographic a axis, form a continuous, contiguous sheet through the crystal. In the orthorhombic crystals, two molecules of APOC1 are related by a noncrystallographic 2-fold axis to create an arc of about 120 Å length. This symmetrical dimer utilizes interactions not present in dimers of the monoclinic crystals. Versatility of APOC1 monomer association shown by these crystals is suggestive of physiological function.
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Affiliation(s)
- Alexander McPherson
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900
| | - Steven B Larson
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900
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20
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Prentoe J, Bukh J. Hypervariable Region 1 in Envelope Protein 2 of Hepatitis C Virus: A Linchpin in Neutralizing Antibody Evasion and Viral Entry. Front Immunol 2018; 9:2146. [PMID: 30319614 PMCID: PMC6170631 DOI: 10.3389/fimmu.2018.02146] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 08/30/2018] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is the cause of about 400,000 annual liver disease-related deaths. The global spread of this important human pathogen can potentially be prevented through the development of a vaccine, but this challenge has proven difficult, and much remains unknown about the multitude of mechanisms by which this heterogeneous RNA virus evades inactivation by neutralizing antibodies (NAbs). The N-terminal motif of envelope protein 2 (E2), termed hypervariable region 1 (HVR1), changes rapidly in immunoglobulin-competent patients due to antibody-driven antigenic drift. HVR1 contains NAb epitopes and is directly involved in protecting diverse antibody-specific epitopes on E1, E2, and E1/E2 through incompletely understood mechanisms. The ability of HVR1 to protect HCV from NAbs appears linked with modulation of HCV entry co-receptor interactions. Thus, removal of HVR1 increases interaction with CD81, while altering interaction with scavenger receptor class B, type I (SR-BI) in a complex fashion, and decreasing interaction with low-density lipoprotein receptor. Despite intensive efforts this modulation of receptor interactions by HVR1 remains incompletely understood. SR-BI has received the most attention and it appears that HVR1 is involved in a multimodal HCV/SR-BI interaction involving high-density-lipoprotein associated ApoCI, which may prime the virus for later entry events by exposing conserved NAb epitopes, like those in the CD81 binding site. To fully elucidate the multifunctional role of HVR1 in HCV entry and NAb evasion, improved E1/E2 models and comparative studies with other NAb evasion strategies are needed. Derived knowledge may be instrumental in the development of a prophylactic HCV vaccine.
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Affiliation(s)
- Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark.,Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark.,Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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21
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Andrade VGD, Yamashiro FDS, Oliveira CV, Kurozawa LL, Moreira A, Silva GF. INCREASE OF LIPIDS DURING HCV TREATMENT: VIRUS ACTION OR MEDICATION? ARQUIVOS DE GASTROENTEROLOGIA 2018; 55:184-187. [PMID: 30043871 DOI: 10.1590/s0004-2803.201800000-33] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 01/09/2018] [Indexed: 11/22/2022]
Abstract
BACKGROUND The interaction between serum lipids and C virus infection is well known, as are serum lipid levels in the Peg-IFN / RBV-based treatment. However, with direct action antivirals (DAAs) this behavior is still unclear. OBJECTIVE To compare serum lipids levels between patients treated with Peg-IFN/RBV and DAAs and to evaluate lipids in sustained virological response (SVR) with DAAs. METHODS Retro prospective study comparing the behavior of total cholesterol (TC), low-density lipoprotein (LDL) and triglycerides (TG) serum levels during treatment with DAAs (G-DAAs) and a control historic group Peg-IFN/RBV (G-PR). Coorte, prospective study, to study the behavior of lipids in the SVR with DAAs. Data were collected at the beginning of treatment (baseline: t-base) and at week 12 of treatment (t-12) for G-DAAs and at week 24 (t-24) for G-PR, groups. In the cohort evaluation, the samples at t-base and at week 12 after the end of treatment (t-SVR). Delta lipids: difference between lipids in t-12 / t-24 minus t-base for comparison between G-PR and G-AADs groups and t-SVR minus t-base for lipid analysis in SVR. Analysis with Kruskal Wallis and Wilcoxon tests to compare the delta lipids of the groups. The P value was 0.05. RESULTS In the assessment between G-PR and G-DAAs groups, we included 63 and 121 patients, respectively. The groups did not differ one from the other (BMI, sex, genotype, fibrosis, total cholesterol, LDL, and TG) except by age (50.38±10.44 vs 56±9.69, P=0.0006). We observed a decrease in levels of TC and LDL and an increase in TG, in G-PR, and in G-DAAs the opposite (Δ TC -13.9±34.5 vs 4.12±34.3 P=0.0005, Δ LDL -7.16±32 vs 10.13±29.92, P=0.003, Δ TG 4.51±53.7 vs -8.24±49.93, P=0.0025). In the coorte analysis, we included 102 patients, 70% men and 56% F4, 95 of them reached SVR. We observed an increase of TC and LDL and a decrease of TG in both groups (SVR and non SVR), with no statistical difference (Δ TC P=0.68; Δ LDL P=0.69; Δ TG P=0.43). We did not find significant difference in delta evaluation by genotype 1 and 3 (Δ TC +29.7±40.2 vs +13.4±30.3, P=0.06; Δ LDL +21.4±28.6 vs +16.6±31.3, P=0.41; Δ TG -3.6±60.6 vs -0.7±40, P=0.91). CONCLUSION Serum lipids level differed during treatment with Peg-IFN and DAAs. Treatment with DAAs was associated with an increase of TC and LDL and a decrease of TG, independently of SVR.
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Affiliation(s)
- Vanessa Gutierrez de Andrade
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
| | - Fabio da Silva Yamashiro
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
| | - Cassio Vieira Oliveira
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
| | - Leticia Lastória Kurozawa
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
| | - Alecsandro Moreira
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
| | - Giovanni Faria Silva
- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Campus Botucatu, Faculdade de Medicina, Departamento de Clínica Médica, Botucatu, SP, Brasil
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22
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Zhang H, Qiao L, Luo G. Characterization of apolipoprotein C1 in hepatitis C virus infection and morphogenesis. Virology 2018; 524:1-9. [PMID: 30130702 DOI: 10.1016/j.virol.2018.08.004] [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: 05/23/2018] [Revised: 08/05/2018] [Accepted: 08/05/2018] [Indexed: 12/12/2022]
Abstract
Previous studies have shown that apolipoprotein C1 (apoC1)-specific antibodies precipitated hepatitis C virus (HCV) and neutralized HCV infectivity, suggesting that apoC1 is a HCV component. However, the importance of apoC1 in the HCV life cycle has not been experimentally examined. In the present study, we sought to determine the role of apoC1 in the HCV infection and morphogenesis by knocking out the apoC1 gene using the CRISPR/Cas9 system. Strikingly, apoC1 gene knockout markedly enhanced apoE expression. As a result, apoC1 gene knockout per se didn't significantly affect HCV infection or morphogenesis, probably ascribing to its redundant functions with apoE. However, knockout of apoC1 gene potentiated the impairment of HCV infection and/or morphogenesis by apoE-specific small interfering RNAs. Additionally, a recombinant apoC1 protein efficiently blocked HCV infection. Collectively, these findings suggest that apoC1 and apoE have redundant functions in the HCV infection and morphogenesis.
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Affiliation(s)
- Han Zhang
- Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing 100191, China
| | - Luhua Qiao
- Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, United States
| | - Guangxiang Luo
- Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing 100191, China; Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, United States.
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23
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Exploring lipid and apolipoprotein levels in chronic hepatitis C patients according to their response to antiviral treatment. Clin Biochem 2018; 60:17-23. [PMID: 30030979 DOI: 10.1016/j.clinbiochem.2018.07.007] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2018] [Revised: 07/17/2018] [Accepted: 07/17/2018] [Indexed: 02/07/2023]
Abstract
BACKGROUND Hepatitis C virus is known to be highly dependent of lipid metabolism to infect new cells and replicate. AIMS To investigate lipid and apolipoprotein profile in chronic HCV patients according to treatment response. METHODS Patients recruited from the Hepatitis Treatment Center at Niteroi (Brazil) who received interferon (IFN)-based therapies were separated into two groups, those who achieved sustained virological response (SVR) or not (non-SVR). Another group of patients treated with IFN-free direct-acting antiviral (DAA) therapies was followed from before starting the treatment until one year after therapy. Triglycerides, total cholesterol and fractions were determined by colorimetric and/or electrophoresis techniques. Lecithin cholesterol acyltransferase (LCAT) activity and serum levels of apolipoproteins A1, A2, B, C2, C3 and E were assessed by enzymatic and multiplex assays, respectively. RESULTS We studied 114 patients, and SVR was reached in 28 (39.4%) patients treated with IFN-therapy and in all (100%) patients who received DAA. Non-SVR patients (n = 43) presented altered liver parameters post-treatment. Levels of total cholesterol, LDL-C, VLDL-C and triglycerides were significant higher in SVR group. In contrast, LCAT activity and HDL-C levels were elevated in non-SVR patients. Only apolipoproteins B, C2 and C3 levels were increased in SVR group. The follow-up of SVR-DAA patients (n = 43) revealed a significant and progressive increase in serum levels of total cholesterol, LDL-C, VLDL-C and triglycerides. CONCLUSIONS After a successful treatment, chronic hepatitis C patients experienced a reestablishment of lipid metabolism. Our results suggest that the monitoring of serum lipids could be a practical and routine laboratory tool to be applied during the treatment follow-up.
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24
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Sun HY, Cheng PN, Tseng CY, Tsai WJ, Chiu YC, Young KC. Favouring modulation of circulating lipoproteins and lipid loading capacity by direct antiviral agents grazoprevir/elbasvir or ledipasvir/sofosbuvir treatment against chronic HCV infection. Gut 2018; 67:1342-1350. [PMID: 28615303 DOI: 10.1136/gutjnl-2017-313832] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Revised: 04/28/2017] [Accepted: 04/30/2017] [Indexed: 02/07/2023]
Abstract
OBJECTIVE Lipid homoeostasis is disturbed in patients with HCV infection. Direct-acting antiviral agent (DAA) treatment eradicates chronic HCV viraemia, but the dynamics of lipid components remain elusive. This study investigates the clinical manifestation and mechanistic relevance of plasma triglyceride (TG), cholesterol (Chol), lipoproteins and apolipoproteins (apos) after DAA treatment. DESIGN Twenty-four patients with chronic genotype 1 (GT1) HCV treated with elbasvir/grazoprevir or ledipasvir/sofosbuvir for 12 weeks, and followed-up thereafter, were recruited. Their TG, Chol, apoAI and apoB levels were quantified in plasma samples and individually fractionated lipoprotein of various classes. Liver fibrosis was evaluated using the FIB-4 Score. The TG and Chol loading capacities were calculated with normalisation to apoB, which represents per very low density lipoprotein (VLDL) and LDL particle unit RESULTS: DAA treatment achieved a sustained virological response rate of 91.7% and reduced the FIB-4 Score. Relative to the baseline, the plasma TG level was reduced but the Chol level increased gradually. Plasma apoB levels and apoB/apoAI ratio were transiently downregulated as early as the first 4 weeks of treatment. The TG and Chol loading capacities in VLDL were elevated by ~20% during the period of DAA treatment and had steadily increased by 100% at follow-up. Furthermore, the TG-to-Chol ratio in VLDL was increased, while the ratio in LDL was reduced, indicating an efficient catabolism. CONCLUSION The DAA treatment of patients with chronic hepatitis C might lead to efficient HCV eradication and hepatic improvement concomitantly evolving with favouring lipoprotein/apo metabolisms.
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Affiliation(s)
- Hung-Yu Sun
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Pin-Nan Cheng
- Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Chiung-Ying Tseng
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Wei-Jen Tsai
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Yen-Cheng Chiu
- Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Kung-Chia Young
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
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25
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Wrensch F, Crouchet E, Ligat G, Zeisel MB, Keck ZY, Foung SKH, Schuster C, Baumert TF. Hepatitis C Virus (HCV)-Apolipoprotein Interactions and Immune Evasion and Their Impact on HCV Vaccine Design. Front Immunol 2018; 9:1436. [PMID: 29977246 PMCID: PMC6021501 DOI: 10.3389/fimmu.2018.01436] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2018] [Accepted: 06/11/2018] [Indexed: 12/15/2022] Open
Abstract
With more than 71 million people chronically infected, hepatitis C virus (HCV) is one of the leading causes of liver disease and hepatocellular carcinoma. While efficient antiviral therapies have entered clinical standard of care, the development of a protective vaccine is still elusive. Recent studies have shown that the HCV life cycle is closely linked to lipid metabolism. HCV virions associate with hepatocyte-derived lipoproteins to form infectious hybrid particles that have been termed lipo-viro-particles. The close association with lipoproteins is not only critical for virus entry and assembly but also plays an important role during viral pathogenesis and for viral evasion from neutralizing antibodies. In this review, we summarize recent findings on the functional role of apolipoproteins for HCV entry and assembly. Furthermore, we highlight the impact of HCV-apolipoprotein interactions for evasion from neutralizing antibodies and discuss the consequences for antiviral therapy and vaccine design. Understanding these interactions offers novel strategies for the development of an urgently needed protective vaccine.
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Affiliation(s)
- Florian Wrensch
- INSERM, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Emilie Crouchet
- INSERM, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Gaetan Ligat
- 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.,INSERM U1052, CNRS UMR 5286, Cancer Research Center of Lyon (CRCL), Université de Lyon (UCBL), Lyon, France
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
| | - Catherine Schuster
- 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.,Institut Universitaire de France, Paris, France
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26
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Bankwitz D, Doepke M, Hueging K, Weller R, Bruening J, Behrendt P, Lee JY, Vondran FWR, Manns MP, Bartenschlager R, Pietschmann T. Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity. J Hepatol 2017; 67:480-489. [PMID: 28438690 DOI: 10.1016/j.jhep.2017.04.010] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2017] [Revised: 04/03/2017] [Accepted: 04/11/2017] [Indexed: 12/19/2022]
Abstract
BACKGROUND & AIMS Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles. METHODS ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted. RESULTS Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs). CONCLUSIONS Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of the availability of ApoE likely influences HCV infectivity, antibody escape and transmission.
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Affiliation(s)
- Dorothea Bankwitz
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Mandy Doepke
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Kathrin Hueging
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Romy Weller
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Janina Bruening
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Patrick Behrendt
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Florian W R Vondran
- Regenerative Medicine & Experimental Surgery (ReMediES), Department of General, Visceral and Transplant Surgery, Hannover Medical School, Germany; German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 30625 Hannover, Germany
| | - Michael P Manns
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 30625 Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg University, Heidelberg, Germany
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 30625 Hannover, Germany.
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27
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Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes. J Virol 2017; 91:JVI.00478-17. [PMID: 28659476 DOI: 10.1128/jvi.00478-17] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2017] [Accepted: 06/22/2017] [Indexed: 12/23/2022] Open
Abstract
Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells. The relevance of cell polarity in the HCV life cycle has been addressed only in distantly related models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins typical of columnar polarized epithelia, which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used, and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on a semipermeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture-derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density were secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.IMPORTANCE Hepatitis C is a major health burden, with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distantly related models and remains unclear. Hepatocyte organization is complex, with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with unique apical and basolateral domains was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower-density viral particles were produced under conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.
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28
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Coto-Llerena M, Koutsoudakis G, Boix L, López-Oliva JM, Caro-Pérez N, Fernández-Carrillo C, González P, Gastaminza P, Bruix J, Forns X, Pérez-Del-Pulgar S. Permissiveness of human hepatocellular carcinoma cell lines for hepatitis C virus entry and replication. Virus Res 2017; 240:35-46. [PMID: 28751105 DOI: 10.1016/j.virusres.2017.07.018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 07/20/2017] [Accepted: 07/20/2017] [Indexed: 12/28/2022]
Abstract
Hepatitis C virus (HCV) is a globally prevalent pathogen and is associated with high death rates and morbidity. Since its discovery in 1989, HCV research has been impeded by the lack of a robust infectious cell culture system and thus in vitro studies on diverse genetic backgrounds are hampered because of the limited number of hepatoma cell lines which are able to support different aspects of the HCV life cycle. In the current study, we sought to expand the limited number of permissive cells capable of supporting the diverse phases of the HCV life cycle. Initially, we screened a panel of new hepatoma-derived cell lines, designated BCLC-1, -2, -3, -4, -5, -6, -9 and -10 cells, for their ability to express essential HCV receptors and subsequently to support HCV entry by using the well-characterized HCV pseudoparticle system (HCVpp). Apart from BCLC-9, all BCLC cell lines were permissive for HCVpp infection. Next, BCLC cells were subjected to short- and long-term HCV RNA replication studies using HCV subgenomic replicons. Interestingly, only BCLC-1, -5 and -9 cells, supported short-term HCV RNA replication, but the latter were excluded from further studies since they were refractory for HCV entry. BCLC-1, -5 were able to support long-term HCV replication too; yet BCLC-5 cells supported the highest long-term HCV RNA replication levels. Furthermore, cured BCLC-5 clones from HCV subgenomic replicon, showed increased permissiveness for HCV RNA replication. Strikingly, we were unable to detect endogenous BCLC-5 miR122 expression - an important HCV host factor- and as expected, the exogenous expression of miR122 in BCLC-5 cells increased their permissiveness for HCV RNA replication. However, this cell line was unable to produce HCV infectious particles despite ectopic expression of apolipoprotein E, which in other hepatoma cell lines has been shown to be sufficient to enable the HCV secretion process, suggesting a lack of other host cellular factor(s) and/or the presence of inhibitory factor(s). In conclusion, the establishment of these new permissive cell lines for HCV entry and replication, which possess a different genetic background compared to the well-established models, expands the current repertoire of hepatoma cell lines susceptible to the study of the HCV life cycle and also will aid to further elucidate the cellular determinants that modulate HCV replication, assembly and egress.
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Affiliation(s)
| | | | - Loreto Boix
- Barcelona Clínic Liver Cancer (BCLC) Group, Hospital Clínic, IDIBAPS, CIBERehd, Spain
| | | | | | | | | | - Pablo Gastaminza
- Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus Cantoblanco, Madrid, Spain
| | - Jordi Bruix
- Barcelona Clínic Liver Cancer (BCLC) Group, Hospital Clínic, IDIBAPS, CIBERehd, Spain
| | - Xavier Forns
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Barcelona, Spain
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29
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Andreo U, de Jong YP, Scull MA, Xiao JW, Vercauteren K, Quirk C, Mommersteeg MC, Bergaya S, Menon A, Fisher EA, Rice CM. Analysis of Hepatitis C Virus Particle Heterogeneity in Immunodeficient Human Liver Chimeric fah-/- Mice. Cell Mol Gastroenterol Hepatol 2017; 4:405-417. [PMID: 28936471 PMCID: PMC5602752 DOI: 10.1016/j.jcmgh.2017.07.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Accepted: 07/10/2017] [Indexed: 12/21/2022]
Abstract
BACKGROUND & AIMS Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. METHODS To analyze the evolution of the buoyant density of in vivo-derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. RESULTS Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. CONCLUSIONS Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet.
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Key Words
- Alb-uPA, Albumin-urokinase plasminogen activator
- CETP, cholesterol ester transfer protein
- FAH, fumaryl acetoacetate hydrolase
- FNRG, absence of fumaryl acetoacetate hydrolase on a immunodeficient NOD Rag gamma IL2 deficient mouse background
- FPLC, fast-performance liquid chromatography
- HCV
- HCV, hepatitis C virus
- HCVcc, cell culture–derived hepatitis C virus
- HDL, high-density lipoprotein
- Human Liver Chimeric Mice
- LVP, lipoviroparticle
- Lipoprotein
- Mouse Model
- NRG, nod rag γ
- NTBC, nitisinone
- PBS, phosphate-buffered saline
- SCID, severe combined immunodeficiency disease
- VLDL, very low density lipoprotein
- apo, apolipoprotein
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Affiliation(s)
- Ursula Andreo
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
- Correspondence Address correspondence to: Ursula Andreo, PhD, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, Box 64, New York, New York 10065. fax: (212) 327-7048.Center for the Study of Hepatitis CThe Rockefeller University1230 York AvenueBox 64New YorkNew York 10065
| | - Ype P. de Jong
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
- Division of Gastroenterology and Hepatology, Center for the Study of Hepatitis C, Weill Cornell Medical College, New York, New York
| | - Margaret A. Scull
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
| | - Jing W. Xiao
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
| | - Koen Vercauteren
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
| | - Corrine Quirk
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
| | | | - Sonia Bergaya
- Division of Cardiology, Department of Medicine, New York University Langone Medical Center, New York, New York
| | - Arjun Menon
- Division of Cardiology, Department of Medicine, New York University Langone Medical Center, New York, New York
| | - Edward A. Fisher
- Division of Cardiology, Department of Medicine, New York University Langone Medical Center, New York, New York
| | - Charles M. Rice
- Center for the Study of Hepatitis C, The Rockefeller University, New York, New York
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30
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Crouchet E, Baumert TF, Schuster C. Hepatitis C virus-apolipoprotein interactions: molecular mechanisms and clinical impact. Expert Rev Proteomics 2017; 14:593-606. [PMID: 28625086 PMCID: PMC6138823 DOI: 10.1080/14789450.2017.1344102] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
INTRODUCTION Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. Moreover, chronic HCV infection is associated with liver steatosis and metabolic disorders. With 130-150 million people chronically infected in the world, HCV infection represents a major public health problem. One hallmark on the virus is its close link with hepatic lipid and lipoprotein metabolism. Areas covered: HCV is associated with lipoprotein components such as apolipoproteins. These interactions play a key role in the viral life cycle, viral persistence and pathogenesis of liver disease. This review introduces first the role of apolipoproteins in lipoprotein metabolism, then highlights the molecular mechanisms of HCV-lipoprotein interactions and finally discusses their clinical impact. Expert commentary: While the study of virus-host interactions has resulted in a improvement of the understanding of the viral life cycle and the development of highly efficient therapies, major challenges remain: access to therapy is limited and an urgently needed HCV vaccine remains still elusive. Furthermore, the pathogenesis of disease biology is still only partially understood. The investigation of HCV-lipoproteins interactions offers new perspectives for novel therapeutic approaches, contribute to HCV vaccine design and understand virus-induced liver disease and cancer.
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Affiliation(s)
- Emilie Crouchet
- 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
- Pôle hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - Catherine Schuster
- Inserm, U1110: Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
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31
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Lavie M, Dubuisson J. Interplay between hepatitis C virus and lipid metabolism during virus entry and assembly. Biochimie 2017. [PMID: 28630011 DOI: 10.1016/j.biochi.2017.06.009] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis C virus (HCV) infection is a major public health problem worldwide. In most cases, HCV infection becomes chronic, leading to the development of liver diseases that range from fibrosis to cirrhosis and hepatocellular carcinoma. Due to its medical importance, the HCV life cycle has been deeply characterized, and a unique feature of this virus is its interplay with lipids. Accordingly, all the steps of the virus life cycle are influenced by the host lipid metabolism. Indeed, due to their association with host lipoproteins, HCV particles have a unique lipid composition. Furthermore, the biogenesis pathway of very low density lipoproteins has been shown to be involved in HCV morphogenesis with apolipoprotein E being an essential element for the production of infectious HCV particles. Association of viral components with host cytoplasmic lipid droplets is also central to the HCV morphogenesis process. Finally, due to its close connection with host lipoproteins, HCV particle also uses several lipoprotein receptors to initiate its infectious cycle. In this review, we outline the way host lipoproteins participate to HCV particle composition, entry and assembly.
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Affiliation(s)
- Muriel Lavie
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, F-59000, Lille, France
| | - Jean Dubuisson
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, F-59000, Lille, France.
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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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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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Falcón V, Acosta-Rivero N, González S, Dueñas-Carrera S, Martinez-Donato G, Menéndez I, Garateix R, Silva JA, Acosta E, Kourı J. Ultrastructural and biochemical basis for hepatitis C virus morphogenesis. Virus Genes 2017; 53:151-164. [PMID: 28233195 DOI: 10.1007/s11262-017-1426-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2016] [Accepted: 01/06/2017] [Indexed: 12/16/2022]
Abstract
Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.
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Affiliation(s)
- Viviana Falcón
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba.
| | - Nelson Acosta-Rivero
- National Center for Scientific Research, P.O. Box 6414, 10600, Havana, Cuba.
- Centre for Protein Studies, Faculty of Biology, University of Havana, 10400, Havana, Cuba.
| | | | | | | | - Ivon Menéndez
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
| | - Rocio Garateix
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
| | - José A Silva
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
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Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System. J Virol 2017; 91:JVI.01720-16. [PMID: 27852847 DOI: 10.1128/jvi.01720-16] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2016] [Accepted: 11/07/2016] [Indexed: 12/29/2022] Open
Abstract
Hepatitis C virus (HCV) infects 2 to 3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology and antiviral drug development to drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, the HCV-dependent fluorescence relocalization (HDFR) system allowed live-cell visualization of infection without modifying the viral genome, but this strategy required careful recognition of the fluorescence relocalization pattern for its high fluorescence background in the cytoplasm. In this study, to achieve background-free visualization of HCV infection, a viral infection-activated split-intein-mediated reporter system (VISI) was devised. Uninfected Huh7.5.1-VISI cells show no background signal, while HCV infection specifically illuminates the nuclei of infected Huh7.5.1-VISI cells with either green fluorescent protein (GFP) or mCherry. Combining VISI-GFP and VISI-mCherry systems, we revisited HCV cell-to-cell transmission with clear-cut distinction of donor and recipient cells in a live-cell manner. Independently of virion assembly, exosomes have been reported to transfer HCV subgenomic RNA to initiate replication in uninfected cells, which suggested an assembly-free pathway. However, our data demonstrated that HCV structural genes and the p7 gene were essential for not only cell-free infectivity but also cell-to-cell transmission. Additionally, depletion of apolipoprotein E (ApoE) from donor cells but not from recipient cells significantly reduced HCV cell-to-cell transmission efficiency. In summary, we developed a background-free cell-based reporter system for convenient live-cell visualization of HCV infection, and our data indicate that complete HCV virion assembly machinery is essential for both cell-free and cell-to-cell transmission. IMPORTANCE Hepatitis C virus (HCV) infects hepatocytes via two pathways: cell-free infection and cell-to-cell transmission. Structural modules of the HCV genome are required for production of infectious cell-free virions; however, the role of specific genes within the structural module in cell-to-cell transmission is not clearly defined. Our data demonstrate that deletion of core, E1E2, and p7 genes individually results in no HCV cell-to-cell transmission and that ApoE knockdown from donor cells causes less-efficient cell-to-cell transmission. Thus, this work indicates that the complete HCV assembly machinery is required for HCV cell-to-cell transmission. At last, this work presents an optimized viral infection-activated split-intein-mediated reporter system for easy live-cell monitoring of HCV infection.
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Wasilewski LN, Ray SC, Bailey JR. Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles. J Gen Virol 2016; 97:2883-2893. [PMID: 27667373 DOI: 10.1099/jgv.0.000608] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.
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Affiliation(s)
- Lisa N Wasilewski
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Stuart C Ray
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.,Department of Oncology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Justin R Bailey
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
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Kinoshita C, Nagano T, Seki N, Tomita Y, Sugita T, Aida Y, Itagaki M, Satoh K, Sutoh S, Abe H, Tsubota A, Aizawa Y. Hepatitis C virus G1b infection decreases the number of small low-density lipoprotein particles. World J Gastroenterol 2016; 22:6716-6725. [PMID: 27547014 PMCID: PMC4970482 DOI: 10.3748/wjg.v22.i29.6716] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 06/11/2016] [Accepted: 07/06/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate how hepatitis C virus (HCV) G1b infection influences the particle number of lipoproteins. METHODS The numbers of lipoprotein particles in fasting sera from 173 Japanese subjects, 82 with active HCV G1b infection (active HCV group) and 91 with cleared HCV infection (SVR group), were examined. Serum lipoprotein was fractionated by high-performance liquid chromatography into twenty fractions. The cholesterol and triglyceride concentrations in each fraction were measured using LipoSEARCH. The number of lipoprotein particles in each fraction was calculated using a newly developed algorithm, and the relationship between chronic HCV G1b infection and the lipoprotein particle number was determined by multiple linear regression analysis. RESULTS The median number of low-density lipoprotein (LDL) particles was significantly lower in the active HCV group [1182 nmol/L, interquartile range (IQR): 444 nmol/L] than in the SVR group (1363 nmol/L, IQR: 472 nmol/L, P < 0.001), as was that of high-density lipoprotein (HDL) particles (14168 nmol/L vs 15054 nmol/L, IQR: 4114 nmol/L vs 3385 nmol/L, P = 0.042). The number of very low-density lipoprotein (VLDL) particles was similar between the two groups. Among the four LDL sub-fractions, the number of large LDL particles was similar between the two groups. However, the numbers of medium (median: 533.0 nmol/L, IQR: 214.7 nmol/L vs median: 633.5 nmol/L, IQR: 229.6 nmol/L, P < 0.001), small (median: 190.9 nmol/L, IQR: 152.4 nmol/L vs median: 263.2 nmol/L, IQR: 159.9 nmol/L; P < 0.001), and very small LDL particles (median: 103.5 nmol/L, IQR: 66.8 nmol/L vs median: 139.3 nmol/L, IQR: 67.3 nmol/L, P < 0.001) were significantly lower in the active HCV group than in the SVR group, respectively. Multiple linear regression analysis indicated an association between HCV G1b infection and the decreased numbers of medium, small, and very small LDL particles. However, active HCV infection did not affect the number of large LDL particles or any sub-fractions of VLDL and HDL particles. CONCLUSION HCV G1b infection decreases the numbers of medium, small, and very small LDL particles.
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Earnest-Silveira L, Chua B, Chin R, Christiansen D, Johnson D, Herrmann S, Ralph SA, Vercauteren K, Mesalam A, Meuleman P, Das S, Boo I, Drummer H, Bock CT, Gowans EJ, Jackson DC, Torresi J. Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line. J Gen Virol 2016; 97:1865-1876. [PMID: 27147296 DOI: 10.1099/jgv.0.000493] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.
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Affiliation(s)
- L Earnest-Silveira
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
| | - B Chua
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
| | - R Chin
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
| | - D Christiansen
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
- Department of Surgery, Austin Hospital, University of Melbourne, Australia
| | - D Johnson
- Department of Infectious Diseases, Austin Hospital, Heidelberg, Victoria 3084, Australia
| | - S Herrmann
- Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Australia
| | - S A Ralph
- Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Australia
| | - K Vercauteren
- Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185 9000, Ghent, Belgium
| | - A Mesalam
- Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185 9000, Ghent, Belgium
| | - P Meuleman
- Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185 9000, Ghent, Belgium
| | - S Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - I Boo
- Centre for Biomedical Research, Burnet Institute, Melbourne, Australia
| | - H Drummer
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
- Centre for Biomedical Research, Burnet Institute, Melbourne, Australia
- Department of Microbiology, Monash University, Clayton, Australia
| | - C-T Bock
- Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany
| | - E J Gowans
- The Basil Hetzel Institute and Queen Elizabeth Hospital, University of Adelaide, Australia
| | - D C Jackson
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
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Cell-death-inducing DFFA-like Effector B Contributes to the Assembly of Hepatitis C Virus (HCV) Particles and Interacts with HCV NS5A. Sci Rep 2016; 6:27778. [PMID: 27282740 PMCID: PMC4901263 DOI: 10.1038/srep27778] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Accepted: 05/23/2016] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) uses components of the very-low-density lipoprotein (VLDL) pathway for assembly/release. We previously reported that hepatocyte nuclear factor 4α (HNF4α) participates in HCV assembly/release through downstream factors those participate in VLDL assembly/secretion. Cell-death-inducing DFFA-like effector B (CIDEB) is an important regulator of the VLDL pathway. CIDEB is required for entry of HCV particles from cell culture (HCVcc), but the effects of CIDEB on the post-entry steps of the HCV lifecycle are unclear. In the present study, we determined that CIDEB is required for HCV assembly in addition to HCVcc entry. Furthermore, CIDEB interacts with the HCV NS5A protein, and the N terminus of CIDEB and the domain I of NS5A are involved in this interaction. Moreover, CIDEB silencing impairs the association of apolipoprotein E (ApoE) with HCV particles. Interestingly, CIDEB is also required for the post-entry stages of the dengue virus (DENV) life cycle. Collectively, these results indicate that CIDEB is a new host factor that is involved in HCV assembly, presumably by interacting with viral protein, providing new insight into the exploitation of the VLDL regulator CIDEB by HCV.
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40
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Yamamoto S, Fukuhara T, Ono C, Uemura K, Kawachi Y, Shiokawa M, Mori H, Wada M, Shima R, Okamoto T, Hiraga N, Suzuki R, Chayama K, Wakita T, Matsuura Y. Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus. PLoS Pathog 2016; 12:e1005610. [PMID: 27152966 PMCID: PMC4859476 DOI: 10.1371/journal.ppat.1005610] [Citation(s) in RCA: 68] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 04/12/2016] [Indexed: 02/07/2023] Open
Abstract
Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV. Hepatitis C virus (HCV) utilizes several receptors to enter hepatocytes, including scavenger receptor class B type 1 (SR-B1) receptor and low-density lipoprotein receptor (LDLR). HCV particles interact with lipoprotein and apolipoproteins to form complexes termed lipoviroparticles. Several reports have shown that SR-B1 and LDLR participate in the entry of lipoviroparticles through interaction with lipoproteins. However, the precise roles of SR-B1 and LDLR in HCV entry have not been fully clarified. In this study, we showed that SR-B1 and LDLR have a redundant role in HCV entry. In addition, we showed that very low-density lipoprotein receptor (VLDLR) played a role in HCV entry similar to the roles of SR-B1 and LDLR. Interestingly, VLDLR expression was low in the liver in contrast to the abundant expressions of SR-B1 and LDLR, but high in several extrahepatic tissues. Our data suggest that lipoprotein receptors participate in the entry of HCV particles associated with various lipoproteins.
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Affiliation(s)
- Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Kentaro Uemura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yukako Kawachi
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Mai Shiokawa
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Masami Wada
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Ryoichi Shima
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Nobuhiko Hiraga
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- * E-mail:
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Fukuhara T, Ono C, Puig-Basagoiti F, Matsuura Y. Roles of Lipoproteins and Apolipoproteins in Particle Formation of Hepatitis C Virus. Trends Microbiol 2016; 23:618-629. [PMID: 26433694 DOI: 10.1016/j.tim.2015.07.007] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2015] [Revised: 07/07/2015] [Accepted: 07/20/2015] [Indexed: 02/06/2023]
Abstract
More than 160 million people worldwide are infected with hepatitis C virus (HCV), and cirrhosis and hepatocellular carcinoma induced by HCV infection are life-threatening diseases. HCV takes advantage of many aspects of lipid metabolism for an efficient propagation in hepatocytes. Due to the morphological and physiological similarities of HCV particles to lipoproteins, lipid-associated HCV particles are named lipoviroparticles. Recent analyses have revealed that exchangeable apolipoproteins directly interact with the viral membrane to generate infectious HCV particles. In this review, we summarize the roles of lipid metabolism in the life cycle of HCV.
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Affiliation(s)
- Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Francesc Puig-Basagoiti
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Grassi G, Di Caprio G, Fimia GM, Ippolito G, Tripodi M, Alonzi T. Hepatitis C virus relies on lipoproteins for its life cycle. World J Gastroenterol 2016; 22:1953-1965. [PMID: 26877603 PMCID: PMC4726671 DOI: 10.3748/wjg.v22.i6.1953] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2015] [Revised: 10/19/2015] [Accepted: 12/21/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infects over 150 million people worldwide. In most cases, HCV infection becomes chronic causing liver disease ranging from fibrosis to cirrhosis and hepatocellular carcinoma. Viral persistence and pathogenesis are due to the ability of HCV to deregulate specific host processes, mainly lipid metabolism and innate immunity. In particular, HCV exploits the lipoprotein machineries for almost all steps of its life cycle. The aim of this review is to summarize current knowledge concerning the interplay between HCV and lipoprotein metabolism. We discuss the role played by members of lipoproteins in HCV entry, replication and virion production.
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43
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Zhou LY, Zhang LL. Host restriction factors for hepatitis C virus. World J Gastroenterol 2016; 22:1477-1486. [PMID: 26819515 PMCID: PMC4721981 DOI: 10.3748/wjg.v22.i4.1477] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Revised: 09/30/2015] [Accepted: 11/13/2015] [Indexed: 02/06/2023] Open
Abstract
Host-hepatitis C virus (HCV) interactions have both informed fundamental concepts of viral replication and pathogenesis and provided novel insights into host cell biology. These findings are illustrated by the recent discovery of host-encoded factors that restrict HCV infection. In this review, we briefly discuss these restriction factors in different steps of HCV infection. In each case, we discuss how these restriction factors were identified, the mechanisms by which they inhibit HCV infection and their potential contribution to viral pathogenesis.
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Zayas M, Long G, Madan V, Bartenschlager R. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A. PLoS Pathog 2016; 12:e1005376. [PMID: 26727512 PMCID: PMC4699712 DOI: 10.1371/journal.ppat.1005376] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Accepted: 12/09/2015] [Indexed: 12/19/2022] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. Hepatitis C virus (HCV) nonstructural protein (NS)5A is an enigmatic RNA-binding protein that appears to regulate the different steps from RNA replication to the assembly of infectious virus particles by yet unknown mechanisms. Assembly requires delivery of the viral RNA genome from the replication machinery to the capsid protein to ensure genome packaging into nucleocapsids that acquire a membranous envelope by budding into the lumen of the endoplasmic reticulum. In this study, we provide genetic and biochemical evidence that the viral nonstructural protein (NS)5A contains two regulatory determinants in its domain (D)III that orchestrate virus assembly at two closely linked steps: (i) recruitment of replication complexes to core protein requiring a serine cluster in the C-terminal region of DIII and (ii) RNA genome delivery to core protein requiring a basic cluster in the N-terminal region of DIII. This RNA transfer most likely triggers encapsidation, which is tightly coupled to particle envelopment. These results provide a striking example for a multi-purpose viral protein exerting several distinct functions in the viral replication cycle, thus reflecting genetic economy.
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Affiliation(s)
- Margarita Zayas
- Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- * E-mail: (MZ); (RB)
| | - Gang Long
- Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Vanesa Madan
- Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Ralf Bartenschlager
- Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- * E-mail: (MZ); (RB)
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Cottarel J, Plissonnier ML, Kullolli M, Pitteri S, Clément S, Millarte V, Si-Ahmed SN, Farhan H, Zoulim F, Parent R. FIG4 is a hepatitis C virus particle-bound protein implicated in virion morphogenesis and infectivity with cholesteryl ester modulation potential. J Gen Virol 2015; 97:69-81. [PMID: 26519381 DOI: 10.1099/jgv.0.000331] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
There is growing evidence that virus particles also contain host cell proteins, which provide viruses with certain properties required for entry and release. A proteomic analysis performed on double-gradient-purified hepatitis C virus (HCV) from two highly viraemic patients identified the phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (KIAA0274) as part of the viral particles. We validated the association using immunoelectron microscopy, immunoprecipitation and neutralization assays in vitro as well as patient-derived virus particles. RNA interference-mediated reduction of FIG4 expression decreased cholesteryl ester (CE) levels along with intra- and extracellular viral infectivity without affecting HCV RNA levels. Likewise, overexpressing FIG4 increased intracellular CE levels as well as intra- and extracellular viral infectivity without affecting viral RNA levels. Triglyceride levels and lipid droplet (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found to strongly condition these results. Whilst FIG4 was found to localize to areas corresponding to viral assembly sites, at the immediate vicinity of LDs in calnexin-positive and HCV core-positive regions, no implication of FIG4 in the secretory pathway of the hepatocytes could be found using either FIG4-null mice, in vitro morphometry or functional assays of the ERGIC/Golgi compartments. This indicates that FIG4-dependent modulation of HCV infectivity is unrelated to alterations in the functionality of the secretory pathway. As a result of the documented implication of CE in the composition and infectivity of HCV particles, these results suggest that FIG4 binds to HCV and modulates particle formation in a CE-related manner.
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Affiliation(s)
- Jessica Cottarel
- Pathogenesis of Hepatitis B and C - DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, 69008 Lyon, France
| | - Marie-Laure Plissonnier
- Pathogenesis of Hepatitis B and C - DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, 69008 Lyon, France
| | - Majlinda Kullolli
- Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA 94304, USA
| | - Sharon Pitteri
- Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA 94304, USA
| | - Sophie Clément
- Department of Clinical Pathology, University of Geneva, Geneva, Switzerland
| | | | | | - Hesso Farhan
- Department of Biology, University of Konstanz, Germany.,Hôpital d'Orléans, 45000 Orléans, France
| | - Fabien Zoulim
- Pathogenesis of Hepatitis B and C - DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, 69008 Lyon, France.,Hospices Civils de Lyon, Service d'Hépatogastroentérologie, 69001 Lyon, France
| | - Romain Parent
- Pathogenesis of Hepatitis B and C - DEVweCAN LabEx, INSERM U1052-CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, 69008 Lyon, France
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Stefas I, Tigrett S, Dubois G, Kaiser M, Lucarz E, Gobby D, Bray D, Ellerbrok H, Zarski JP, Veas F. Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus. PLoS One 2015; 10:e0140900. [PMID: 26502286 PMCID: PMC4621047 DOI: 10.1371/journal.pone.0140900] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2015] [Accepted: 10/01/2015] [Indexed: 12/20/2022] Open
Abstract
The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient’s sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient’s prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient’s samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.
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Affiliation(s)
- Ilias Stefas
- ApoH-Technologies, Faculté de Pharmacie, Université de Montpellier, Montpellier, France
| | - Sylvia Tigrett
- ApoH-Technologies, Faculté de Pharmacie, Université de Montpellier, Montpellier, France; Institut de Recherche pour le Développement, UMR-Ministère de la Défense 3, Laboratoire d'Immuno-Physiopathologie Moléculaire Comparée, Faculté de Pharmacie, Montpellier, France
| | - Grégor Dubois
- Institut de Recherche pour le Développement, UMR-Ministère de la Défense 3, Laboratoire d'Immuno-Physiopathologie Moléculaire Comparée, Faculté de Pharmacie, Montpellier, France
| | | | - Estelle Lucarz
- ApoH-Technologies, Faculté de Pharmacie, Université de Montpellier, Montpellier, France
| | - Delphine Gobby
- ApoH-Technologies, Faculté de Pharmacie, Université de Montpellier, Montpellier, France
| | - Dorothy Bray
- Immunoclin Corporation, Washington, DC, United States of America
| | - Heinz Ellerbrok
- Robert Koch-Institute, Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses, Berlin, Germany
| | - Jean Pierre Zarski
- Clinique d'Hépato-gastroentérologie, Centre Hospitalier Universitaire de Grenoble, IAB, INSERM U823, Grenoble, France
| | - Francisco Veas
- Institut de Recherche pour le Développement, UMR-Ministère de la Défense 3, Laboratoire d'Immuno-Physiopathologie Moléculaire Comparée, Faculté de Pharmacie, Montpellier, France
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Del Campo JA, Romero-Gómez M. Modulation of host lipid metabolism by hepatitis C virus: Role of new therapies. World J Gastroenterol 2015; 21:10776-10782. [PMID: 26478669 PMCID: PMC4600579 DOI: 10.3748/wjg.v21.i38.10776] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2015] [Revised: 07/07/2015] [Accepted: 09/14/2015] [Indexed: 02/06/2023] Open
Abstract
It is well established that hepatitis C virus (HCV) infection and replication relies on host lipid metabolism. HCV proteins interact and associate with lipid droplets to facilitate virion assembly and production. Besides, circulating infective particles are associated with very low-density lipoprotein. On the other hand, higher serum lipid levels have been associated with sustained viral response to pegylated interferon and ribavirin therapy in chronic HCV infection, suggesting a relevant role in viral clearance for host proteins. Host and viral genetic factors play an essential role in chronic infection. Lipid metabolism is hijacked by viral infection and could determine the success of viral replication. Recently development of direct acting antiviral agents has shown a very high efficacy (> 90%) in sustained viral response rates even for cirrhotic patients and most of the viral genotypes. HCV RNA clearance induced by Sofosbuvir has been associated with an increased concentration and size of the low-density lipoprotein particles. In this review, host genetic factors, viral factors and the interaction between them will be depicted to clarify the major issues involved in viral infection and lipid metabolism.
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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: 46] [Impact Index Per Article: 4.6] [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: 08/30/2015] [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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Hueging K, Weller R, Doepke M, Vieyres G, Todt D, Wölk B, Vondran FWR, Geffers R, Lauber C, Kaderali L, Penin F, Pietschmann T. Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses. PLoS One 2015; 10:e0134529. [PMID: 26226615 PMCID: PMC4520612 DOI: 10.1371/journal.pone.0134529] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 07/09/2015] [Indexed: 02/08/2023] Open
Abstract
Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.
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Affiliation(s)
- Kathrin Hueging
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Romy Weller
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Mandy Doepke
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Daniel Todt
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Benno Wölk
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Florian W. R. Vondran
- Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
- Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany
- German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
| | - Robert Geffers
- Research Group Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Chris Lauber
- Institute for Medical Informatics and Biometry, Medical Faculty, Technische Universität Dresden, Dresden, Germany
| | - Lars Kaderali
- Institute for Medical Informatics and Biometry, Medical Faculty, Technische Universität Dresden, Dresden, Germany
| | - François Penin
- Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, CNRS, Labex Ecofect, University of Lyon, Lyon, France
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
- German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
- * E-mail:
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The Serum Very-Low-Density Lipoprotein Serves as a Restriction Factor against Hepatitis C Virus Infection. J Virol 2015; 89:6782-91. [PMID: 25903344 DOI: 10.1128/jvi.00194-15] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Accepted: 04/10/2015] [Indexed: 02/06/2023] Open
Abstract
UNLABELLED Recent studies demonstrated that transgenic mice expressing key human hepatitis C virus (HCV) receptors are susceptible to HCV infection, albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or the presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effects of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. The results also imply that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication. IMPORTANCE HCV is a major cause of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in the mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines.
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