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Entry Inhibitors of Hepatitis C Virus. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1366:207-222. [DOI: 10.1007/978-981-16-8702-0_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Augestad EH, Bukh J, Prentoe J. Hepatitis C virus envelope protein dynamics and the link to hypervariable region 1. Curr Opin Virol 2021; 50:69-75. [PMID: 34403905 DOI: 10.1016/j.coviro.2021.07.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2021] [Revised: 07/22/2021] [Accepted: 07/22/2021] [Indexed: 12/11/2022]
Abstract
Conformational dynamics of viral envelope proteins seem to be involved in mediating evasion from neutralizing antibodies (NAbs) by mechanisms that limit exposure of conserved protein motifs. For hepatitis C virus (HCV), molecular studies have only recently begun to unveil how such dynamics of the envelope protein heterodimer, E1/E2, are linked to viral entry and NAb evasion. Here, we review data suggesting that E1/E2 exists in an equilibrium between theoretical 'open' (NAb-sensitive) and 'closed' (NAb-resistant) conformational states. We describe how this equilibrium is influenced by viral sequence polymorphisms and that it is critically dependent on the N-terminal region of E2, termed hypervariable region 1 (HVR1). Finally, we discuss how it appears that the virus binding site for the HCV entry co-receptor CD81 is less available in 'closed' E1/E2 states and that NAb-resistant viruses require a more intricate entry pathway involving also the entry co-receptor, SR-BI.
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Affiliation(s)
- Elias H Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark; Copenhagen Hepatitis C Program (CO-HEP), 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, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark; Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark; Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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Joshita S, Yamashita Y, Okamoto T, Usami Y, Sugiura A, Yamazaki T, Kakino A, Ota M, Sawamura T, Umemura T. Quantitative and qualitative lipid improvement with chronic hepatitis C virus eradication using direct-acting antivirals. Hepatol Res 2021; 51:758-766. [PMID: 33982310 DOI: 10.1111/hepr.13666] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 04/29/2021] [Accepted: 05/08/2021] [Indexed: 02/08/2023]
Abstract
AIM Direct-acting antivirals have revolutionized hepatitis C virus (HCV) therapy by providing a high sustained virological response (SVR) rate and subsequent favorable lipid increases. Proprotein convertase subtilisin-kexin like-9 (PCSK9) plays an important role in regulating quantitative lipid levels. This study examined the interactions between quantitative PCSK9 and lipid changes, as well as qualitative lipid changes in terms of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligand containing apolipoprotein B (LAB) and high-density lipoprotein (HDL) cholesterol uptake capacity (HDL-CUC). METHODS Patients with chronic HCV infection (N = 231) who achieved an SVR by direct-acting antivirals without lipid-lowering therapy were included for comparisons of PCSK9, LAB, HDL-CUC, and other clinical indices between pretreatment and SVR12 time points. RESULTS LDL (LDL) cholesterol and HDL cholesterol levels were quantitatively increased at SVR12, along with higher PCSK9 (all p < 0.0001). PCSK9 was significantly correlated with LDL cholesterol (r = 0.244, p = 0.0003) and apolipoprotein B (r = 0.222, p = 0.0009) at SVR12. Regarding qualitative LDL changes, LAB was significantly decreased and LAB/LDL cholesterol and LAB/apolipoprotein B proportions were improved at SVR12 (all p < 0.0001). In terms of qualitative HDL changes, HDL-CUC was significantly ameliorated, along with HDL-CUC/HDL cholesterol, HDL-CUC/ apolipoprotein A1, and HDL-CUC/ apolipoprotein A2 at SVR12 (all p < 0.0001). CONCLUSIONS HCV eradication by direct-acting antivirals may produce quantitative lipid profile changes, along with PCSK9 production recovery in addition to qualitative lipid improvement, which possibly confers the additional secondary benefits of atherosclerosis improvement and cardiovascular disease event reduction.
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Affiliation(s)
- Satoru Joshita
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Yuki Yamashita
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Toru Okamoto
- Institute for Advanced Co-Creation Studies Research, Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yoko Usami
- Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan
| | - Ayumi Sugiura
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Tomoo Yamazaki
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Akemi Kakino
- Department of Molecular Pathophysiology, Shinshu University School of Medicine, Matsumoto, Japan.,Department of Life Innovation, Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan
| | - Masao Ota
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Tatsuya Sawamura
- Department of Molecular Pathophysiology, Shinshu University School of Medicine, Matsumoto, Japan.,Department of Life Innovation, Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan
| | - Takeji Umemura
- Department of Medicine, Division of Gastroenterology and Hepatology, Shinshu University School of Medicine, Matsumoto, Japan.,Department of Life Innovation, Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan
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Ahmed W, Neelakanta G, Sultana H. Tetraspanins as Potential Therapeutic Candidates for Targeting Flaviviruses. Front Immunol 2021; 12:630571. [PMID: 33968023 PMCID: PMC8097176 DOI: 10.3389/fimmu.2021.630571] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 04/06/2021] [Indexed: 12/18/2022] Open
Abstract
Tetraspanin family of proteins participates in numerous fundamental signaling pathways involved in viral transmission, virus-specific immunity, and virus-mediated vesicular trafficking. Studies in the identification of novel therapeutic candidates and strategies to target West Nile virus, dengue and Zika viruses are highly warranted due to the failure in development of vaccines. Recent evidences have shown that the widely distributed tetraspanin proteins may provide a platform for the development of novel therapeutic approaches. In this review, we discuss the diversified and important functions of tetraspanins in exosome/extracellular vesicle biology, virus-host interactions, virus-mediated vesicular trafficking, modulation of immune mechanism(s), and their possible role(s) in host antiviral defense mechanism(s) through interactions with noncoding RNAs. We also highlight the role of tetraspanins in the development of novel therapeutics to target arthropod-borne flaviviral diseases.
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Affiliation(s)
- Waqas Ahmed
- Department of Biological Sciences, Old Dominion University, Norfolk, VA, United States
| | - Girish Neelakanta
- Department of Biological Sciences, Old Dominion University, Norfolk, VA, United States.,Center for Molecular Medicine, Old Dominion University, Norfolk, VA, United States
| | - Hameeda Sultana
- Department of Biological Sciences, Old Dominion University, Norfolk, VA, United States.,Center for Molecular Medicine, Old Dominion University, Norfolk, VA, United States
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Al-Ansari MM, Ranjit Singh AJA, Al-Khattaf FS, Michael JS. Nano-formulation of herbo-mineral alternative medicine from linga chenduram and evaluation of antiviral efficacy. Saudi J Biol Sci 2021; 28:1596-1606. [PMID: 33732045 PMCID: PMC7938193 DOI: 10.1016/j.sjbs.2020.12.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 11/29/2020] [Accepted: 12/01/2020] [Indexed: 12/18/2022] Open
Abstract
Traditional medicine is becoming a primary source of health care in many countries in recent years. The current study proposes a new dimension of understanding a traditional origin treatment, using herbo-mineral preparations in nanoform. The herbo-mineral preparation, Linga chenduram [HMLC], was prepared according to the ancient palm script protocol dates back to 1000 years. In search of alternative therapy for the coronavirus, an attempt was made to determine this ethnic medicine formulation's therapeutic potential for viral hepatitis infection. The Hepatitis C virus [HCV] has several genomic similarities with SARS-CoV-2 viruses. The herbo-mineral formulation (HMLC) were analyzed using UV–vis, EDAX, FTIR, XRD, SEM, and TEM studies. SEM images confirmed the ' presence of nanoparticles with agglomerated conditions having an average grain size of 18 to 25 nm. EDAX studies showed the presence of metallic components in oxide or sulfide form in HMLC. The HCV inhibitory effects of HMLC indicated a good response. The cytotoxicity of this preparation against the Huh-7 human hepatoma cell line was significant. The HMLC showed a strong inhibitory effect on HCV replication in a dose-dependent manner. The genomic component of HCV is similar to COVID −19 virus. The Hepatitis C virus (HCV) NS3/4A protease has a striking three-dimensional structural similarity to the SARS-CoV2 Mpro protease, particularly in the arrangement of key active site residues. So HMLC can be tried to treat coronavirus infection. At higher concentrations, HMLC exhibited over 100-fold inhibition. In the MTT assay, HMLC did not show any apparent cytotoxic effect on cell viability at the concentrations 1–100 µg. Histological studies indicated that the liver and kidney did not experience any toxicity by 7 and 15 consecutive days of administration of HMLC on experimental Wistar rats. Hence, the HMLC can be tried as a therapy for COVID −19 infections using the preparations strictly according to ethnopharmacological protocol and optimum doses.
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Affiliation(s)
- Mysoon M Al-Ansari
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
| | - A J A Ranjit Singh
- Department of Biotechnology, Prathyusha Engineering College, Chennai 600056, India
| | - Fatimah S Al-Khattaf
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
| | - J S Michael
- Department of Biotechnology, Prathyusha Engineering College, Chennai 600056, India
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Aftab A, Afzal S, Idrees M, Shahid AA. p53 and rb promoter methylation in hepatitis C virus-related chronic hepatitis and hepatocellular carcinoma. Future Virol 2021. [DOI: 10.2217/fvl-2020-0154] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Aim: To identify methylation in p53 and rb during hepatitis C virus (HCV) infection in individuals in Pakistan. Materials & methods: Methylation-specific PCR was used on liver biopsies from hepatocellular carcinoma and chronic hepatitis C patients and on blood samples from healthy individuals. Real-time PCR was used to assess changes in the expression of p53 and rb in Huh-7 cells transfected with HCV-3a. Results: The p53 and rb promoters were methylated in hepatocellular carcinoma patients. The presence of HCV-3a- Core (p = 0.03), HCV-3a- NS-3 (p = 0.01) and HCV-3a- NS-5a (p = 0.02) downregulated p53 expression. Exposure to HCV-3a- Core (p = 0.04) downregulated rb expression. Conclusion: It can be hypothesized that HCV-induced epigenetic modifications may lead to the development of hepatic cancer that in turn inactivates p53 and rb.
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Affiliation(s)
- Ayma Aftab
- Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Samia Afzal
- Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Muhammad Idrees
- Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Ahmad Ali Shahid
- Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
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7
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Bove G, Mehnert AK, Dao Thi VL. iPSCs for modeling hepatotropic pathogen infections. IPSCS FOR STUDYING INFECTIOUS DISEASES 2021:149-213. [DOI: 10.1016/b978-0-12-823808-0.00013-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process. Int J Mol Sci 2020; 21:ijms21062091. [PMID: 32197477 PMCID: PMC7140000 DOI: 10.3390/ijms21062091] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 03/15/2020] [Accepted: 03/16/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. Its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. This review provides an overview of the viral and host factors involved in HCV entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors.
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Aljowaie RM, Almajhdi FN, Ali HH, El-Wetidy MS, Shier MK. Inhibition of hepatitis C virus genotype 4 replication using siRNA targeted to the viral core region and the CD81 cellular receptor. Cell Stress Chaperones 2020; 25:345-355. [PMID: 32060691 PMCID: PMC7058739 DOI: 10.1007/s12192-020-01077-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Revised: 01/21/2020] [Accepted: 01/24/2020] [Indexed: 01/12/2023] Open
Abstract
Hepatitis C virus (HCV) is one of the most important causative agents of hepatitis worldwide. The current study aimed to evaluate the silencing effect of the small interference RNA (siRNA) molecules designed against the core region of HCV genotype 4 (HCV-4) and the CD81 gene, which is the cellular receptor for HCV in the human hepatocytes. RT-PCR was used to measure the changes in both the viral HCV core and the cellular CD81 genes induced by the specific siRNA molecules. Additionally, the fluctuations in either the viral or the cellular proteins of the target regions were tested by flow cytometry and immunofluorescence. The results showed the effectiveness of the used siRNA molecules against the target genes in either RNA or protein levels. The effect of 100 nM of siCD81 and 40 nM of siCore was more evident at 24 and 48 h post-transfection. The combination of the two siRNA molecules resulted in an extra inhibitory effect of the HCV core at both the RNA (85.6%) and protein (98.5%) levels. The current study suggested that targeting of the CD81 cellular receptor and/or the viral HCV core region by the small interference molecules might be a suitable choice in the suppression of HCV-4 replication. This might assist the development of new antiviral medications and provides a new alternative strategy for the targeting and treatment of HCV genotype 4.
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Affiliation(s)
- Reem M Aljowaie
- Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia.
| | - Fahad N Almajhdi
- Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia
| | - Hebatallh H Ali
- College of Medicine Research Center, King Saud University, Riyadh, Saudi Arabia
| | | | - Medhat K Shier
- College of Medicine Research Center, King Saud University, Riyadh, Saudi Arabia
- Department of Medical Microbiology and Immunology, College of Medicine, Menofia University, Shibin el Kom, Egypt
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10
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Mounir M, Ibrahim MK, Dawood RM, Barakat AB, El Awady MK. Establishment of serum derived infectivity coculture model for enhancement of hepatitis C virus replication in vitro. Hum Antibodies 2020; 27:185-191. [PMID: 30958341 DOI: 10.3233/hab-190370] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND AND AIMS Although HCV is one of the major health problems worldwide with the highest prevalence of genotype 4a in Egypt, it is poorly understood because of the limitations of having a robust in vitro model that allows the investigation and understanding of viral pathogenesis and life cycle. Genomic replicons for HCV are widely used and proved to have strong replication efficiency in cell culture, however, they are not able to produce infectious particles to enable the investigation of the whole viral life cycle and they mostly represent few sub-genomic classes for HCV. Hence, Genotype specific replication system is necessary to address specific sub-genomic phenotypes related to Hepatitis C pathogenicity. METHODS In this study we attempt to develop a sustainable co-culture model, which potentially provides essential route of infection for HCV by using HCV-positive sera from infected patients. In this novel in vitro model, we tested the viral replication in co-cultured Huh 7.5 and HepG2 cells in order to sustain full viral replication cycle. We used high viral load serum of HCV-infected patients (10 × 106 to 20 × 106 IU/ml) as a source for HCV particles to infect co-cultured cells for 7 days. RESULTS AND CONCLUSIONS Viral replication capacity was increased 3-5 folds in the coculture condition compared to the individual cell lines, which indicates an improvement to viral infectivity in vitro. SIGNIFICANCE STATEMENT This novel coculture system represents a new in vitro model that will help study the underlying mechanisms of HCV pathogenicity.
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Affiliation(s)
- Mariam Mounir
- Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - Marwa Khalil Ibrahim
- Genetic Engineering Division, Department of Microbial Biotechnology, National Research Center, Giza, Egypt.,Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - Reham M Dawood
- Genetic Engineering Division, Department of Microbial Biotechnology, National Research Center, Giza, Egypt.,Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - Ahmed B Barakat
- Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - Mostafa K El Awady
- Genetic Engineering Division, Department of Microbial Biotechnology, National Research Center, Giza, Egypt
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Gerold G, Moeller R, Pietschmann T. Hepatitis C Virus Entry: Protein Interactions and Fusion Determinants Governing Productive Hepatocyte Invasion. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a036830. [PMID: 31427285 DOI: 10.1101/cshperspect.a036830] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) entry is among the best-studied uptake processes for human pathogenic viruses. Uptake follows a spatially and temporally tightly controlled program. Numerous host factors including proteins, lipids, and glycans promote productive uptake of HCV particles into human liver cells. The virus initially attaches to surface proteoglycans, lipid receptors such as the scavenger receptor BI (SR-BI), and to the tetraspanin CD81. After lateral translocation of virions to tight junctions, claudin-1 (CLDN1) and occludin (OCLN) are essential for entry. Clathrin-mediated endocytosis engulfs HCV particles, which fuse with endosomal membranes after pH drop. Uncoating of the viral RNA genome in the cytoplasm completes the entry process. Here we systematically review and classify HCV entry factors by their mechanistic role, relevance, and level of evidence. Finally, we report on more recent knowledge on determinants of membrane fusion and close with an outlook on future implications of HCV entry research.
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Affiliation(s)
- Gisa Gerold
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany.,Department of Clinical Microbiology, Virology & Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, 901 85 Umeå, Sweden
| | - Rebecca Moeller
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany
| | - Thomas Pietschmann
- TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany
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Shahid I, Almalki WH, Ibrahim MM, Alghamdi SA, Mukhtar MH, Almalki SSR, Alkahtani SA, Alhaidari MS. Characterization of In vitro inhibitory effects of consensus short interference RNAs against non-structural 5B gene of hepatitis C virus 1a genotype. Indian J Med Microbiol 2019; 36:494-503. [PMID: 30880695 DOI: 10.4103/ijmm.ijmm_17_146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Purpose Chronic hepatitis C has infected approximately 170 million people worldwide. The novel direct-acting antivirals have proven their clinical efficacy to treat hepatitis C infection but still very expensive and beyond the financial range of most infected patients in low income and even resource replete nations. This study was conducted to establish an in vitro stable human hepatoma 7 (Huh-7) cell culture system with consistent expression of the non-structural 5B (NS5B) protein of hepatitis C virus (HCV) 1a genotype and to explore inhibitory effects of sequence-specific short interference RNA (siRNA) targeting NS5B in stable cell clones, and against viral replication in serum-inoculated Huh-7 cells. Materials and Methods In vitro stable Huh-7 cells with persistent expression of NS5B protein was produced under gentamycin (G418) selection. siRNAs inhibitory effects were determined by analysing NS5B expression at mRNA and protein level through reverse transcription-polymerase chain reaction (PCR), quantitative real-time PCR, and Western blot, respectively. Statistical significance of data (NS5B gene suppression) was performed using SPSS software (version 16.0, SPSS Inc.). Results siRNAs directed against NS5B gene significantly decreased NS5B expression at mRNA and protein levels in stable Huh-7 cells, and a vivid decrease in viral replication was also exhibited in serum-infected Huh-7 cells. Conclusions Stable Huh-7 cells persistently expressing NS5B protein should be helpful for molecular pathogenesis of HCV infection and development of anti-HCV drug screening assays. The siRNA was effective against NS5B and could be considered as an adjuvant therapy along with other promising anti-HCV regimens.
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Affiliation(s)
- Imran Shahid
- Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia
| | - Waleed Hassan Almalki
- Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia
| | - Munjed M Ibrahim
- Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia
| | - Sultan Ahmad Alghamdi
- Infection Control Department, King Fahd Hospital, Ministry of Health, Jeddah, Saudi Arabia
| | - Mohammed H Mukhtar
- Department of Biochemistry, College of Medicine, Umm Al-Qura Univeristy, Makkah, Saudi Arabia
| | - Shaia Saleh R Almalki
- Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al Baha University, Al Baha, Saudi Arabia
| | - Saad Ahmed Alkahtani
- Department of Clinical Pharmacy, College of Pharmacy, Najran University, Najran, Saudi Arabia
| | - Mohammad S Alhaidari
- Pharmaceutical Care Department, King Fahad Hospital, Ministry of Health, Madinah, Saudi Arabia
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Wang J, Qu B, Zhang F, Zhang C, Deng W, Dao Thi VL, Xia Y. Stem Cell-Derived Hepatocyte-Like Cells as Model for Viral Hepatitis Research. Stem Cells Int 2019; 2019:9605252. [PMID: 31281392 PMCID: PMC6594266 DOI: 10.1155/2019/9605252] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Accepted: 05/09/2019] [Indexed: 02/06/2023] Open
Abstract
Viral hepatitis, the leading cause of liver diseases worldwide, is induced upon infection with hepatotropic viruses, including hepatitis A, B, C, D, and E virus. Due to their obligate intracellular lifestyles, culture systems for efficient viral replication are vital. Although basic and translational research on viral hepatitis has been performed for many years, conventional hepatocellular culture systems are not optimal. These studies have greatly benefited from recent efforts on improving cell culture models for virus replication and infection studies. Here we summarize the use of human stem cell-derived hepatocyte-like cells for hepatotropic virus infection studies, including the dissection of virus-host interactions and virus-induced pathogenesis as well as the identification and validation of novel antiviral agents.
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Affiliation(s)
- Jingjing Wang
- State Key Laboratory of Virology, School of Basic Medical Sciences, Wuhan University, Wuhan, China
| | - Bingqian Qu
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Fang Zhang
- Department of Translational Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
| | - Cindy Zhang
- Schaller Research Group at Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), Heidelberg, Germany
| | - Wanyan Deng
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
| | - Viet Loan Dao Thi
- Schaller Research Group at Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
| | - Yuchen Xia
- State Key Laboratory of Virology, School of Basic Medical Sciences, Wuhan University, Wuhan, China
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Ramirez S, Bukh J. Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies. Antiviral Res 2018; 158:264-287. [PMID: 30059723 DOI: 10.1016/j.antiviral.2018.07.014] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 07/17/2018] [Accepted: 07/20/2018] [Indexed: 02/08/2023]
Abstract
In this review, we summarize the relevant scientific advances that led to the development of infectious cell culture systems for hepatitis C virus (HCV) with the corresponding challenges and successes. We also provide an overview of how these systems have contributed to the study of antiviral compounds and their relevance for the development of a much-needed vaccine against this major human pathogen. An efficient infectious system to study HCV in vitro, using human hepatoma derived cells, has only been available since 2005, and was limited to a single isolate, named JFH1, until 2012. Successive developments have been slow and cumbersome, as each available system has been the result of a systematic effort for discovering adaptive mutations conferring culture replication and propagation to patient consensus clones that are inherently non-viable in vitro. High genetic heterogeneity is a paramount characteristic of this virus, and as such, it should preferably be reflected in basic, translational, and clinical studies. The limited number of efficient viral culture systems, in the context of the vast genetic diversity of HCV, continues to represent a major hindrance for the study of this virus, posing a significant barrier towards studies of antivirals (particularly of resistance) and for advancing vaccine development. Intensive research efforts, driven by isolate-specific culture adaptation, have only led to efficient full-length infectious culture systems for a few strains of HCV genotypes 1, 2, 3, and 6. Hence research aimed at identifying novel strategies that will permit universal culture of HCV will be needed to further our understanding of this unique virus causing 400 thousand deaths annually.
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Affiliation(s)
- Santseharay Ramirez
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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Maryam M, Idrees M. Study of promoter hypomethylation profiles ofRASoncogenes in hepatocellular carcinoma derived from hepatitis C virus genotype 3a in Pakistani population. J Med Virol 2018; 90:1516-1523. [DOI: 10.1002/jmv.25221] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 05/03/2018] [Indexed: 02/06/2023]
Affiliation(s)
- Maria Maryam
- National Centre of Excellence in Molecular Biology; University of the Punjab; Lahore Pakistan
| | - Muhammad Idrees
- National Centre of Excellence in Molecular Biology; University of the Punjab; Lahore Pakistan
- Vice Chancellor Office, Hazara University; Mansehra Pakistan
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16
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Nelson B, Adams J, Kuglstatter A, Li Z, Harris SF, Liu Y, Bohini S, Ma H, Klumpp K, Gao J, Sidhu SS. Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies. J Mol Biol 2018; 430:2139-2152. [PMID: 29778602 DOI: 10.1016/j.jmb.2018.05.018] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2018] [Revised: 05/08/2018] [Accepted: 05/10/2018] [Indexed: 12/12/2022]
Abstract
Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.
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Affiliation(s)
- Bryce Nelson
- Banting and Best Department of Medical Research and Department of Medical Genetics, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada
| | - Jarrett Adams
- Banting and Best Department of Medical Research and Department of Medical Genetics, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada
| | | | - Zhijian Li
- Banting and Best Department of Medical Research and Department of Medical Genetics, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada
| | | | - Yang Liu
- Hoffmann-La Roche Inc., Palo Alto, 94304, CA, USA
| | | | - Han Ma
- Hoffmann-La Roche Inc., Palo Alto, 94304, CA, USA
| | - Klaus Klumpp
- Hoffmann-La Roche Inc., Palo Alto, 94304, CA, USA
| | - Junjun Gao
- Hoffmann-La Roche Inc., Palo Alto, 94304, CA, USA.
| | - Sachdev S Sidhu
- Banting and Best Department of Medical Research and Department of Medical Genetics, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
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17
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Guo X, Wang S, Qiu ZG, Dou YL, Liu WL, Yang D, Shen ZQ, Chen ZL, Wang JF, Zhang B, Wang XW, Guo XF, Zhang XL, Jin M, Li JW. Efficient replication of blood-borne hepatitis C virus in human fetal liver stem cells. Hepatology 2017; 66:1045-1057. [PMID: 28407288 DOI: 10.1002/hep.29211] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2017] [Accepted: 04/05/2017] [Indexed: 02/06/2023]
Abstract
UNLABELLED The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).
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Affiliation(s)
- Xuan Guo
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Shu Wang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Zhi-Gang Qiu
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Ya-Ling Dou
- Peking Union Medical College Hospital, Chinese Medical Academy, Beijing, China
| | - Wei-Li Liu
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Dong Yang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Zhi-Qiang Shen
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Zhao-Li Chen
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Jing-Feng Wang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Bin Zhang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Xin-Wei Wang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Xiang-Fei Guo
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Xue-Lian Zhang
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Min Jin
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
| | - Jun-Wen Li
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin, China
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18
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Shahid I, AlMalki WH, AlRabia MW, Mukhtar MH, Almalki SSR, Alkahtani SA, Ashgar SS, Faidah HS, Hafeez MH. In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype. ASIAN PAC J TROP MED 2017; 10:701-709. [PMID: 28870347 DOI: 10.1016/j.apjtm.2017.07.011] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Revised: 05/25/2017] [Accepted: 06/20/2017] [Indexed: 11/17/2022] Open
Abstract
OBJECTIVE To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. METHODS Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. RESULTS RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05). CONCLUSIONS siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.
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Affiliation(s)
- Imran Shahid
- Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, P.O. Box 13578, Makkah, Saudi Arabia.
| | - Waleed Hassan AlMalki
- Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, P.O. Box 13578, Makkah, Saudi Arabia
| | - Mohammed Wanees AlRabia
- Department of Medical Microbiology, College of Medicine, King Abdul Aziz University, Jeddah, Saudi Arabia
| | - Mohammed Hasan Mukhtar
- Department of Biochemistry, Faculty of Medicine, Umm Al Qura University, Makkah, Saudi Arabia
| | - Shaia Saleh R Almalki
- Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Albaha, Saudi Arabia
| | | | - Sami S Ashgar
- Department of Microbiology, College of Medicine, Umm Al Qura University, P.O. Box. 13765, Makkah, Saudi Arabia
| | - Hani S Faidah
- Department of Microbiology, College of Medicine, Umm Al Qura University, P.O. Box. 13765, Makkah, Saudi Arabia
| | - Muhammad Hassan Hafeez
- Department of Gastroenterology and Hepatology, Fatima Memorial College of Medicine and Dentistry, Shadman, Lahore 54000, Pakistan
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19
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Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition. Proc Natl Acad Sci U S A 2016; 113:7620-5. [PMID: 27298373 DOI: 10.1073/pnas.1602701113] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.
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20
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Rajalakshmy AR, Malathi J, Madhavan HN. Serum-derived hepatitis C virus 1a infection of human astrocyte cell line SVG. J Viral Hepat 2016; 23:211-6. [PMID: 26469941 DOI: 10.1111/jvh.12480] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Accepted: 09/04/2015] [Indexed: 01/26/2023]
Abstract
Neuroinvasion of hepatitis C virus (HCV) is evidenced by recent clinical studies. In this study, serum-derived HCV infection of astrocytes was analysed. Astrocytes were infected with HCV-positive serum, and viral replication was assessed on different days postinfection. RT-PCR was positive for HCV-negative strand on 5th and 7th day postinfection in the HCV-positive serum-infected astrocytes. Real-time RNA count in the cell culture supernatant was steadily increasing from day 3 to day 7. To reconfirm the viral replication, astrocytes were treated with an antiviral before the serum infection, and the antiviral treatment significantly reduced the viral RNA count. Further, the virus-infected cells stained positive for the presence of viral core protein. Electron microscopy revealed the presence of HCV-like particles in the astrocyte cell culture supernatant. In conclusion, serum-derived HCV replicates in human astrocyte cell line SVG.
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Affiliation(s)
- A R Rajalakshmy
- L & T Microbiology Research Centre, Vision Research Foundation, Chennai, 600006, India.,Centre for Nanotechnology and Advanced Biomaterials, SASTRA, Thanjavur, 612001, India
| | - J Malathi
- L & T Microbiology Research Centre, Vision Research Foundation, Chennai, 600006, India
| | - H N Madhavan
- L & T Microbiology Research Centre, Vision Research Foundation, Chennai, 600006, India
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21
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Liu X, Chen N, Lin S, Liu M. Synthesized peptide 710-725 from HCV subtype 1a E2 glycoprotein blocks HCV infection through competitive binding of CD81. Int J Mol Med 2016; 37:836-42. [PMID: 26796693 DOI: 10.3892/ijmm.2016.2459] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Accepted: 01/12/2016] [Indexed: 11/05/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a significant public health problem worldwide. However, there is still a lack of effective therapeutic drugs which could be used for the interruption of the disease. In the present study, for the first time, we reported that a synthesized peptide, which was synthesized by solid phase peptide synthesis and derived from the amino acids 710 to 725 of the HCV E2, functioned as an inhibitor of HCV infection. Using an MTT assay, we found that the E2 (710-725) peptide exerted no specific cytotoxicity on Huh7.5 cells and primary human hepatocytes (pHH). Interestingly, E2 (710-725) peptide blocked the entry of cell culture‑derived HCV (HCVcc) into hepatocytes. Moreover, it suppressed HCV RNA replication and HCV-specific protein NS3 and NS5B expression, as shown by western blot analysis. Moreover, E2 (710-725) markedly attenuated the inhibitory effect of HCVcc on hepatocyte viability. Additionally, a co-immunino-precipitation assay demonstrated that E2 (710-725) abrogated the interaction between CD81 and HCV E2 envelope protein through competitive binding of CD81. Overall, our results revealed that the synthesized peptide E2 (710-725) blocked CD81‑mediated HCV entry and possessed the potential to treat HCV infection. Thus, the present study provided novel insights into the development of new drugs for preventing HCV infection.
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Affiliation(s)
- Xiaojing Liu
- Department of Infectious Diseases, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Na Chen
- Department of Infectious Diseases, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Shumei Lin
- Department of Infectious Diseases, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Min Liu
- Department of Infectious Diseases, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
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22
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Qian XJ, Zhu YZ, Zhao P, Qi ZT. Entry inhibitors: New advances in HCV treatment. Emerg Microbes Infect 2016; 5:e3. [PMID: 26733381 PMCID: PMC4735057 DOI: 10.1038/emi.2016.3] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Revised: 10/28/2015] [Accepted: 11/02/2015] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus (HCV) infection affects approximately 3% of the world's population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry.
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Affiliation(s)
- Xi-Jing Qian
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Yong-Zhe Zhu
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Ping Zhao
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
| | - Zhong-Tian Qi
- Shanghai Key Laboratory of Medical Biodefense, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
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23
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Park GB, Kim D, Park SJ, Lee HK, Kim JH, Kim YS, Park SG, Choi IH, Yoon SH, Lee YJ, Paeng S, Hur DY. Pre-stimulation of CD81 expression by resting B cells increases proliferation following EBV infection, but the overexpression of CD81 induces the apoptosis of EBV-transformed B cells. Int J Mol Med 2015; 36:1464-78. [PMID: 26498453 PMCID: PMC4678167 DOI: 10.3892/ijmm.2015.2372] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2015] [Accepted: 09/23/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.
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Affiliation(s)
- Ga Bin Park
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Daejin Kim
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Sung Jae Park
- Department of Internal Medicine, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Hyun-Kyung Lee
- Department of Internal Medicine, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Ji Hyun Kim
- Department of Internal Medicine, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Yeong Seok Kim
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Sae-Gwang Park
- Department of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - In-Hak Choi
- Department of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Sung Ho Yoon
- Department of Plastic Surgery, Inje University Haeundae Paik Hospital, Busan 614-735, Republic of Korea
| | - Youn Jae Lee
- Department of Internal Medicine, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Sunghwa Paeng
- Department of Neurosurgery, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Dae Young Hur
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
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24
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Aoudjehane L, Bisch G, Scatton O, Granier C, Gaston J, Housset C, Roingeard P, Cosset FL, Perdigao F, Balladur P, Wakita T, Calmus Y, Conti F. Infection of Human Liver Myofibroblasts by Hepatitis C Virus: A Direct Mechanism of Liver Fibrosis in Hepatitis C. PLoS One 2015. [PMID: 26214688 PMCID: PMC4516308 DOI: 10.1371/journal.pone.0134141] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Background Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities. Methods We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture–derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction. Results We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients. Conclusion These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.
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Affiliation(s)
- Lynda Aoudjehane
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
- Human HepCell, Hôpital Saint-Antoine, F-75012 Paris, France
- * E-mail:
| | - Grégoire Bisch
- Human HepCell, Hôpital Saint-Antoine, F-75012 Paris, France
| | - Olivier Scatton
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
- AP-HP, Hôpital Pitié-Salpêtrière, Unité de Transplantation Hépatique, F-75013, Paris, France
| | - Christelle Granier
- CIRI–International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France
- CNRS, UMR5308, Lyon, France
- LabEx Ecofect, Université de Lyon, Lyon, France
| | - Jesintha Gaston
- Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), INSERM U1016, Paris, France
| | - Chantal Housset
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
| | | | - François-Loïc Cosset
- CIRI–International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France
- CNRS, UMR5308, Lyon, France
- LabEx Ecofect, Université de Lyon, Lyon, France
| | - Fabiano Perdigao
- AP-HP, Hôpital Pitié-Salpêtrière, Unité de Transplantation Hépatique, F-75013, Paris, France
| | - Pierre Balladur
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
- AP-HP, Hôpital Saint Antoine, Département de la chirurgie digestive, F-75012, Paris, France
| | - Takaji Wakita
- National Institute of Infectious Diseases, Department of Virology II, Tokyo, Japan
| | - Yvon Calmus
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
- AP-HP, Hôpital Pitié-Salpêtrière, Unité de Transplantation Hépatique, F-75013, Paris, France
| | - Filomena Conti
- Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint-Antoine, F-75005, Paris, France
- INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France
- AP-HP, Hôpital Pitié-Salpêtrière, Unité de Transplantation Hépatique, F-75013, Paris, France
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Abstract
The past decade has witnessed steady and rapid progress in HCV research, which has led to the recent breakthrough in therapies against this significant human pathogen. Yet a deeper understanding of the life cycle of the virus is required to develop more affordable treatments and to advance vaccine design. HCV entry presents both a challenge for scientific research and an opportunity for alternative intervention approaches, owning to its highly complex nature and the myriad of players involved. More than half a dozen cellular proteins are implicated in HCV entry; and a more definitive picture regarding the structures of the glycoproteins is emerging. A role of apolipoproteins in HCV entry has also been established. Still, major questions remain, and the answers to these, which we summarize in this review, will hopefully close the gaps in our understanding and complete the puzzle that is HCV entry.
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Affiliation(s)
- Sarah C Ogden
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, USA
| | - Hengli Tang
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, USA ; Institute of Health Sciences, Anhui University, Hefei, 230601, PR China
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Ji C, Liu Y, Pamulapati C, Bohini S, Fertig G, Schraeml M, Rubas W, Brandt M, Ries S, Ma H, Klumpp K. Prevention of hepatitis C virus infection and spread in human liver chimeric mice by an anti-CD81 monoclonal antibody. Hepatology 2015; 61:1136-44. [PMID: 25417967 DOI: 10.1002/hep.27603] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2014] [Accepted: 11/13/2014] [Indexed: 01/01/2023]
Abstract
UNLABELLED CD81 is a required receptor for hepatitis C virus (HCV) infection of human hepatocytes in vitro. We generated several high-affinity anti-human CD81 monoclonal antibodies (mAbs) that demonstrated potent, specific, and cross-genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively. All vehicle control mice established HCV infection, reaching steady-state levels of serum HCV RNA by day 21. Pretreatment of mice with K04 prevented HCV infection in all mice (n = 5). Treatment of mice with mAb K04 every 3 days for 21 days, starting at 6 hours postinfection, resulted in effective inhibition of virus spread. In 3 mice that were sacrificed on day 24, serum HCV levels remained detectable, below the limit of quantification (LOQ), indicating that infection was established, but virus spread was blocked, by the anti-CD81 mAb. In 5 additional mice that were followed for a longer time, virus remained detectable, below LOQ, until days 24 and 30 in 4 of 5 mice. In the fifth mouse, viral load was quantifiable, but reduced to 64-fold below the mean viral load in vehicle control at day 24. In addition, 2 of 5 mice cleared the infection by day 30 and 1 mouse had undetectable virus load from day 6 onward. CONCLUSION These results demonstrate that CD81 is required for HCV infection and virus spread in vivo, and that anti-CD81 antibodies such as K04 may have potential as broad-spectrum antiviral agents for prevention and treatment of HCV infection.
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Shahid I, AlMalki WH, R. Almalki SS, AlTurkestany IM, AlGhamdi HA, AlMenshawi SA. Inhibition of Hepatitis C Virus Genotype 1a Non-Structural Proteins by Small Interference RNA in Human Hepatoma Cell Lines. ACTA ACUST UNITED AC 2015. [DOI: 10.4236/pp.2015.611053] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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The mechanism of HCV entry into host cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2014; 129:63-107. [PMID: 25595801 DOI: 10.1016/bs.pmbts.2014.10.003] [Citation(s) in RCA: 83] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus classified within the Flaviviridae family and is a major cause of liver disease worldwide. HCV life cycle and propagation are tightly linked to several aspects of lipid metabolism. HCV propagation depends on and also shapes several aspects of lipid metabolism such as cholesterol uptake and efflux through different lipoprotein receptors during its entry into cells, lipid metabolism modulating HCV genome replication, lipid droplets acting as a platform for recruitment of viral components, and very low density lipoprotein assembly pathway resulting in incorporation of neutral lipids and apolipoproteins into viral particles. During the first steps of infection, HCV enters hepatocytes through a multistep and slow process. The initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I, a major receptor of high-density lipoprotein, the CD81 tetraspanin, and the tight junction proteins Claudin-1 and Occludin. This tight concert of receptor interactions ultimately leads to uptake and cellular internalization of HCV through a process of clathrin-dependent endocytosis. Over the years, the identification of the HCV entry receptors and cofactors has led to a better understanding of HCV entry and of the narrow tropism of HCV for the liver. Yet, the role of the two HCV envelope glycoproteins, E1 and E2, remains ill-defined, particularly concerning their involvement in the membrane fusion process. Here, we review the current knowledge and advances addressing the mechanism of HCV cell entry within hepatocytes and we highlight the challenges that remain to be addressed.
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Vercauteren K, Mesalam AA, Leroux-Roels G, Meuleman P. Impact of lipids and lipoproteins on hepatitis C virus infection and virus neutralization. World J Gastroenterol 2014; 20:15975-91. [PMID: 25473151 PMCID: PMC4239485 DOI: 10.3748/wjg.v20.i43.15975] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2014] [Revised: 07/09/2014] [Accepted: 09/05/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infections represent a major global health problem. End-stage liver disease caused by chronic HCV infection is a major indication for liver transplantation. However, after transplantation the engrafted liver inevitably becomes infected by the circulating virus. Direct acting antivirals are not yet approved for use in liver transplant patients, and limited efficacy and severe side effects hamper the use of pegylated interferon combined with ribavirin in a post-transplant setting. Therefore, alternative therapeutic options need to be explored. Viral entry represents an attractive target for such therapeutic intervention. Understanding the mechanisms of viral entry is essential to define the viral and cellular factors involved. The HCV life cycle is dependent of and associated with lipoprotein physiology and the presence of lipoproteins has been correlated with altered antiviral efficacy of entry inhibitors. In this review, we summarise the current knowledge on how lipoprotein physiology influences the HCV life cycle. We focus especially on the influence of lipoproteins on antibodies that target HCV envelope proteins or antibodies that target the cellular receptors of the virus. This information can be particularly relevant for the prevention of HCV re-infection after liver transplantation.
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Gondeau C, Briolotti P, Razafy F, Duret C, Rubbo PA, Helle F, Rème T, Ripault MP, Ducos J, Fabre JM, Ramos J, Pécheur EI, Larrey D, Maurel P, Daujat-Chavanieu M. In vitro infection of primary human hepatocytes by HCV-positive sera: insights on a highly relevant model. Gut 2014; 63:1490-500. [PMID: 24153249 DOI: 10.1136/gutjnl-2013-304623] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
OBJECTIVE Adult primary human hepatocytes (PHHs) support the complete infection cycle of natural HCV from patients' sera. The molecular details underlying sera infectivity towards these cells remain largely unknown. Therefore, we sought to gain a deeper comprehension of these features in the most physiologically relevant culture system. DESIGN Using kinetic experiments, we defined the optimal conditions to infect PHH and explored the link between cell organisation and permissivity. Based on their infectivity, about 120 sera were classified in three groups. Concentration of 52 analytes was measured in 79 selected sera using multiplexed immunobead-based analyte profiling. RESULTS PHH permissivity towards HCV infection negatively correlated with cell polarisation and formation of functional bile canaliculi. PHH supported HCV replication for at least 2 weeks with de novo virus production. Depending on their reactivity, sera could be classified in three groups of high, intermediate or low infectivity toward PHH. Infectivity could not be predicted based on the donors' clinical characteristics, viral load or genotype. Interestingly, highly infectious sera displayed a specific cytokine profile with low levels of most of the 52 tested analytes. Among them, 24 cytokines/growth factors could impact hepatocyte biology and infection efficiency. CONCLUSIONS We identified critical factors leading to efficient PHH infection by HCV sera in vitro. Overall, we showed that this cellular model provides a useful tool for studying the mechanism of HCV infection in its natural host cell, selecting highly infectious isolates, and determining the potency of drugs towards various HCV strains.
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Affiliation(s)
- Claire Gondeau
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Philippe Briolotti
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Francia Razafy
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Cédric Duret
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Pierre-Alain Rubbo
- Université Montpellier 1, Montpellier, France INSERM U1058, Montpellier, France
| | - François Helle
- EA4294, Laboratoire de Virologie, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - Thierry Rème
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Marie-Pierre Ripault
- Department of Hepato-gastroenterology A, Hospital Saint Eloi, CHU Montpellier, Montpellier, France
| | - Jacques Ducos
- INSERM U1058, Montpellier, France Département de Bactériologie-Virologie, CHU de Montpellier, Montpellier, France
| | - Jean-Michel Fabre
- Department of Digestive Surgery, Hospital Saint Eloi, CHU Montpellier, France
| | - Jeanne Ramos
- Pathological anatomy department, CHU Gui de Chauliac, Montpellier, France
| | - Eve-Isabelle Pécheur
- UMR INSERM 1052/CNRS 5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Lyon, France
| | - Dominique Larrey
- INSERM U1040, Montpellier, France Department of Hepato-gastroenterology A, Hospital Saint Eloi, CHU Montpellier, Montpellier, France
| | - Patrick Maurel
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France
| | - Martine Daujat-Chavanieu
- INSERM U1040, Montpellier, France Université Montpellier 1, Montpellier, France CHU Saint Eloi, Institute of Research in Biotherapy, Montpellier, France
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32
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Abstract
Despite the tremendous hurdles presented by the complexity of the liver's structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near- and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.
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Affiliation(s)
- Sangeeta N Bhatia
- Institute for Medical Engineering & Science at MIT, Department of Electrical Engineering and Computer Science, David H. Koch Institute at MIT, and the Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Division of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
| | - Gregory H Underhill
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Kenneth S Zaret
- Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Ira J Fox
- Department of Surgery, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, and McGowan Institute for Regenerative Medicine, Pittsburgh, PA 15224, USA
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Unexpected structural features of the hepatitis C virus envelope protein 2 ectodomain. J Virol 2014; 88:10280-8. [PMID: 24991010 DOI: 10.1128/jvi.00874-14] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV), a member of the family Flaviviridae, is a leading cause of chronic liver disease and cancer. Recent advances in HCV therapeutics have resulted in improved cure rates, but an HCV vaccine is not available and is urgently needed to control the global pandemic. Vaccine development has been hampered by the lack of high-resolution structural information for the two HCV envelope glycoproteins, E1 and E2. Recently, Kong and coworkers (Science 342:1090-1094, 2013, doi:10.1126/science.1243876) and Khan and coworkers (Nature 509[7500]:381-384, 2014, doi:10.1038/nature13117) independently determined the structure of the HCV E2 ectodomain core with some unexpected and informative results. The HCV E2 ectodomain core features a globular architecture with antiparallel β-sheets forming a central β sandwich. The residues comprising the epitopes of several neutralizing and nonneutralizing human monoclonal antibodies were also determined, which is an essential step toward obtaining a fine map of the human humoral response to HCV. Also clarified were the regions of E2 that directly bind CD81, an important HCV cellular receptor. While it has been widely assumed that HCV E2 is a class II viral fusion protein (VFP), the newly determined structure suggests that the HCV E2 ectodomain shares structural and functional similarities only with domain III of class II VFPs. The new structural determinations suggest that the HCV glycoproteins use a different mechanism than that used by class II fusion proteins for cell fusion.
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Yang D, Meng X, Xue B, Liu N, Wang X, Zhu H. MiR-942 mediates hepatitis C virus-induced apoptosis via regulation of ISG12a. PLoS One 2014; 9:e94501. [PMID: 24727952 PMCID: PMC3984147 DOI: 10.1371/journal.pone.0094501] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2014] [Accepted: 03/17/2014] [Indexed: 12/17/2022] Open
Abstract
The interaction between hepatitis C virus (HCV) and human hepatic innate antiviral responses is unclear. The aim of this study was to examine how human hepatocytes respond to HCV infection. An infectious HCV isolate, JFH1, was used to infect a newly established human hepatoma cell line HLCZ01. Viral RNA or NS5A protein was examined by real-time PCR or immunofluorescence respectively. The mechanisms of HCV-induced IFN-β and apoptosis were explored. Our data showed that HLCZ01 cells supported the entire HCV lifecycle and IFN-β and interferon-stimulated genes (ISGs) were induced in HCV-infected cells. Viral infection caused apoptosis of HLCZ01 cells. Silencing of RIG-I, IRF3 or TRAIL inhibited ISG12a expression and blocked apoptosis of viral-infected HLCZ01 cells. Knockdown ISG12a blocked apoptosis of viral-infected cells. MiR-942 is a candidate negative regulator of ISG12a predicted by bioinformatics search. Moreover, HCV infection decreased miR-942 expression in HLCZ01 cells and miR-942 was inversely correlated with ISG12a expression in both HCV-infected cells and liver biopsies. MiR-942 forced expression in HLCZ01 cells decreased ISG12a expression and subsequently suppressed apoptosis triggered by HCV infection. Conversely, silencing of miR-942 expression by anti-miR-942 increased ISG12a expression and enhanced apoptosis in HCV-infected cells. Induction of Noxa by HCV infection contributed to ISG12a-mediated apoptosis. All the data indicated that innate host response is intact in HCV-infected hepatocytes. MiR-942 regulates HCV-induced apoptosis of human hepatocytes by targeting ISG12a. Our study provides a novel mechanism by which human hepatocytes respond to HCV infection.
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Affiliation(s)
- Darong Yang
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Xianghe Meng
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Binbin Xue
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Nianli Liu
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Xiaohong Wang
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Haizhen Zhu
- Department of Molecular Medicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
- Research Center of Cancer Prevention & Treatment, Translational Medicine Research Center of Liver Cancer, Hunan Provincial Tumor Hospital (Affiliated Tumor Hospital of Xiangya Medical School of Central South University), Changsha, China
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Rafique S, Idrees M, Ali A, Sahibzada KI, Iqbal M. Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection. Mol Biol Rep 2014; 41:3813-9. [PMID: 24549717 DOI: 10.1007/s11033-014-3247-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2013] [Accepted: 02/07/2014] [Indexed: 11/24/2022]
Abstract
Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1-E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag-Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.
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Affiliation(s)
- Shazia Rafique
- Centre of Applied Molecular Biology, Ministry of Science & Technology Govt. of Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore, Pakistan,
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Fénéant L, Levy S, Cocquerel L. CD81 and hepatitis C virus (HCV) infection. Viruses 2014; 6:535-72. [PMID: 24509809 PMCID: PMC3939471 DOI: 10.3390/v6020535] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Revised: 01/29/2014] [Accepted: 02/02/2014] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C Virus (HCV) infection is a global public health problem affecting over 160 million individuals worldwide. Its symptoms include chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped RNA virus mainly targeting liver cells and for which the initiation of infection occurs through a complex multistep process involving a series of specific cellular entry factors. This process is likely mediated through the formation of a tightly orchestrated complex of HCV entry factors at the plasma membrane. Among HCV entry factors, the tetraspanin CD81 is one of the best characterized and it is undoubtedly a key player in the HCV lifecycle. In this review, we detail the current knowledge on the involvement of CD81 in the HCV lifecycle, as well as in the immune response to HCV infection.
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Affiliation(s)
- Lucie Fénéant
- Center for Infection and Immunity of Lille, CNRS-UMR8204, Inserm-U1019, Institut Pasteur de Lille, Université Lille Nord de France, Institut de Biologie de Lille, 1 rue du Pr Calmette, CS50447, 59021 Lille Cedex, France.
| | - Shoshana Levy
- Department of Medicine, Division of Oncology, CCSR, Stanford University Medical Center, Stanford, CA 94305, USA.
| | - Laurence Cocquerel
- Center for Infection and Immunity of Lille, CNRS-UMR8204, Inserm-U1019, Institut Pasteur de Lille, Université Lille Nord de France, Institut de Biologie de Lille, 1 rue du Pr Calmette, CS50447, 59021 Lille Cedex, France.
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Gerbal-Chaloin S, Funakoshi N, Caillaud A, Gondeau C, Champon B, Si-Tayeb K. Human induced pluripotent stem cells in hepatology: beyond the proof of concept. THE AMERICAN JOURNAL OF PATHOLOGY 2013; 184:332-47. [PMID: 24269594 DOI: 10.1016/j.ajpath.2013.09.026] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/12/2013] [Revised: 09/20/2013] [Accepted: 09/26/2013] [Indexed: 02/08/2023]
Abstract
The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.
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Affiliation(s)
- Sabine Gerbal-Chaloin
- INSERM, U1087, Montpellier, France; UMR 1040, Université Montpellier 1, Montpellier, France
| | - Natalie Funakoshi
- INSERM, U1087, Montpellier, France; UMR 1040, Université Montpellier 1, Montpellier, France; Hepato-Gastroenterology Service B, Saint Eloi Hospital, CHU Montpellier, Montpellier, France
| | - Amandine Caillaud
- INSERM, UMR 1087, the Institute of the Thorax, Nantes, France; CNRS, UMR 6291, Nantes, France; School of Medicine, University of Nantes, Nantes, France
| | - Claire Gondeau
- INSERM, U1087, Montpellier, France; UMR 1040, Université Montpellier 1, Montpellier, France
| | - Benoite Champon
- INSERM, UMR 1087, the Institute of the Thorax, Nantes, France; CNRS, UMR 6291, Nantes, France; School of Medicine, University of Nantes, Nantes, France
| | - Karim Si-Tayeb
- INSERM, UMR 1087, the Institute of the Thorax, Nantes, France; CNRS, UMR 6291, Nantes, France; School of Medicine, University of Nantes, Nantes, France.
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Padovani JL, Corvino SM, Drexler JF, Silva GF, Pardini MIDMC, Grotto RMT. In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus. Rev Soc Bras Med Trop 2013; 46:154-5. [PMID: 23559343 DOI: 10.1590/0037-8682-1627-2013] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2012] [Accepted: 01/16/2013] [Indexed: 12/29/2022] Open
Abstract
INTRODUCTION Despite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus. METHODS Platelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR. RESULTS After incubation in the presence of virus, all samples of platelets showed HCV RNA. CONCLUSIONS The results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.
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Affiliation(s)
- Juliana Lara Padovani
- Laboratório de Biologia Molecular do Hemocentro de Botucatu, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista -UNESP, Botucatu, SP, Brasil
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Blackard JT, Kong L, Huber AK, Tomer Y. Hepatitis C virus infection of a thyroid cell line: implications for pathogenesis of hepatitis C virus and thyroiditis. Thyroid 2013; 23:863-70. [PMID: 23259732 PMCID: PMC3704108 DOI: 10.1089/thy.2012.0507] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Autoimmune and non-autoimmune thyroiditis frequently occur in persons with hepatitis C virus (HCV) infection. Treatment with interferon alpha (IFNα) is also associated with significant risk for the development of thyroiditis. To explore HCV-thyroid interactions at a cellular level, we evaluated whether a human thyroid cell line (ML1) could be infected productively with HCV in vitro. METHODS AND RESULTS ML1 cells showed robust surface expression of the major HCV receptor CD81. Using a highly sensitive, strand-specific reverse transcription polymerase chain reaction assay, positive-sense and negative-sense HCV RNA were detected in ML1 cell lysates at days 3, 7, and 14 postinfection with HCV. HCV core protein was expressed at high levels in ML1 supernatants at days 1, 3, 5, 7, and 14 postinfection. The nonstructural protein NS5A was also detected in ML1 cell lysates by Western blotting. HCV entry into ML1 cells was shown to be dependent on the HCV entry factors CD81 and SR-B1/CLA1, while IFNα inhibited HCV replication in ML1 cells in a dose-dependent manner. Supernatants from HCV-infected ML1 cells were able to infect fresh ML1 cells productively, suggesting that infectious virions could be transferred from infected to naïve thyroid cells in vivo. Additionally, HCV infection of ML1 cells led to increased expression of the pro-inflammatory cytokine IL-8. CONCLUSIONS For the first time, we have demonstrated that HCV can infect human thyroid cells in vitro. These findings strongly suggest that HCV infection of thyrocytes may play a role in the association between chronic HCV infection and thyroid autoimmunity. Furthermore, the thyroid may serve as an extrahepatic reservoir for HCV viral replication, thus contributing to the persistence of viral infection and to the development of thyroid autoimmunity.
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Affiliation(s)
- Jason T Blackard
- Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
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Shahid I, Gull S, Ijaz B, Ahmad W, Ansar M, Asad S, Kausar H, Sarwar MT, Khan MK, Hassan S. Stable Huh-7 cell lines expressing non-structural proteins of genotype 1a of hepatitis C virus. J Virol Methods 2013; 189:65-9. [PMID: 23352716 DOI: 10.1016/j.jviromet.2013.01.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2012] [Revised: 12/25/2012] [Accepted: 01/14/2013] [Indexed: 01/03/2023]
Abstract
Hepatitis C virus (HCV) infection has infected approximately 3% of the world population. HCV genotype 1a is distributed throughout the world, and along with genotype 1b, is relatively resistant to current standards of therapy compared to other HCV genotypes. The present study was designed to produce stable Huh-7 cell lines expressing non-structural proteins of HCV genotype la, representing an in vitro system to facilitate the development of new antiviral drugs against chronic HCV infection. The non-structural genes of HCV genotype 1a were amplified and cloned in a mammalian expression vector pCR 3.1/FIagTag. Huh-7 cells were transfected with one of two expression plasmids, the first containing the NS2, NS3, and NS4a cassette, and second containing the NS5a and NS5b genes. Stable cell lines were produced under the selection of gentamycin (G418). mRNA and protein expression analysis was performed by RT-PCR and Western blotting. The RT-PCR and Western blot results confirmed the stable expression of each of the gene products. Stable Huh-7 cell lines with HCV la non-structural proteins may be helpful for evaluating the role of HCV proteins in molecular pathogenesis, and could facilitate the development of new therapeutic drugs.
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Affiliation(s)
- Imran Shahid
- Applied and Functional Genomics Laboratory, Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, 53700 Lahore, Pakistan.
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Flego M, Ascione A, Cianfriglia M, Vella S. Clinical development of monoclonal antibody-based drugs in HIV and HCV diseases. BMC Med 2013; 11:4. [PMID: 23289632 PMCID: PMC3565905 DOI: 10.1186/1741-7015-11-4] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2012] [Accepted: 01/04/2013] [Indexed: 12/21/2022] Open
Abstract
Today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. Compared to conventional antiviral drugs, monoclonal antibodies (mAbs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. Despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. The current high cost of mAbs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mAbs' clinical advantages. These are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses.This review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mAb-based drugs in clinical trials for HIV and HCV diseases. For each clinical trial the available data are reported and the emerging conceptual problems of the employed mAbs are highlighted.This overview helps to give a clear picture of the efficacy and challenges of the mAbs in the field of these two infectious diseases which have such a global impact.
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Affiliation(s)
- Michela Flego
- Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
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Abstract
Due to the obligatory intracellular lifestyle of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus-host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models continues to be a challenging task. The first efforts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry, and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple complementary cell-based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact, drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this groundwork and further refining our cellular models to better mimic the architecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may explain the variable natural course of hepatitis C.
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Affiliation(s)
- Eike Steinmann
- Helmholtz Centre for Infection Research, Hannover, Germany
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Samreen B, Khaliq S, Ashfaq UA, Khan M, Afzal N, Shahzad MA, Riaz S, Jahan S. Hepatitis C virus entry: role of host and viral factors. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2012; 12:1699-1709. [PMID: 22878095 DOI: 10.1016/j.meegid.2012.07.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2012] [Revised: 07/13/2012] [Accepted: 07/16/2012] [Indexed: 12/24/2022]
Abstract
Hepatitis C virus (HCV) has been considered to be a significant risk factor in developing liver associated diseases including hepatocellular carcinoma all over the world. HCV is an enveloped positive strand virus comprising a complex between genomic RNA and viral envelope glycoproteins (E1 and E2), which are anchored within host derived double-layered lipid membrane surrounding the nucleocapsid composed of several copies of core protein. HCV cell entry is the first step in infection and viral replication into host cells mainly hepatocytes. HCV cell entry is a complex process involving both the viral (envelope glycoproteins E1/E2) and host factors (cellular receptors and associated factors i.e. CD81, SR-BI, LDL-R, CLDN1, Occludin, DC-SIGN, L-SIGN and Glycosaminoglycans). Besides these the expression of certain other conditions such as polarization and EWI-2 expression inhibits the viral cell entry. Exploring the mechanism of HCV entry will help to better understand the viral life cycle and possible therapeutic targets against HCV infection including viral and host factors involved in this process. New strategies such as RNAi represents a new option for targeting the host or viral factors for prevention and therapeutic against HCV infection. In the current review we try to summarize the current knowledge about mechanism and interaction of cellular and viral factors involved in HCV cell entry and its implication as therapeutic target to inhibit HCV infection.
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Affiliation(s)
- Baila Samreen
- National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
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Roque-Cuéllar MC, Sánchez B, García-Lozano JR, Garrido-Serrano A, Sayago M, Praena-Fernández JM, Núñez-Roldán A, Aguilar-Reina J. Expression of CD81, SR-BI and LDLR in lymphocytes and monocytes from patients with classic and occult hepatitis C virus infection. J Med Virol 2012; 84:1727-36. [DOI: 10.1002/jmv.23345] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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Lagaye S, Shen H, Saunier B, Nascimbeni M, Gaston J, Bourdoncle P, Hannoun L, Massault PP, Vallet-Pichard A, Mallet V, Pol S. Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection. Hepatology 2012; 56:861-72. [PMID: 22454196 DOI: 10.1002/hep.25738] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2011] [Accepted: 03/13/2012] [Indexed: 12/16/2022]
Abstract
UNLABELLED The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2-specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. CONCLUSION This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs.
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Affiliation(s)
- Sylvie Lagaye
- Equipe Cycle cellulaire, Régénération et Hépatopathies, Institut Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Centre National de la Recherche Scientifique (CNRS) UMR8104, Université Paris Descartes (UMR S1016), Paris, France.
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Hussain S, Barretto N, Uprichard SL. New hepatitis C virus drug discovery strategies and model systems. Expert Opin Drug Discov 2012; 7:849-59. [PMID: 22861052 DOI: 10.1517/17460441.2012.711312] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
INTRODUCTION Hepatitis C virus (HCV) is a major cause of liver disease worldwide and the leading indication for liver transplantation in the United States. Current treatment options are expensive, not effective in all patients and are associated with serious side effects. Although preclinical, anti-HCV drug screening is still hampered by the lack of readily infectable small animal models, the development of cell culture HCV experimental model systems has driven a promising new wave of HCV antiviral drug discovery. AREAS COVERED This review contains a concise overview of current HCV treatment options and limitations with a subsequent in-depth focus on the available experimental models and novel strategies that have, and continue to enable, important advances in HCV drug development. EXPERT OPINION With a large cohort of chronically HCV-infected patients progressively developing liver disease that puts them at risk for hepatocellular carcinoma and hepatic decompensation, there is an urgent need to develop effective therapeutics that are well tolerated and effective in all patients and against all HCV genotypes. Significant advances in HCV experimental model development have expedited drug discovery; however, additional progress is needed. Importantly, the current trends and momentum in the field suggests that we will continue to overcome critical experimental challenges to reach this end goal.
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Affiliation(s)
- Snawar Hussain
- University of Illinois at Chicago, Department of Medicine, Chicago, IL 60612, USA
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Abstract
Owing to the tremendous effort from both academia and industry, drug development for hepatitis C virus (HCV) infection has been flourishing, with a range of pipeline compounds at various stages of development. Although combination of the recently launched serine protease inhibitors will further improve the response rate of current interferon-based therapy, some intrinsic limitations of these compounds and the tendency of resistance development by the virus, urge the development of alternative or additional therapeutic strategies. In this article we provide an overview of different host and viral factors which have emerged as new potential targets for therapeutic intervention using state-of-the-art technologies.
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Vercauteren K, Leroux-Roels G, Meuleman P. Blocking HCV entry as potential antiviral therapy. Future Virol 2012. [DOI: 10.2217/fvl.12.47] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Zhu YZ, Luo Y, Cao MM, Liu Y, Liu XQ, Wang W, Wu DG, Guan M, Xu QQ, Ren H, Zhao P, Qi ZT. Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry. Virology 2012; 429:112-23. [PMID: 22560863 DOI: 10.1016/j.virol.2012.03.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2012] [Accepted: 03/03/2012] [Indexed: 12/15/2022]
Abstract
CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.
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Affiliation(s)
- Yong-Zhe Zhu
- Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, China
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