|
1
|
Zhang F, Xu LD, Wu S, Wang B, Xu P, Huang YW. Deciphering the hepatitis E virus ORF1: Functional domains, protein processing, and patient-derived mutations. Virology 2025; 603:110350. [PMID: 39675187 DOI: 10.1016/j.virol.2024.110350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Revised: 11/25/2024] [Accepted: 12/05/2024] [Indexed: 12/17/2024]
Abstract
Hepatitis E virus (HEV) is a major cause of acute and chronic hepatitis in humans. The HEV open reading frames (ORF1) encodes a large non-structural protein essential for viral replication, which contains several functional domains, including helicase and RNA-dependent RNA polymerase. A confusing aspect is that, while RNA viruses typically encode large polyproteins that rely on their enzymatic activity for processing into functional units, the processing of the ORF1 protein and the mechanisms involved remain unclear. The ORF1 plays a pivotal role in the viral life cycle, thus mutations in this region, especially those occurring under environmental pressures such as during antiviral drug treatment, could significantly affect viral replication and survival. Here, we summarize the recent advances in the functional domains, processing, and mutations of ORF1. Gaining a deeper understanding of HEV biology, particularly focusing on ORF1, could facilitate the development of new strategies to control HEV infections.
Collapse
Affiliation(s)
- Fei Zhang
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China; ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311215, China; MOE Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China
| | - Ling-Dong Xu
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China; MOE Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Laboratory Animal Center, Zhejiang University, Hangzhou, 310058, China
| | - Shiying Wu
- MOE Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
| | - Bin Wang
- Guangdong Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, 510642, China
| | - Pinglong Xu
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China; ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311215, China; MOE Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, China.
| | - Yao-Wei Huang
- Guangdong Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, 510642, China.
| |
Collapse
|
|
2
|
Letafati A, Taghiabadi Z, Roushanzamir M, Memarpour B, Seyedi S, Farahani AV, Norouzi M, Karamian S, Zebardast A, Mehrabinia M, Ardekani OS, Fallah T, Khazry F, Daneshvar SF, Norouzi M. From discovery to treatment: tracing the path of hepatitis E virus. Virol J 2024; 21:194. [PMID: 39180020 PMCID: PMC11342613 DOI: 10.1186/s12985-024-02470-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2024] [Accepted: 08/14/2024] [Indexed: 08/26/2024] Open
Abstract
The hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. HEV is classified into eight genotypes, labeled HEV-1 through HEV-8. Genotypes 1 and 2 exclusively infect humans, while genotypes 3, 4, and 7 can infect both humans and animals. In contrast, genotypes 5, 6, and 8 are restricted to infecting animals. While most individuals with a strong immune system experience a self-limiting infection, those who are immunosuppressed may develop chronic hepatitis. Pregnant women are particularly vulnerable to severe illness and mortality due to HEV infection. In addition to liver-related complications, HEV can also cause extrahepatic manifestations, including neurological disorders. The immune response is vital in determining the outcome of HEV infection. Deficiencies in T cells, NK cells, and antibody responses are linked to poor prognosis. Interestingly, HEV itself contains microRNAs that regulate its replication and modify the host's antiviral response. Diagnosis of HEV infection involves the detection of HEV RNA and anti-HEV IgM/IgG antibodies. Supportive care is the mainstay of treatment for acute infection, while chronic HEV infection may be cleared with the use of ribavirin and pegylated interferon. Prevention remains the best approach against HEV, focusing on sanitation infrastructure improvements and vaccination, with one vaccine already licensed in China. This comprehensive review provides insights into the spread, genotypes, prevalence, and clinical effects of HEV. Furthermore, it emphasizes the need for further research and attention to HEV, particularly in cases of acute hepatitis, especially among solid-organ transplant recipients.
Collapse
Affiliation(s)
- Arash Letafati
- Department of Virology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran.
| | - Zahra Taghiabadi
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Mahshid Roushanzamir
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
- Department of Pharmacological and Biomolecular Science, University of Milan, Milan, Italy
| | - Bahar Memarpour
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
- Shahid Chamran University of Ahvaz, Ahvaz, Iran
| | - Saba Seyedi
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | | | - Masoomeh Norouzi
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Saeideh Karamian
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Arghavan Zebardast
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Marzieh Mehrabinia
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Omid Salahi Ardekani
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Tina Fallah
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Fatemeh Khazry
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Samin Fathi Daneshvar
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| | - Mehdi Norouzi
- Department of Virology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran
- Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
| |
Collapse
|
|
3
|
Orosz L, Sárvári KP, Dernovics Á, Rosztóczy A, Megyeri K. Pathogenesis and clinical features of severe hepatitis E virus infection. World J Virol 2024; 13:91580. [PMID: 38984076 PMCID: PMC11229844 DOI: 10.5501/wjv.v13.i2.91580] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2023] [Revised: 02/08/2024] [Accepted: 04/15/2024] [Indexed: 06/24/2024] Open
Abstract
The hepatitis E virus (HEV), a member of the Hepeviridae family, is a small, non-enveloped icosahedral virus divided into eight distinct genotypes (HEV-1 to HEV-8). Only genotypes 1 to 4 are known to cause diseases in humans. Genotypes 1 and 2 commonly spread via fecal-oral transmission, often through the consumption of contaminated water. Genotypes 3 and 4 are known to infect pigs, deer, and wild boars, often transferring to humans through inadequately cooked meat. Acute hepatitis caused by HEV in healthy individuals is mostly asymptomatic or associated with minor symptoms, such as jaundice. However, in immunosuppressed individuals, the disease can progress to chronic hepatitis and even escalate to cirrhosis. For pregnant women, an HEV infection can cause fulminant liver failure, with a potential mortality rate of 25%. Mortality rates also rise amongst cirrhotic patients when they contract an acute HEV infection, which can even trigger acute-on-chronic liver failure if layered onto pre-existing chronic liver disease. As the prevalence of HEV infection continues to rise worldwide, highlighting the particular risks associated with severe HEV infection is of major medical interest. This text offers a brief summary of the characteristics of hepatitis developed by patient groups at an elevated risk of severe HEV infection.
Collapse
Affiliation(s)
- László Orosz
- Department of Medical Microbiology, University of Szeged, Szeged 6720, Csongrád-Csanád, Hungary
| | - Károly Péter Sárvári
- Department of Medical Microbiology, University of Szeged, Szeged 6720, Csongrád-Csanád, Hungary
| | - Áron Dernovics
- Department of Medical Microbiology, University of Szeged, Szeged 6720, Csongrád-Csanád, Hungary
| | - András Rosztóczy
- Department of Internal Medicine, Division of Gastroenterology, University of Szeged, Szeged 6725, Csongrád-Csanád, Hungary
| | - Klára Megyeri
- Department of Medical Microbiology, University of Szeged, Szeged 6720, Csongrád-Csanád, Hungary
| |
Collapse
|
|
4
|
Wißing MH, Meister TL, Nocke MK, Gömer A, Masovic M, Knegendorf L, Brüggemann Y, Bader V, Siddharta A, Bock CT, Ploss A, Kenney SP, Winklhofer KF, Behrendt P, Wedemeyer H, Steinmann E, Todt D. Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication. Nat Commun 2024; 15:4855. [PMID: 38844458 PMCID: PMC11156872 DOI: 10.1038/s41467-024-49219-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2023] [Accepted: 05/24/2024] [Indexed: 06/09/2024] Open
Abstract
Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.
Collapse
Affiliation(s)
| | - Toni Luise Meister
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany
- Institute for Infection Research and Vaccine Development (IIRVD), Centre for Internal Medicine, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, Germany
- Department for Clinical Immunology of Infectious Diseases, Bernhard Nocht Institute for Tropical Medicine (BNITM), Hamburg, Germany
- German Centre for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel-Riems, Hamburg, Germany
| | - Maximilian Klaus Nocke
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany
- European Virus Bioinformatics Center (EVBC), Jena, Germany
| | - André Gömer
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany
| | - Mejrema Masovic
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany
| | - Leonard Knegendorf
- Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- Hannover Medical School, Institute for Medical Microbiology and Hospital Epidemiology, Hannover, Germany
| | - Yannick Brüggemann
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany
| | - Verian Bader
- Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany
- Department of Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany
| | - Anindya Siddharta
- Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Claus-Thomas Bock
- Division of Viral Gastroenteritis and Hepatitis Pathogens and Enteroviruses, Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany
| | - Alexander Ploss
- Department of Molecular Biology, Princeton University, Princeton, NJ, USA
| | - Scott P Kenney
- Center for Food Animal Health, Departments of Animal Sciences and Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, 43210, USA
| | - Konstanze F Winklhofer
- Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany
- Cluster of Excellence RESOLV, Bochum, Germany
| | - Patrick Behrendt
- Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
- German Center for Infectious Disease Research (DZIF); Partner Sites Hannover-Braunschweig, Braunschweig, Germany
| | - Heiner Wedemeyer
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
- German Center for Infectious Disease Research (DZIF); Partner Sites Hannover-Braunschweig, Braunschweig, Germany
- Excellence Cluster 2155 RESIST, Hannover Medical School, Hannover, Germany, Braunschweig, Germany
| | - Eike Steinmann
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany.
- German Centre for Infection Research (DZIF), External Partner Site, Bochum, Germany.
| | - Daniel Todt
- Department for Molecular and Medical Medicine, Ruhr University Bochum, Bochum, Germany.
- European Virus Bioinformatics Center (EVBC), Jena, Germany.
| |
Collapse
|
|
5
|
Zahmanova G, Takova K, Tonova V, Koynarski T, Lukov LL, Minkov I, Pishmisheva M, Kotsev S, Tsachev I, Baymakova M, Andonov AP. The Re-Emergence of Hepatitis E Virus in Europe and Vaccine Development. Viruses 2023; 15:1558. [PMID: 37515244 PMCID: PMC10383931 DOI: 10.3390/v15071558] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 07/11/2023] [Accepted: 07/13/2023] [Indexed: 07/30/2023] Open
Abstract
Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis. Transmission of HEV mainly occurs via the fecal-oral route (ingesting contaminated water or food) or by contact with infected animals and their raw meat products. Some animals, such as pigs, wild boars, sheep, goats, rabbits, camels, rats, etc., are natural reservoirs of HEV, which places people in close contact with them at increased risk of HEV disease. Although hepatitis E is a self-limiting infection, it could also lead to severe illness, particularly among pregnant women, or chronic infection in immunocompromised people. A growing number of studies point out that HEV can be classified as a re-emerging virus in developed countries. Preventative efforts are needed to reduce the incidence of acute and chronic hepatitis E in non-endemic and endemic countries. There is a recombinant HEV vaccine, but it is approved for use and commercially available only in China and Pakistan. However, further studies are needed to demonstrate the necessity of applying a preventive vaccine and to create conditions for reducing the spread of HEV. This review emphasizes the hepatitis E virus and its importance for public health in Europe, the methods of virus transmission and treatment, and summarizes the latest studies on HEV vaccine development.
Collapse
Affiliation(s)
- Gergana Zahmanova
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria
- Department of Technology Transfer and IP Management, Center of Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria
| | - Katerina Takova
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria
| | - Valeria Tonova
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria
| | - Tsvetoslav Koynarski
- Department of Animal Genetics, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
| | - Laura L Lukov
- Faculty of Sciences, Brigham Young University-Hawaii, Laie, HI 96762, USA
| | - Ivan Minkov
- Department of Technology Transfer and IP Management, Center of Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria
- Institute of Molecular Biology and Biotechnologies, 4108 Markovo, Bulgaria
| | - Maria Pishmisheva
- Department of Infectious Diseases, Pazardzhik Multiprofile Hospital for Active Treatment, 4400 Pazardzhik, Bulgaria
| | - Stanislav Kotsev
- Department of Infectious Diseases, Pazardzhik Multiprofile Hospital for Active Treatment, 4400 Pazardzhik, Bulgaria
| | - Ilia Tsachev
- Department of Microbiology, Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
| | - Magdalena Baymakova
- Department of Infectious Diseases, Military Medical Academy, 1606 Sofia, Bulgaria
| | - Anton P Andonov
- Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
| |
Collapse
|
|
6
|
Kumar A, Subramani C, Raj S, Ranjith-Kumar CT, Surjit M. Hepatitis E Virus Protease Inhibits the Activity of Eukaryotic Initiation Factor 2-Alpha Kinase 4 and Promotes Virus Survival. J Virol 2023; 97:e0034723. [PMID: 37199644 PMCID: PMC10308950 DOI: 10.1128/jvi.00347-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 05/02/2023] [Indexed: 05/19/2023] Open
Abstract
Multiple mechanisms exist in a cell to cope with stress. Four independent stress-sensing kinases constitute the integrated stress response machinery of the mammalian cell, and they sense the stress signals and act by phosphorylating the eukaryotic initiation factor 2α (eIF2α) to arrest cellular translation. Eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4) is one of the four kinases and is activated under conditions of amino acid starvation, UV radiation, or RNA virus infection, resulting in shutdown of global translation. An earlier study in our laboratory constructed the protein interaction network of the hepatitis E virus (HEV) and identified eIF2AK4 as a host interaction partner of the genotype 1 (g1) HEV protease (PCP). Here, we report that PCP's association with the eIF2AK4 results in inhibition of self-association and concomitant loss of kinase activity of eIF2AK4. Site-directed mutagenesis of the 53rd phenylalanine residue of PCP abolishes its interaction with the eIF2AK4. Further, a genetically engineered HEV-expressing F53A mutant PCP shows poor replication efficiency. Collectively, these data identify an additional property of the g1-HEV PCP protein, through which it helps the virus in antagonizing eIF2AK4-mediated phosphorylation of the eIF2α, thus contributing to uninterrupted synthesis of viral proteins in the infected cells. IMPORTANCE Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. It causes chronic infection in organ transplant patients. Although the disease is self-limiting in normal individuals, it is associated with high mortality (~30%) in pregnant women. In an earlier study, we identified the interaction between the genotype 1 HEV protease (PCP) and cellular eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4). Since eIF2AK4 is a sensor of the cellular integrated stress response machinery, we evaluated the significance of the interaction between PCP and eIF2AK4. Here, we show that PCP competitively associates with and interferes with self-association of the eIF2AK4, thereby inhibiting its kinase activity. Lack of eIF2AK4 activity prevents phosphorylation-mediated inactivation of the cellular eIF2α, which is essential for initiation of cap-dependent translation. Thus, PCP behaves as a proviral factor, promoting uninterrupted synthesis of viral proteins in infected cells, which is crucial for survival and proliferation of the virus.
Collapse
Affiliation(s)
- Amit Kumar
- Virology Laboratory, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Chandru Subramani
- Virology Laboratory, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Shivani Raj
- University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi, India
| | - C. T. Ranjith-Kumar
- University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi, India
| | - Milan Surjit
- Virology Laboratory, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| |
Collapse
|
|
7
|
Songtanin B, Molehin AJ, Brittan K, Manatsathit W, Nugent K. Hepatitis E Virus Infections: Epidemiology, Genetic Diversity, and Clinical Considerations. Viruses 2023; 15:1389. [PMID: 37376687 DOI: 10.3390/v15061389] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2023] [Revised: 06/13/2023] [Accepted: 06/15/2023] [Indexed: 06/29/2023] Open
Abstract
According to the World Health Organization, approximately 20 million people worldwide are infected annually with the hepatitis E virus (HEV). There are four main genotypes of HEV. Genotype 1 and genotype 2 are common in developing countries and are transmitted by contaminated water from a fecal-oral route. Genotype 3 and genotype 4 are common in developed countries and can lead to occasional transmission to humans via undercooked meat. Hepatitis E virus 1 and HEV3 can lead to fulminant hepatitis, and HEV3 can lead to chronic hepatitis and cirrhosis in immunocompromised patients. The majority of patients with HEV infection are asymptomatic and usually have spontaneous viral clearance without treatment. However, infection in immunocompromised individuals can lead to chronic HEV infection. Both acute and chronic HEV infections can have extrahepatic manifestations. No specific treatment is required for acute HEV infection, no treatment has been approved in chronic infection, and no HEV vaccine has been approved by the (United States) Food and Drug Administration. This review focuses on the molecular virology (HEV life cycle, genotypes, model systems, zoonosis), pathogenesis, clinical manifestation, and treatment of chronic HEV infection, especially in immunocompromised patients, to provide clinicians a better understanding of the global distribution of these infections and the significant effect they can have on immunocompromised patients.
Collapse
Affiliation(s)
- Busara Songtanin
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Adebayo J Molehin
- Department of Microbiology & Immunology, College of Graduate Studies, Midwestern University, Glendale, AZ 85308, USA
| | - Kevin Brittan
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Wuttiporn Manatsathit
- Department of Gastroenterology and Hepatology, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Kenneth Nugent
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| |
Collapse
|
|
8
|
LeDesma R, Heller B, Biswas A, Maya S, Gili S, Higgins J, Ploss A. Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication. eLife 2023; 12:e80529. [PMID: 36852909 PMCID: PMC9977285 DOI: 10.7554/elife.80529] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Accepted: 02/12/2023] [Indexed: 03/01/2023] Open
Abstract
Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the 'pPCP' domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.
Collapse
Affiliation(s)
- Robert LeDesma
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniversityPrincetonUnited States
| | - Brigitte Heller
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniversityPrincetonUnited States
| | - Abhishek Biswas
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniversityPrincetonUnited States
| | - Stephanie Maya
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniversityPrincetonUnited States
| | - Stefania Gili
- Department of Geosciences, Princeton UniversityPrincetonUnited States
| | - John Higgins
- Department of Geosciences, Princeton UniversityPrincetonUnited States
| | - Alexander Ploss
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniversityPrincetonUnited States
| |
Collapse
|
|
9
|
Ju X, Dong L, Ding Q. Hepatitis E Virus Life Cycle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1417:141-157. [PMID: 37223864 DOI: 10.1007/978-981-99-1304-6_10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
Hepatitis E virus (HEV) infects over 20 million people worldwide per year, leading to 30,000-40,000 deaths. In most cases HEV infection in a self-limited, acute illness. However, chronic infections could occur in immunocompromised individuals. Due to scarcity of robust cell culture models in vitro and genetic tractable animal models in vivo, the details of HEV life cycle, as well as its interaction with host cells still remain elusive, which dampens antivirals discovery. In this chapter, we present an update in the HEV infectious cycle steps: entry, genome replication/subgenomic RNA transcription, assembly, and release. Moreover, we discussed the future prospective on HEV research and illustrates important questions urgently to be addressed.
Collapse
Affiliation(s)
- Xiaohui Ju
- School of Medicine, Tsinghua University, Beijing, China
| | - Lin Dong
- School of Medicine, Tsinghua University, Beijing, China
| | - Qiang Ding
- School of Medicine, Tsinghua University, Beijing, China.
| |
Collapse
|
|
10
|
Fieulaine S, Tubiana T, Bressanelli S. De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation. Virology 2023; 578:128-140. [PMID: 36527931 DOI: 10.1016/j.virol.2022.12.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2022] [Revised: 12/01/2022] [Accepted: 12/02/2022] [Indexed: 12/14/2022]
Abstract
Hepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1's N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis.
Collapse
Affiliation(s)
- Sonia Fieulaine
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
| | - Thibault Tubiana
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
| | - Stéphane Bressanelli
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
| |
Collapse
|
|
11
|
Zhou Y, Zhao C, Tian Y, Xu N, Wang Y. Characteristics and Functions of HEV Proteins. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1417:15-32. [PMID: 37223856 DOI: 10.1007/978-981-99-1304-6_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
Hepatitis E virus (HEV) is a non-enveloped virus containing a single-stranded, positive-sense RNA genome of 7.2 kb, which consists of a 5' non-coding region, three open reading frames (ORFs), and a 3' non-coding region. ORF1 is diverse between genotypes and encodes the nonstructural proteins, which include the enzymes needed for virus replication. In addition to its role in virus replication, the function of ORF1 is relevant to viral adaption in culture and may also relate to virus infection and HEV pathogenicity. ORF2 protein is the capsid protein, which is about 660 amino acids in length. It not only protects the integrity of the viral genome, but is also involved in many important physiological activities, such as virus assembly, infection, host interaction, and innate immune response. The main immune epitopes, especially neutralizing epitopes, are located on ORF2 protein, which is a candidate antigen for vaccine development. ORF3 protein is a phosphoprotein of 113 or 114 amino acids with a molecular weight of 13 kDa with multiple functions that can also induce strong immune reactivity. A novel ORF4 has been identified only in genotype 1 HEV and its translation promotes viral replication.
Collapse
Affiliation(s)
- Yan Zhou
- RegCMC, Great Regulatory Affairs, Sanofi (China) Investment Co., Ltd, Beijing, China
| | - Chenyan Zhao
- Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China
| | - Yabin Tian
- Division II of In Vitro Diagnostics for Infectious Diseases, National Institutes for Food and Drug Control, Beijing, China
| | - Nan Xu
- Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China
| | - Youchun Wang
- Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Kunming, China.
| |
Collapse
|
|
12
|
Muñoz-Chimeno M, Rodriguez-Paredes V, García-Lugo MA, Avellon A. Hepatitis E genotype 3 genome: A comprehensive analysis of entropy, motif conservation, relevant mutations, and clade-associated polymorphisms. Front Microbiol 2022; 13:1011662. [PMID: 36274715 PMCID: PMC9582770 DOI: 10.3389/fmicb.2022.1011662] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 09/20/2022] [Indexed: 11/13/2022] Open
Abstract
Hepatitis E virus genotype 3 (HEV-3) is an EU/EEA emergent zoonosis. HEV-3 clades/subtypes have been described. Its genome contains ORF1, which encodes nonstructural proteins for virus replication, ORF2, the capsid protein, and ORF3, a multifunctional protein involved in virion pathogenesis. The study aims with respect to HEV-3 are to: (1) calculate genome entropy (excluding hypervariable region); (2) analyze the described motifs/mutations; (3) characterize clade/subtype genome polymorphisms. Seven hundred and five sequences from the GenBank database were used. The highest entropies were identified in zoonotic genotypes (HEV-3 and HEV-4) with respect to HEV-1 in X domain, RdRp, ORF2, and ORF3. There were statistically significant differences in the entropy between proteins, protease and ORF3 being the most variable and Y domain being the most conserved. Methyltransferase and Y domain motifs were completely conserved. By contrast, essential protease H581 residue and catalytic dyad exhibited amino acid changes in 1.8% and 0.4% of sequences, respectively. Several X domain amino acids were associated with clades. We found sequences with mutations in all helicase motifs except number IV. Helicase mutations related to increased virulence and/or fulminant hepatitis were frequent, the 1,110 residue being a typical HEV-3e and HEV-3f-A2 polymorphism. RdRp motifs III, V, VII also had high mutation rates. Motif III included residues that are polymorphisms of HEV-3e (F1449) and HEV-3 m (D1451). RdRp ribavirin resistance mutations were frequent, mainly 1479I (67.4, 100% in HEV-3efglmk) and 1634R/K (10.0%, almost 100% in HEV-3e). With respect to ORF2, 19/27 neutralization epitopes had mutations. The S80 residue in ORF3 presented mutations in 3.5% of cases. Amino acids in the ORF3-PSAP motif had high substitution rates, being more frequent in the first PSAP (44.8%) than in the second (1.5%). This is the first comprehensive analysis of the HEV-3 genome, aimed at improving our knowledge of the genome, and establishing the basis for future genotype-to-phenotype analysis, given that viral features associated with severity have not been explored in depth. Our results demonstrate there are important genetic differences in the studied genomes that sometimes affect significant viral structures, and constitute clade/subtype polymorphisms that may affect the clinical course or treatment efficacy.
Collapse
Affiliation(s)
- Milagros Muñoz-Chimeno
- Hepatitis Unit, National Center of Microbiology, Carlos III Institute of Health, Madrid, Spain
- Alcalá de Henares University, Madrid, Spain
| | | | | | - Ana Avellon
- Hepatitis Unit, National Center of Microbiology, Carlos III Institute of Health, Madrid, Spain
- CIBERESP Epidemiology and Public Health, Madrid, Spain
- *Correspondence: Ana Avellon,
| |
Collapse
|
|
13
|
Sathanantham P, Zhao W, He G, Murray A, Fenech E, Diaz A, Schuldiner M, Wang X. A conserved viral amphipathic helix governs the replication site-specific membrane association. PLoS Pathog 2022; 18:e1010752. [PMID: 36048900 PMCID: PMC9473614 DOI: 10.1371/journal.ppat.1010752] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 09/14/2022] [Accepted: 07/19/2022] [Indexed: 11/18/2022] Open
Abstract
Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their destinations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a’s localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the Alsuviricetes class targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies. Positive-strand RNA viruses [(+)RNA viruses] are the largest viral class that include numerous pathogens causing important diseases in humans, animals, and plants. During their infections, (+)RNA viruses assemble their viral replication complexes (VRCs), where they multiply themselves, at specific organelle membranes. An initial step to form VRCs is to target viral replication proteins to the designated organelle membranes. For brome mosaic virus (BMV), its replication protein 1a is responsible for the VRC formation at the nuclear endoplasmic reticulum (ER) membrane. We show that an amphipathic alpha-helix, helix B, in BMV 1a is necessary for the association of BMV 1a with the nuclear ER membrane and for BMV genome amplification. In addition, Helix B is sufficient to target several soluble proteins to the nuclear ER membrane in yeast and plant cells. BMV belongs to the Alsuviricetes class that includes viruses infecting humans, animals, and plants. We further show that the helix B across members of the Alsuviricetes class is sufficient to target fluorescence proteins to the designated organelle membranes. Our results reveal a conserved feature among a group of viruses in governing the associations with replication site-specific organelle membranes and point to a target to develop broad-spectrum antivirals.
Collapse
Affiliation(s)
- Preethi Sathanantham
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America
| | - Wenhao Zhao
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America
- Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing, China
| | - Guijuan He
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America
| | - Austin Murray
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America
| | - Emma Fenech
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Arturo Diaz
- Department of Biology, La Sierra University, Riverside, California, United States of America
| | - Maya Schuldiner
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Xiaofeng Wang
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America
- * E-mail:
| |
Collapse
|
|
14
|
Hooda P, Chaudhary M, Parvez MK, Sinha N, Sehgal D. Inhibition of Hepatitis E Virus Replication by Novel Inhibitor Targeting Methyltransferase. Viruses 2022; 14:v14081778. [PMID: 36016400 PMCID: PMC9415367 DOI: 10.3390/v14081778] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 08/07/2022] [Accepted: 08/11/2022] [Indexed: 11/17/2022] Open
Abstract
Hepatitis E Virus (HEV) is a quasi-enveloped virus having a single-stranded, positive-sense RNA genome (~7.2 kb), flanked with a 5′ methylated cap and a 3′ polyadenylated tail. The HEV open reading frame 1 (ORF1) encodes a 186-kDa polyprotein speculated to get processed and produce Methyltransferase (MTase), one of the four essential replication enzymes. In this study, we report the identification of the MTase inhibitor, which may potentially deplete its enzymatic activity, thus causing the cessation of viral replication. Using in silico screening through docking, we identified ten putative compounds, which were tested for their anti-MTase activity. This resulted in the identification of 3-(4-Hydroxyphenyl)propionic acid (HPPA), with an IC50 value of 0.932 ± 0.15 μM, which could be perceived as an effective HEV inhibitor. Furthermore, the compound was tested for inhibition of HEV replication in the HEV culture system. The viral RNA copies were markedly decreased from ~3.2 × 106 in untreated cells to ~4.3 × 102.8 copies in 800 μM HPPA treated cells. Therefore, we propose HPPA as a potential drug-like inhibitor against HEV-MTase, which would need further validation through in vivo analysis using animal models and the administration of Pharmacokinetic and Pharmacodynamic (PK/PD) studies.
Collapse
Affiliation(s)
- Preeti Hooda
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Gautam Budh Nagar, Greater Noida 201314, India
| | - Meenakshi Chaudhary
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Gautam Budh Nagar, Greater Noida 201314, India
| | - Mohammad K. Parvez
- Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
- Correspondence: (M.K.P.); (D.S.)
| | - Neha Sinha
- Department of Infectious Diseases and Microbiology, School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Deepak Sehgal
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Gautam Budh Nagar, Greater Noida 201314, India
- Correspondence: (M.K.P.); (D.S.)
| |
Collapse
|
|
15
|
Cierniak F, Ulrich RG, Groschup MH, Eiden M. A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains. Pathogens 2022; 11:pathogens11030355. [PMID: 35335679 PMCID: PMC8948863 DOI: 10.3390/pathogens11030355] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2022] [Revised: 03/04/2022] [Accepted: 03/13/2022] [Indexed: 12/02/2022] Open
Abstract
Zoonotic hepatitis E virus (HEV) infection is an emerging cause of acute viral hepatitis in developed countries. Known reservoirs of zoonotic genotype 3 (HEV-3) are mainly pigs and wild boar, and to a lesser extent rabbits and deer. Rabbit hepatitis E virus (HEV-3ra) is prevalent in rabbits worldwide and represents a particular risk for zoonotic infection. Current understanding of the molecular mechanisms of HEV pathogenesis is incomplete, particularly due to the limited availability of efficient and reliable cell culture systems. In order to identify genomic regions responsible for HEV propagation in cell culture, we developed a modular chimeric reporter replicon system based on cell culture-adapted (Kernow-C1/p6 and 47832mc) and rabbit-derived HEV strains. Replication in HepG2 cells was monitored on the basis of a Gaussia luciferase reporter gene that was inserted in place of the open reading frame (ORF) 2 of the HEV genome. Luciferase activity of rabbit HEV-derived replicons was significantly lower than that of Kernow-C1/p6 and 47832mc replicons. Serial exchanges of defined ORF1 segments within the Kernow-C1/p6 replicon backbone indicated that HEV replication in HepG2 cells is not determined by a single domain but rather by an interplay of longer segments of the ORF1-derived nonstructural polyprotein. This implies that a specific combination of viral factors is required for efficient HEV propagation in cell culture.
Collapse
Affiliation(s)
- Filip Cierniak
- Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; (F.C.); (R.G.U.); (M.H.G.)
| | - Rainer G. Ulrich
- Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; (F.C.); (R.G.U.); (M.H.G.)
- Partner Site Hamburg-Lübeck-Borstel-Riems, German Centre for Infection Research (DZIF), 17493 Greifswald-Insel Riems, Germany
| | - Martin H. Groschup
- Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; (F.C.); (R.G.U.); (M.H.G.)
- Partner Site Hamburg-Lübeck-Borstel-Riems, German Centre for Infection Research (DZIF), 17493 Greifswald-Insel Riems, Germany
| | - Martin Eiden
- Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; (F.C.); (R.G.U.); (M.H.G.)
- Correspondence:
| |
Collapse
|
|
16
|
Hooda P, Ishtikhar M, Saraswat S, Bhatia P, Mishra D, Trivedi A, Kulandaisamy R, Aggarwal S, Munde M, Ali N, AlAsmari AF, Rauf MA, Inampudi KK, Sehgal D. Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase. Molecules 2022; 27:1505. [PMID: 35268608 PMCID: PMC8911963 DOI: 10.3390/molecules27051505] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 02/09/2022] [Accepted: 02/18/2022] [Indexed: 11/21/2022] Open
Abstract
Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.
Collapse
Affiliation(s)
- Preeti Hooda
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Mohd Ishtikhar
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Shweta Saraswat
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Pooja Bhatia
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Deepali Mishra
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Aditya Trivedi
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| | - Rajkumar Kulandaisamy
- Department of Biophysics, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India;
| | - Soumya Aggarwal
- School of Physical Sciences, Jawaharlal Nehru University (JNU), New Delhi 110067, India; (S.A.); (M.M.)
| | - Manoj Munde
- School of Physical Sciences, Jawaharlal Nehru University (JNU), New Delhi 110067, India; (S.A.); (M.M.)
| | - Nemat Ali
- Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 55760, Riyadh 11451, Saudi Arabia; (N.A.); (A.F.A.)
| | - Abdullah F. AlAsmari
- Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 55760, Riyadh 11451, Saudi Arabia; (N.A.); (A.F.A.)
| | - Mohd A. Rauf
- Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201, USA;
| | - Krishna K. Inampudi
- Department of Biophysics, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India;
| | - Deepak Sehgal
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida 201314, India; (P.H.); (M.I.); (S.S.); (P.B.); (D.M.); (A.T.)
| |
Collapse
|
|
17
|
El-Kafrawy SA, El-Daly MM. Hepatitis E virus in Saudi Arabia: more surveillance needed. Future Virol 2022. [DOI: 10.2217/fvl-2021-0320] [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
Hepatitis E virus (HEV) is a small quasi-enveloped ssRNA causing acute hepatitis. HEV is the leading cause of intermittent acute hepatitis and fulminant hepatic failure. Risk factors include drinking contaminated water in developing countries and consumption of infected animal products in developed countries. Previous reports on HEV prevalence in Saudi Arabia had small sample sizes. Nationwide systematic seroprevalence studies are needed to investigate risk factors and annual incidence. Camels play a cultural and economic role in the life of Saudi citizens with frequent human contact and potential role in zoonotic transmission. Future research needs to include larger sample-sizes and nationwide studies. Future studies should also focus on raising awareness of HEV infection and the need for wider population testing and screening.
Collapse
Affiliation(s)
- Sherif Aly El-Kafrawy
- Special Infectious Agents Unit-BSL3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
- Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
| | - Mai Mohamed El-Daly
- Special Infectious Agents Unit-BSL3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
- Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
| |
Collapse
|
|
18
|
Cancela F, Noceti O, Arbiza J, Mirazo S. Structural aspects of hepatitis E virus. Arch Virol 2022; 167:2457-2481. [PMID: 36098802 PMCID: PMC9469829 DOI: 10.1007/s00705-022-05575-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Accepted: 06/04/2022] [Indexed: 12/14/2022]
Abstract
Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Hepatitis E is an enterically transmitted zoonotic disease that causes large waterborne epidemic outbreaks in developing countries and has become an increasing public-health concern in industrialized countries. In this setting, the infection is usually acute and self-limiting in immunocompetent individuals, although chronic cases in immunocompromised patients have been reported, frequently associated with several extrahepatic manifestations. Moreover, extrahepatic manifestations have also been reported in immunocompetent individuals with acute HEV infection. HEV belongs to the alphavirus-like supergroup III of single-stranded positive-sense RNA viruses, and its genome contains three partially overlapping open reading frames (ORFs). ORF1 encodes a nonstructural protein with eight domains, most of which have not been extensively characterized: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region, proline-rich region, X domain, Hel domain, and RNA-dependent RNA polymerase. ORF2 and ORF3 encode the capsid protein and a multifunctional protein believed to be involved in virion release, respectively. The novel ORF4 is only expressed in HEV genotype 1 under endoplasmic reticulum stress conditions, and its exact function has not yet been elucidated. Despite important advances in recent years, the biological and molecular processes underlying HEV replication remain poorly understood, primarily due to a lack of detailed information about the functions of the viral proteins and the mechanisms involved in host-pathogen interactions. This review summarizes the current knowledge concerning HEV proteins and their biological properties, providing updated detailed data describing their function and focusing in detail on their structural characteristics. Furthermore, we review some unclear aspects of the four proteins encoded by the ORFs, highlighting the current key information gaps and discussing potential novel experimental strategies for shedding light on those issues.
Collapse
Affiliation(s)
- Florencia Cancela
- grid.11630.350000000121657640Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Ofelia Noceti
- grid.414402.70000 0004 0469 0889Programa Nacional de Trasplante Hepático y Unidad Docente Asistencial Centro Nacional de Tratamiento Hepatobiliopancreatico. Hospital Central de las Fuerzas Armadas, Montevideo, Uruguay
| | - Juan Arbiza
- grid.11630.350000000121657640Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Santiago Mirazo
- grid.11630.350000000121657640Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay ,grid.11630.350000000121657640Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay ,Av. Alfredo Navarro 3051, PC 11600 Montevideo, Uruguay
| |
Collapse
|
|
19
|
Wang B, Meng XJ. Structural and molecular biology of hepatitis E virus. Comput Struct Biotechnol J 2021; 19:1907-1916. [PMID: 33995894 PMCID: PMC8079827 DOI: 10.1016/j.csbj.2021.03.038] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 03/26/2021] [Accepted: 03/29/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is one of the most common causes of acute viral hepatitis, mainly transmitted by fecal-oral route but has also been linked to fulminant hepatic failure, chronic hepatitis, and extrahepatic neurological and renal diseases. HEV is an emerging zoonotic pathogen with a broad host range, and strains of HEV from numerous animal species are known to cross species barriers and infect humans. HEV is a single-stranded, positive-sense RNA virus in the family Hepeviridae. The genome typically contains three open reading frames (ORFs): ORF1 encodes a nonstructural polyprotein for virus replication and transcription, ORF2 encodes the capsid protein that elicits neutralizing antibodies, and ORF3, which partially overlaps ORF2, encodes a multifunctional protein involved in virion morphogenesis and pathogenesis. HEV virions are non-enveloped spherical particles in feces but exist as quasi-enveloped particles in circulating blood. Two types of HEV virus-like particles (VLPs), small T = 1 (270 Å) and native virion-sized T = 3 (320-340 Å) have been reported. There exist two distinct forms of capsid protein, the secreted form (ORF2S) inhibits antibody neutralization, whereas the capsid-associated form (ORF2C) self-assembles to VLPs. Four cis-reactive elements (CREs) containing stem-loops from secondary RNA structures have been identified in the non-coding regions and are critical for virus replication. This mini-review discusses the current knowledge and gaps regarding the structural and molecular biology of HEV with emphasis on the virion structure, genomic organization, secondary RNA structures, viral proteins and their functions, and life cycle of HEV.
Collapse
Affiliation(s)
- Bo Wang
- Center for Emerging, Zoonotic and Arthropod-borne Pathogens, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
| | - Xiang-Jin Meng
- Center for Emerging, Zoonotic and Arthropod-borne Pathogens, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
| |
Collapse
|
|
20
|
Advances in Hepatitis E Virus Biology and Pathogenesis. Viruses 2021; 13:v13020267. [PMID: 33572257 PMCID: PMC7915517 DOI: 10.3390/v13020267] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Revised: 01/21/2021] [Accepted: 02/02/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.
Collapse
|
|
21
|
Yadav KK, Boley PA, Fritts Z, Kenney SP. Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture. Viruses 2021; 13:v13010075. [PMID: 33430442 PMCID: PMC7827316 DOI: 10.3390/v13010075] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2020] [Revised: 01/05/2021] [Accepted: 01/05/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.
Collapse
Affiliation(s)
- Kush K. Yadav
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, OH 44691, USA; (K.K.Y.); (P.A.B.)
| | - Patricia A. Boley
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, OH 44691, USA; (K.K.Y.); (P.A.B.)
| | - Zachary Fritts
- Department of Electrical and Computer Engineering, University of Michigan, Ann Arbor, MI 48105, USA;
| | - Scott P. Kenney
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, OH 44691, USA; (K.K.Y.); (P.A.B.)
- Correspondence:
| |
Collapse
|
|
22
|
Tian D, Yugo DM, Kenney SP, Lynn Heffron C, Opriessnig T, Karuppannan AK, Bayne J, Halbur PG, Meng XJ. Dissecting the potential role of hepatitis E virus ORF1 nonstructural gene in cross-species infection by using intergenotypic chimeric viruses. J Med Virol 2020; 92:3563-3571. [PMID: 32589758 DOI: 10.1002/jmv.26226] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 06/22/2020] [Accepted: 06/23/2020] [Indexed: 12/18/2022]
Abstract
Hepatitis E virus (HEV) infects humans and more than a dozen other animal species. We previously showed that open reading frame 2 (ORF2) and ORF3 are apparently not involved in HEV cross-species infection, which infers that the ORF1 may contribute to host tropism. In this study, we utilize the genomic backbone of HEV-1 which only infects humans to construct a panel of intergenotypic chimeras in which the entire ORF1 gene or its functional domains were swapped with the corresponding regions from HEV-3 that infects both humans and pigs. We demonstrated that the chimeric HEVs were replication competent in human liver cells. Subsequently, we intrahepatically inoculated the RNA transcripts of chimeras into pigs to determine if the swapped ORF1 regions confer the chimeras' ability to infect pigs. We showed that there was no evidence of infectivity in pigs for any of the chimeras. We also investigated the role of human ribosome protein sequence S17, which expanded host range in cultured cells, in HEV cross-species infection. We demonstrated that S17 insertion in HEV ORF1 did not abolish HEV replication competency in vitro, but also did not expand HEV host tropism in vivo. The results highlight the complexity of the underlying mechanism of HEV cross-species infection.
Collapse
Affiliation(s)
- Debin Tian
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Danielle M Yugo
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Scott P Kenney
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University College of Veterinary Medicine, Wooster, Ohio
| | - C Lynn Heffron
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Tanja Opriessnig
- Infection and Immunity Division, The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK
- Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa
| | - Anbu K Karuppannan
- Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa
| | - Jenna Bayne
- Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa
| | - Patrick G Halbur
- Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa
| | - Xiang-Jin Meng
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| |
Collapse
|
|
23
|
Lin MH, Chang SC, Chiu YC, Jiang BC, Wu TH, Hsu CH. Structural, Biophysical, and Biochemical Elucidation of the SARS-CoV-2 Nonstructural Protein 3 Macro Domain. ACS Infect Dis 2020; 6:2970-2978. [PMID: 32946224 PMCID: PMC7537548 DOI: 10.1021/acsinfecdis.0c00441] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2020] [Indexed: 12/16/2022]
Abstract
The pandemic outbreak of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has threatened the global public health and economy since late December 2019. SARS-CoV-2 encodes the conserved macro domain within nonstructural protein 3, which may reverse cellular ADP-ribosylation and potentially cut the signal of a viral infection in the cell. Herein, we report that the SARS-CoV-2 macro domain was examined as a poly-ADP-ribose (ADPR) binding module and possessed mono-ADPR cleavage enzyme activity. After confirming the ADPR binding ability via a biophysical approach, the X-ray crystal structure of the SARS-CoV-2 macro domain was determined and structurally compared with those of other viruses. This study provides structural, biophysical, and biochemical bases to further evaluate the role of the SARS-CoV-2 macro domain in the host response via ADP-ribose binding but also as a potential target for drug design against COVID-19.
Collapse
Affiliation(s)
- Meng-Hsuan Lin
- Genome and Systems Biology Degree
Program, National Taiwan University and Academia
Sinica, Taipei 10617,
Taiwan
| | - San-Chi Chang
- Department of Agricultural Chemistry,
National Taiwan University, Taipei
10617, Taiwan
| | - Yi-Chih Chiu
- Genome and Systems Biology Degree
Program, National Taiwan University and Academia
Sinica, Taipei 10617,
Taiwan
| | - Bo-Chen Jiang
- Department of Agricultural Chemistry,
National Taiwan University, Taipei
10617, Taiwan
| | - Tsung-Han Wu
- Genome and Systems Biology Degree
Program, National Taiwan University and Academia
Sinica, Taipei 10617,
Taiwan
| | - Chun-Hua Hsu
- Genome and Systems Biology Degree
Program, National Taiwan University and Academia
Sinica, Taipei 10617,
Taiwan
- Department of Agricultural Chemistry,
National Taiwan University, Taipei
10617, Taiwan
- Institute of Biochemical Sciences,
National Taiwan University, Taipei
10617, Taiwan
| |
Collapse
|
|
24
|
Pallerla SR, Harms D, Johne R, Todt D, Steinmann E, Schemmerer M, Wenzel JJ, Hofmann J, Shih JWK, Wedemeyer H, Bock CT, Velavan TP. Hepatitis E Virus Infection: Circulation, Molecular Epidemiology, and Impact on Global Health. Pathogens 2020; 9:E856. [PMID: 33092306 PMCID: PMC7589794 DOI: 10.3390/pathogens9100856] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 10/09/2020] [Accepted: 10/16/2020] [Indexed: 12/11/2022] Open
Abstract
Infection with hepatitis E virus (HEV) represents the most common source of viral hepatitis globally. Although infecting over 20 million people annually in endemic regions, with major outbreaks described since the 1950s, hepatitis E remains an underestimated disease. This review gives a current view of the global circulation and epidemiology of this emerging virus. The history of HEV, from the first reported enteric non-A non-B hepatitis outbreaks, to the discovery of the viral agent and the molecular characterization of the different human pathogenic genotypes, is discussed. Furthermore, the current state of research regarding the virology of HEV is critically assessed, and the challenges towards prevention and diagnosis, as well as clinical risks of the disease described. Together, these points aim to underline the significant impact of hepatitis E on global health and the need for further in-depth research to better understand the pathophysiology and its role in the complex disease manifestations of HEV infection.
Collapse
Affiliation(s)
- Srinivas Reddy Pallerla
- Institute of Tropical Medicine, University of Tübingen, 72074 Tübingen, Germany; (S.R.P.); (T.P.V.)
- Vietnamese-German Center for Medical Research (VG-CARE), Hanoi 100000, Vietnam
| | - Dominik Harms
- Division of Viral Gastroenteritis and Hepatitis Pathogens and Enteroviruses, Department of Infectious Diseases, Robert Koch Institute, 13353 Berlin, Germany;
| | - Reimar Johne
- Unit Viruses in Food, Department Biological Safety, German Federal Institute for Risk Assessment, 10589 Berlin, Germany;
| | - Daniel Todt
- Department of Molecular and Medical Virology, Ruhr University Bochum, 44801 Bochum, Germany; (D.T.); (E.S.)
- European Virus Bioinformatics Center (EVBC), 07743 Jena, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr University Bochum, 44801 Bochum, Germany; (D.T.); (E.S.)
| | - Mathias Schemmerer
- Institute of Clinical Microbiology and Hygiene, National Consultant Laboratory for HAV and HEV, University Medical Center Regensburg, 93053 Regensburg, Germany; (M.S.); (J.J.W.)
| | - Jürgen J. Wenzel
- Institute of Clinical Microbiology and Hygiene, National Consultant Laboratory for HAV and HEV, University Medical Center Regensburg, 93053 Regensburg, Germany; (M.S.); (J.J.W.)
| | - Jörg Hofmann
- Institute of Virology, Charité Universitätsmedizin Berlin, Labor Berlin-Charité-Vivantes GmbH, 13353 Berlin, Germany;
| | | | - Heiner Wedemeyer
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30623 Hannover, Germany;
- German Center for Infection Research, Partner Hannover-Braunschweig, 38124 Braunschweig, Germany
| | - C.-Thomas Bock
- Institute of Tropical Medicine, University of Tübingen, 72074 Tübingen, Germany; (S.R.P.); (T.P.V.)
- Division of Viral Gastroenteritis and Hepatitis Pathogens and Enteroviruses, Department of Infectious Diseases, Robert Koch Institute, 13353 Berlin, Germany;
| | - Thirumalaisamy P. Velavan
- Institute of Tropical Medicine, University of Tübingen, 72074 Tübingen, Germany; (S.R.P.); (T.P.V.)
- Vietnamese-German Center for Medical Research (VG-CARE), Hanoi 100000, Vietnam
- Faculty of Medicine, Duy Tan University, Da Nang 550000, Vietnam
| |
Collapse
|
|
25
|
Zhang W, Ami Y, Suzaki Y, Doan YH, Jirintai S, Takahashi M, Okamoto H, Takeda N, Muramatsu M, Li TC. Persistent infection with a rabbit hepatitis E virus created by a reverse genetics system. Transbound Emerg Dis 2020; 68:615-625. [PMID: 32649803 DOI: 10.1111/tbed.13723] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Revised: 06/24/2020] [Accepted: 07/06/2020] [Indexed: 12/12/2022]
Abstract
Rabbit hepatitis E virus (HEV) is a novel zoonotic infectious agent. Although a cell culture system to grow the virus has been established, there is currently no reverse genetics system for generating the virus. In this study, capped genomic rabbit HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells, and the recovered viruses were subsequently passaged in the cells. The cell culture supernatant was capable of infecting rabbits negative for anti-HEV antibody by intravenous and oral inoculation, indicating that rabbit HEV generated by the reverse genetics system is infectious. Genome-wide analyses indicated that no nucleotide sequence change occurred in the virus genomes that were recovered from the cell culture supernatant after transfection and passaged one time or in the virus genomes recovered from faecal specimens of the infected rabbits. Ribavirin, a broad-spectrum anti-viral inhibitor, efficiently abrogated virus replication ex vivo and transiently suppressed the virus growth in the virus-infected rabbits, suggesting that this reagent is a candidate for therapeutic treatment. In addition, transmission of rabbit HEV to rabbits caused persistent infection, suggesting that the virus-infected rabbit could be an animal model for virus-induced hepatitis. The infectious rabbit HEV produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of viral hepatitis.
Collapse
Affiliation(s)
- Wenjing Zhang
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yasushi Ami
- Division of Experimental Animals Research, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yuriko Suzaki
- Division of Experimental Animals Research, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yen Hai Doan
- Department of Environmental Parasitology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Suljid Jirintai
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Shimotsuke-Shi, Japan
| | - Masaharu Takahashi
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Shimotsuke-Shi, Japan
| | - Hiroaki Okamoto
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Shimotsuke-Shi, Japan
| | - Naokazu Takeda
- Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tian-Cheng Li
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| |
Collapse
|
|
26
|
Kumar M, Hooda P, Khanna M, Patel U, Sehgal D. Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing. Front Microbiol 2020; 11:1347. [PMID: 32625196 PMCID: PMC7315041 DOI: 10.3389/fmicb.2020.01347] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Accepted: 05/26/2020] [Indexed: 01/10/2023] Open
Abstract
The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (-) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy.
Collapse
Affiliation(s)
- Manjeet Kumar
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Preeti Hooda
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Madhu Khanna
- Virology Lab, Department of Virology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi, India
| | - Utkarsh Patel
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Deepak Sehgal
- Virology Laboratory, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| |
Collapse
|
|
27
|
Ju X, Xiang G, Gong M, Yang R, Qin J, Li Y, Nan Y, Yang Y, Zhang QC, Ding Q. Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication. PLoS Pathog 2020; 16:e1008488. [PMID: 32433693 PMCID: PMC7239442 DOI: 10.1371/journal.ppat.1008488] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Accepted: 03/22/2020] [Indexed: 12/29/2022] Open
Abstract
There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5’ and 3’ untranslated regions and three open reading frames (ORF). HEV genome has 5’ cap and 3’ poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as “self” for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific “signal” that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector. Hepatitis E virus (HEV) is an underestimated pathogen, causing approximately 20 million infections worldwide annually and leading to about 60,000 deaths. There are no direct-acting antivirals for treating HEV, and although significant progress has been made to establish robust HEV cell culture models, the life cycle remains poorly characterized. A better understanding of HEV replication could facilitate the development of new drugs targeting this critical process. Our study found that RNA elements in the HEV genome interact with the HEV replicases to promote viral replication, suggesting that these RNA elements function as a scaffold for recruitment and assembly of the viral replication complex. This work furthers our understanding of HEV replication and could inform the generation of RNA-based therapeutics for treating HEV infection.
Collapse
Affiliation(s)
- Xiaohui Ju
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China
| | - Guangtao Xiang
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China
| | - Mingli Gong
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China
| | - Rui Yang
- School of Life Sciences, Tsinghua University, Beijing, China
| | - Jierui Qin
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China
| | - Yafei Li
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Yuchen Nan
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Yonglin Yang
- Department of General Practice, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
| | - Qiangfeng Cliff Zhang
- School of Life Sciences, Tsinghua University, Beijing, China
- Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China
| | - Qiang Ding
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China
- Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China
- * E-mail:
| |
Collapse
|
|
28
|
Saraswat S, Chaudhary M, Sehgal D. Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays. Front Cell Infect Microbiol 2020; 9:478. [PMID: 32039053 PMCID: PMC6989534 DOI: 10.3389/fcimb.2019.00478] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Accepted: 12/27/2019] [Indexed: 12/11/2022] Open
Abstract
Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432–592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of “Papain-like cysteine protease,” as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.
Collapse
Affiliation(s)
- Shweta Saraswat
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Meenakshi Chaudhary
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| | - Deepak Sehgal
- Virology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, India
| |
Collapse
|
|
29
|
Mudgal R, Mahajan S, Tomar S. Inhibition of Chikungunya virus by an adenosine analog targeting the SAM-dependent nsP1 methyltransferase. FEBS Lett 2019; 594:678-694. [PMID: 31623018 PMCID: PMC7164056 DOI: 10.1002/1873-3468.13642] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2019] [Revised: 09/23/2019] [Accepted: 10/02/2019] [Indexed: 01/02/2023]
Abstract
Alphaviruses, including Chikungunya (CHIKV) and Venezuelan equine encephalitis virus (VEEV), are among the leading causes of recurrent epidemics all over the world. Alphaviral nonstructural protein 1 (nsP1) orchestrates the capping of nascent viral RNA via its S-adenosyl methionine-dependent N-7-methyltransferase (MTase) and guanylyltransferase activities. Here, we developed and validated a novel capillary electrophoresis (CE)-based assay for measuring the MTase activity of purified VEEV and CHIKV nsP1. We employed the assay to assess the MTase inhibition efficiency of a few adenosine analogs and identified 5-iodotubercidin (5-IT) as an inhibitor of nsP1. The antiviral potency of 5-IT was evaluated in vitro using a combination of cell-based assays, which suggest that 5-IT is efficacious against CHIKV in cell culture (EC50 : 0.409 µm).
Collapse
Affiliation(s)
- Rajat Mudgal
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
| | - Supreeti Mahajan
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
| | - Shailly Tomar
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
| |
Collapse
|
|
30
|
LeDesma R, Nimgaonkar I, Ploss A. Hepatitis E Virus Replication. Viruses 2019; 11:E719. [PMID: 31390784 PMCID: PMC6723718 DOI: 10.3390/v11080719] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Revised: 07/31/2019] [Accepted: 08/02/2019] [Indexed: 12/19/2022] Open
Abstract
Hepatitis E virus (HEV) is a small quasi-enveloped, (+)-sense, single-stranded RNA virus belonging to the Hepeviridae family. There are at least 20 million HEV infections annually and 60,000 HEV-related deaths worldwide. HEV can cause up to 30% mortality in pregnant women and progress to liver cirrhosis in immunocompromised individuals and is, therefore, a greatly underestimated public health concern. Although a prophylactic vaccine for HEV has been developed, it is only licensed in China, and there is currently no effective, non-teratogenic treatment. HEV encodes three open reading frames (ORFs). ORF1 is the largest viral gene product, encoding the replicative machinery of the virus including a methyltransferase, RNA helicase, and an RNA-dependent RNA polymerase. ORF1 additionally contains a number of poorly understood domains including a hypervariable region, a putative protease, and the so-called 'X' and 'Y' domains. ORF2 is the viral capsid essential for formation of infectious particles and ORF3 is a small protein essential for viral release. In this review, we focus on the domains encoded by ORF1, which collectively mediate the virus' asymmetric genome replication strategy. We summarize what is known, unknown, and hotly debated regarding the coding and non-coding regions of HEV ORF1, and present a model of how HEV replicates its genome.
Collapse
Affiliation(s)
- Robert LeDesma
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA
| | - Ila Nimgaonkar
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA
| | - Alexander Ploss
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA.
| |
Collapse
|
|
31
|
Li TC, Wakita T. Small Animal Models of Hepatitis E Virus Infection. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a032581. [PMID: 29735581 DOI: 10.1101/cshperspect.a032581] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Novel hepeviruses have been recovered from many different animal species in recent years, increasing the diversity known to exist among the Hepeviridae, which now include two genera, Piscihepevirus and Orthohepevirus Multiple viral genotypes in the Orthohepevirus A species are able to replicate and cause acute hepatitis E in humans, and thus represent an important public health problem in industrialized as well as developing countries. Although hepatitis E virus (HEV) infections typically result in acute and self-limited hepatitis, immunocompromised and transplant patients are vulnerable to prolonged infections and to chronic hepatitis. Cell culture systems have been established for several HEV strains and offer new opportunities for the study of HEV biology. Similarly, a variety of new small animal models have been developed, using either nonhuman hepeviruses in their cognate hosts as surrogates for human HEV, or human HEV infection of immunodeficient mice with chimeric livers engrafted with human hepatocytes. These new models provide several advantages over previous nonhuman primate models of hepatitis E infection and will facilitate studies of pathogenicity, cross-species infection, mechanisms of virus replication, and vaccine and antiviral agent development. This article reviews the current understanding of small animal models for HEV.
Collapse
Affiliation(s)
- Tian-Cheng Li
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan
| |
Collapse
|
|
32
|
First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein. J Virol 2019; 93:JVI.00170-19. [PMID: 31019049 DOI: 10.1128/jvi.00170-19] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Accepted: 04/17/2019] [Indexed: 12/14/2022] Open
Abstract
Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.
Collapse
|
|
33
|
Hepatitis E Virus Drug Development. Viruses 2019; 11:v11060485. [PMID: 31141919 PMCID: PMC6631701 DOI: 10.3390/v11060485] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Revised: 05/20/2019] [Accepted: 05/24/2019] [Indexed: 02/08/2023] Open
Abstract
Hepatitis E virus (HEV) is an underestimated disease, leading to estimated 20 million infections and up to 70,000 deaths annually. Infections are mostly asymptomatic but can reach mortality rates up to 25% in pregnant women or become chronic in immunocompromised patients. The current therapy options are limited to the unspecific antivirals Ribavirin (RBV) and pegylated Interferon-α (pegIFN-α). RBV leads to viral clearance in only 80% of patients treated, and is, similar to pegIFN-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. In this review, we focus on the urgent need and current efforts in HEV drug development. We provide an overview of the current status of HEV antiviral research. Furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to HEV.
Collapse
|
|
34
|
Dao Thi VL, Wu X, Rice CM. Stem Cell-Derived Culture Models of Hepatitis E Virus Infection. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a031799. [PMID: 29686039 DOI: 10.1101/cshperspect.a031799] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Similar to other hepatotropic viruses, hepatitis E virus (HEV) has been notoriously difficult to propagate in cell culture, limiting studies to unravel its biology. Recently, major advances have been made by passaging primary HEV isolates and selecting variants that replicate efficiently in carcinoma cells. These adaptations, however, can alter HEV biology. We have explored human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) as an alternative to conventional hepatoma and hepatocyte cell culture systems for HEV studies. HLCs are permissive for nonadapted HEV isolate genotypes (gt)1-4 replication and can be readily genetically manipulated. HLCs, therefore, enable studies of pan-genotype HEV biology and will serve as a platform for testing anti-HEV treatments. Finally, we discuss how hepatocyte polarity is likely an important factor in the maturation and spread of infectious HEV particles.
Collapse
Affiliation(s)
- Viet Loan Dao Thi
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
| | - Xianfang Wu
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
| | - Charles M Rice
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
| |
Collapse
|
|
35
|
Abstract
Hepatitis E virus (HEV) possesses many of the features of other positive-stranded RNA viruses but also adds HEV-specific nuances, making its virus-host interactions unique. Slow virus replication kinetics and fastidious growth conditions, coupled with the historical lack of an efficient cell culture system to propagate the virus, have left many gaps in our understanding of its structure and replication cycle. Recent advances in culturing selected strains of HEV and resolving the 3D structure of the viral capsid are filling in knowledge gaps, but HEV remains an extremely understudied pathogen. Many steps in the HEV life cycle and many aspects of HEV pathogenesis remain unknown, such as the host and viral factors that determine cross-species infection, the HEV-specific receptor(s) on host cells, what determines HEV chronicity and the ability to replicate in extrahepatic sites, and what regulates processing of the open reading frame 1 (ORF1) nonstructural polyprotein.
Collapse
Affiliation(s)
- Scott P Kenney
- Food Animal Health Research Program, The Ohio State University, Wooster, Ohio 44691
| | - Xiang-Jin Meng
- Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
| |
Collapse
|
|
36
|
Bagdassarian E, Doceul V, Pellerin M, Demange A, Meyer L, Jouvenet N, Pavio N. The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response. Viruses 2018; 10:v10120726. [PMID: 30567349 PMCID: PMC6315852 DOI: 10.3390/v10120726] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 12/07/2018] [Accepted: 12/13/2018] [Indexed: 01/09/2023] Open
Abstract
Hepatitis E virus (HEV) is responsible for large waterborne epidemics of hepatitis in endemic countries and is an emerging zoonotic pathogen worldwide. In endemic regions, HEV-1 or HEV-2 genotypes are frequently associated with fulminant hepatitis in pregnant women, while with zoonotic HEV (HEV-3 and HEV-4), chronic cases of hepatitis and severe neurological disorders are reported. Hence, it is important to characterize the interactions between HEV and its host. Here, we investigated the ability of the nonstructural polyprotein encoded by the first open reading frame (ORF1) of HEV to modulate the host early antiviral response and, in particular, the type I interferon (IFN-I) system. We found that the amino-terminal region of HEV-3 ORF1 (MetYPCP), containing a putative methyltransferase (Met) and a papain-like cysteine protease (PCP) functional domain, inhibited IFN-stimulated response element (ISRE) promoter activation and the expression of several IFN-stimulated genes (ISGs) in response to IFN-I. We showed that the MetYPCP domain interfered with the Janus kinase (JAK)/signal transducer and activator of the transcription protein (STAT) signalling pathway by inhibiting STAT1 nuclear translocation and phosphorylation after IFN-I treatment. In contrast, MetYPCP had no effect on STAT2 phosphorylation and a limited impact on the activation of the JAK/STAT pathway after IFN-II stimulation. This inhibitory function seemed to be genotype-dependent, as MetYPCP from HEV-1 had no significant effect on the JAK/STAT pathway. Overall, this study provides evidence that the predicted MetYPCP domain of HEV ORF1 antagonises STAT1 activation to modulate the IFN response.
Collapse
Affiliation(s)
- Eugénie Bagdassarian
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| | - Virginie Doceul
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| | - Marie Pellerin
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| | - Antonin Demange
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| | - Léa Meyer
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| | - Nolwenn Jouvenet
- CNRS-UMR3569, Unité de Génomique Virale et Vaccination, Institut Pasteur, 75015 Paris, France.
| | - Nicole Pavio
- Anses, UMR 1161 Virologie, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France.
- INRA, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
- École Nationale Vétérinaire d'Alfort, UMR 1161 Virologie, 94700 Maisons-Alfort, France.
| |
Collapse
|
|
37
|
Li TC, Bai H, Yoshizaki S, Ami Y, Suzaki Y, Doan YH, Takahashi K, Mishiro S, Takeda N, Wakita T. Genotype 5 Hepatitis E Virus Produced by a Reverse Genetics System Has the Potential for Zoonotic Infection. Hepatol Commun 2018; 3:160-172. [PMID: 30620002 PMCID: PMC6312656 DOI: 10.1002/hep4.1288] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2018] [Accepted: 10/30/2018] [Indexed: 02/05/2023] Open
Abstract
Neither an animal model nor a cell culture system has been established for the genotype 5 hepatitis E virus (G5 HEV), and the pathogenicity, epidemiology, and replication mechanism of the virus remain unclear. In this study, we used a reverse genetics system to generate G5 HEV and examined the possibility of zoonotic infection. Capped and uncapped genomic G5 HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells. Infectious G5 HEV was recovered from the capped G5 HEV RNA–transfected PLC/PRF/5 cells and the subsequently passaged cells. G5 HEV was also recovered from uncapped G5 HEV–transfected PLC/PRF/5 cells after a longer lag phase, suggesting that the 5′‐cap structure is not essential but affected the efficiency of G5 HEV replication. G5 HEV infection was neutralized not only by anti‐G5 HEV‐like particles (HEV‐LPs) antibody, but also by anti‐G1, anti‐G3, anti‐G4, and anti‐G7 HEV‐LPs antibodies. G5 HEV was capable of infecting cynomolgus monkeys negative for anti‐HEV antibody but not animals positive for anti‐G7 HEV immunoglobulin G (IgG), indicating that cynomolgus monkeys were susceptible to G5 HEV, and the serotype of G5 HEV was identical to that of G7 HEV and human HEVs. Moreover, G5 HEV replication was efficiently inhibited by ribavirin and partially inhibited by sofosbuvir. Conclusion: Infectious G5 HEV was produced using a reverse genetics system, and the antigenicity was identical to that of human HEVs and G7 HEV. Transmission of G5 HEV to primates was confirmed by an experimental infection, providing evidence of the possibility of zoonotic infection by G5 HEV.
Collapse
Affiliation(s)
- Tian-Cheng Li
- Department of Virology II National Institute of Infectious Diseases Tokyo Japan
| | - Huimin Bai
- Baotou Medical College Baotou, Inner Mongolia China
| | - Sayaka Yoshizaki
- Department of Virology II National Institute of Infectious Diseases Tokyo Japan
| | - Yasushi Ami
- Division of Experimental Animals Research National Institute of Infectious Diseases Tokyo Japan
| | - Yuriko Suzaki
- Division of Experimental Animals Research National Institute of Infectious Diseases Tokyo Japan
| | - Yen Hai Doan
- Department of Virology II National Institute of Infectious Diseases Tokyo Japan
| | | | - Shunji Mishiro
- Department of Medical Sciences Tokyo-Shinagawa Hospital Tokyo Japan
| | - Naokazu Takeda
- Research Institute for Microbial Diseases, Osaka University Osaka Japan
| | - Takaji Wakita
- Department of Virology II National Institute of Infectious Diseases Tokyo Japan
| |
Collapse
|
|
38
|
Life cycle and morphogenesis of the hepatitis E virus. Emerg Microbes Infect 2018; 7:196. [PMID: 30498191 PMCID: PMC6265337 DOI: 10.1038/s41426-018-0198-7] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2018] [Revised: 11/01/2018] [Accepted: 11/05/2018] [Indexed: 12/19/2022]
Abstract
Hepatitis E virus (HEV) is transmitted primarily via contaminated water and food by the fecal oral route and causes epidemics in developing countries. In industrialized countries, zoonotic transmission of HEV is prevalent. In addition, HEV is the major cause of acute hepatitis in healthy adults and can cause chronic hepatitis in immunocompromised patients, with pregnant HEV-infected women having increased mortality rates of approximately 25%. HEV was once an understudied and neglected virus. However, in recent years, the safety of blood products with respect to HEV has increasingly been considered to be a public health problem. The establishment of HEV infection models has enabled significant progress to be made in understanding its life cycle. HEV infects cells via a receptor (complex) that has yet to be identified. The HEV replication cycle is initiated immediately after the (+) stranded RNA genome is released into the cell cytosol. Subsequently, infectious viral particles are released by the ESCRT complex as quasi-enveloped viruses (eHEVs) into the serum, whereas feces and urine contain only nonenveloped infectious viral progeny. The uncoating of the viral envelope takes place in the biliary tract, resulting in the generation of a nonenveloped virus that is more resistant to environmental stress and possesses a higher infectivity than that of eHEV. This review summarizes the current knowledge regarding the HEV life cycle, viral morphogenesis, established model systems and vaccine development.
Collapse
|
|
39
|
Abstract
Hepatitis E virus (HEV) is an important human pathogen that historically has been difficult to study. Limited levels of replication in vitro hindered our understanding of the viral life cycle. Sporadic and low-level virus shedding, lack of standardized detection methods, and subclinical infections made the development of animal models difficult. Better diagnostic techniques and understanding of the virus increased our ability to identify and characterize animal strains and animals that are amenable to model human-relevant infection. These advances are translating into the development of useful HEV animal models so that some of the greatest concerns associated with HEV infection, including host immunology, chronic infection, severe pregnancy mortality, and extrahepatic manifestations, can now be studied. Continued development of these animal models will be instrumental in understanding the many complex questions associated with HEV infection and for assessing therapeutics and prevention strategies to minimize HEV becoming a greater risk to the human population.
Collapse
Affiliation(s)
- Scott P Kenney
- Food Animal Health Research Program, College of Veterinary Medicine, Ohio State University, Wooster, Ohio 44691, USA;
| | - Xiang-Jin Meng
- Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060, USA;
| |
Collapse
|
|
40
|
Anang S, Kaushik N, Surjit M. Recent Advances Towards the Development of a Potent Antiviral Against the Hepatitis E Virus. J Clin Transl Hepatol 2018; 6:310-316. [PMID: 30271744 PMCID: PMC6160310 DOI: 10.14218/jcth.2018.00005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Revised: 02/19/2018] [Accepted: 03/23/2018] [Indexed: 12/18/2022] Open
Abstract
Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis. It also causes acute liver failure and acute-on-chronic liver failure in many patients, such as those suffering from other infections/liver injuries or organ transplant/chemotherapy recipients. Despite widespread sporadic and epidemic incidents, there is no specific treatment against HEV, justifying an urgent need for developing a potent antiviral against it. This review summarizes the known antiviral candidates and provides an overview of the potential targets for the development of specific antivirals against HEV.
Collapse
Affiliation(s)
- Saumya Anang
- Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, Haryana, India
| | - Nidhi Kaushik
- Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, Haryana, India
| | - Milan Surjit
- Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, Haryana, India
- *Correspondence to: Milan Surjit, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, PO Box No. 04, Faridabad-121001, Haryana, India. Tel: +91-129-2876-318, Fax: +91-129-2876400, E-mail:
| |
Collapse
|
|
41
|
Parvez MK, Subbarao N. Molecular Analysis and Modeling of Hepatitis E Virus Helicase and Identification of Novel Inhibitors by Virtual Screening. BIOMED RESEARCH INTERNATIONAL 2018; 2018:5753804. [PMID: 30246023 PMCID: PMC6136533 DOI: 10.1155/2018/5753804] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Accepted: 08/07/2018] [Indexed: 12/20/2022]
Abstract
The hepatitis E virus- (HEV-) helicase as a novel drug-target was evaluated. While cell culture model was used for mutational characterization of helicase, in silico protein modeling and virtual screening were employed to identify helicase inhibitors. None of the saturation mutant replicons significantly affected RNA replication. Notably, mutants encompassing the Walker motifs replicated as wild-type, showing indispensability of nucleotides conservation in viability compared to known criticality of amino acids. A 3D modeling of HEV-helicase and screening of a compound dataset identified ten most promising inhibitors with drug likeness, notably, JFD02650, RDR03130, and HTS11136 that interacted with Walker A residues Gly975, Gly978, Ser979, and Gly980. Our model building and virtual identification of novel helicase inhibitors warrant further studies towards developing anti-HEV drugs.
Collapse
Affiliation(s)
- Mohammad K. Parvez
- Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
| | - Naidu Subbarao
- School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India
| |
Collapse
|
|
42
|
Vikram T, Kumar P. Analysis of Hepatitis E virus (HEV) X-domain structural model. Bioinformation 2018; 14:398-403. [PMID: 30262978 PMCID: PMC6143357 DOI: 10.6026/97320630014398] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Revised: 06/26/2018] [Accepted: 06/30/2018] [Indexed: 01/22/2023] Open
Abstract
Hepatitis E viral infection is now emerging as a global health concern, which needs to be addressed. Mechanism of viral replication and release is attributed by the different genomic component of HEV. However, few proteins/domain like X and Y domain remain unexplored, so we aim to explore the physiochemical, structural and functional features of HEV ORF-1 X domain. Molecular modeling of the unknown X domain was carried out using Phyre2 and Swiss Model. Active ligand binding sites were predicted using Phyre2. The X-domain protein found to be stable and acidic in nature with high thermostability and better hydrophilic property. Twelve binding sites were predicted along with putative transferase and catalytic functional activity. Homology modeling showed 10 binding sites along with Mg2+ and Zn2+ as metallic heterogen ligands binding to predicted ligand-binding sites. This study may help to decipher the role of this unexplored X-domain of HEV, thereby improving our understanding of the pathogenesis of HEV infection.
Collapse
Affiliation(s)
- Thakur Vikram
- Department of Virology, Postgraduate Institute of Medical Education and Research (PGIMER), Sec-12, Chandigarh, India
| | - Pradeep Kumar
- Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, (HP) India
| |
Collapse
|
|
43
|
Harrison L, DiCaprio E. Hepatitis E Virus: An Emerging Foodborne Pathogen. FRONTIERS IN SUSTAINABLE FOOD SYSTEMS 2018. [DOI: 10.3389/fsufs.2018.00014] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
|
|
44
|
Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription. mBio 2018; 9:mBio.00769-18. [PMID: 29739903 PMCID: PMC5941075 DOI: 10.1128/mbio.00769-18] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis-acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.
Collapse
|
|
45
|
Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing. J Virol 2018; 92:JVI.01853-17. [PMID: 29321328 DOI: 10.1128/jvi.01853-17] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Accepted: 12/19/2017] [Indexed: 01/11/2023] Open
Abstract
Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated in vitro processing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCE Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.
Collapse
|
|
46
|
Abstract
At least 20 million hepatitis E virus (HEV) infections occur annually, with >3 million symptomatic cases and ∼60,000 fatalities. Hepatitis E is generally self-limiting, with a case fatality rate of 0.5-3% in young adults. However, it can cause up to 30% mortality in pregnant women in the third trimester and can become chronic in immunocompromised individuals, such as those receiving organ transplants or chemotherapy and individuals with HIV infection. HEV is transmitted primarily via the faecal-oral route and was previously thought to be a public health concern only in developing countries. It is now also being frequently reported in industrialized countries, where it is transmitted zoonotically or through organ transplantation or blood transfusions. Although a vaccine for HEV has been developed, it is only licensed in China. Additionally, no effective, non-teratogenic and specific treatments against HEV infections are currently available. Although progress has been made in characterizing HEV biology, the scarcity of adequate experimental platforms has hampered further research. In this Review, we focus on providing an update on the HEV life cycle. We will further discuss existing cell culture and animal models and highlight platforms that have proven to be useful and/or are emerging for studying other hepatotropic (viral) pathogens.
Collapse
Affiliation(s)
- Ila Nimgaonkar
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
| | - Qiang Ding
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
| | - Robert E Schwartz
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA
| | - Alexander Ploss
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
| |
Collapse
|
|
47
|
Abstract
Hepatitis E virus (HEV) is a globally important pathogen of acute and chronic hepatitis in humans. The HEV ORF1 gene encodes a nonstructural polyprotein, essential for RNA replication and virus infectivity. Expression and processing of ORF1 polyprotein are shown in prokaryotic and eukaryotic systems, however, its proteolysis into individual proteins is still debated. While molecular or biochemical characterization of methyltransferase, protease, hypervariable region, helicase and RNA polymerase domains in ORF1 has been achieved, the role of the X and Y domains in the HEV life cycle has only been demonstrated very recently. Clinically, detection of a number of ORF1 mutants in infected patients is implicated in disease severity, mortality and drug nonresponse. Moreover, several artificial lethal mutations in ORF1 offer a potential basis for developing live-attenuated vaccines for HEV. This article intends to present the molecular and clinical updates on the HEV ORF1 polyprotein.
Collapse
Affiliation(s)
- Mohammad Khalid Parvez
- Department of Pharmacognosy, King Saud University College of Pharmacy, Riyadh 11451, Saudi Arabia
| |
Collapse
|
|
48
|
Meng M, Lee CC. Function and Structural Organization of the Replication Protein of Bamboo mosaic virus. Front Microbiol 2017; 8:522. [PMID: 28400766 PMCID: PMC5368238 DOI: 10.3389/fmicb.2017.00522] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 03/13/2017] [Indexed: 12/17/2022] Open
Abstract
The genus Potexvirus is one of the eight genera belonging to the family Alphaflexiviridae according to the Virus Taxonomy 2015 released by International Committee on Taxonomy of Viruses (www.ictvonline.org/index.asp). Currently, the genus contains 35 known species including many agricultural important viruses, e.g., Potato virus X (PVX). Members of this genus are characterized by flexuous, filamentous virions of 13 nm in diameter and 470-580 nm in length. A potexvirus has a monopartite positive-strand RNA genome, encoding five open-reading frames (ORFs), with a cap structure at the 5' end and a poly(A) tail at the 3' end. Besides PVX, Bamboo mosaic virus (BaMV) is another potexvirus that has received intensive attention due to the wealth of knowledge on the molecular biology of the virus. In this review, we discuss the enzymatic activities associated with each of the functional domains of the BaMV replication protein, a 155-kDa polypeptide encoded by ORF1. The unique cap formation mechanism, which may be conserved across the alphavirus superfamily, is particularly addressed. The recently identified interactions between the replication protein and the plant host factors are also described.
Collapse
Affiliation(s)
- Menghsiao Meng
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Cheng-Cheng Lee
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| |
Collapse
|
|
49
|
Borkakoti J, Ahmed G, Rai A, Kar P. Report of novel H105R, D29N, V27A mutations in the methyltransferase region of the HEV genome in patients with acute liver failure. J Clin Virol 2017; 91:1-4. [PMID: 28359977 DOI: 10.1016/j.jcv.2017.03.017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Revised: 03/17/2017] [Accepted: 03/18/2017] [Indexed: 10/19/2022]
Abstract
BACKGROUND The Hepatitis E virus (HEV) has been responsible for major outbreaks in the developing countries affecting millions of people and acute sporadic hepatitis worldwide. The HEV methyltransferase is important for capping the 5'-end of the viral pregenomic RNA which is critical for viral infection. OBJECTIVES We aimed to assess the substitutional profile in the HEV methyltransferase region in patients with acute liver failure (ALF) and acute viral hepatitis (AVH) from North Indian population and associate the substitutions with the poor outcome of the disease. STUDY DESIGN HEV RNA was detected and partial region encoding the Methyltransferase domain in the HEV genome was amplified by Reverse Transcriptase(RT-PCR). Viral load of HEV was quantified utilizing Real time PCR.32 representative samples consisting of 16 AVH and 16 ALF were directly sequenced and amino acid changes were compared using Fischer's exact (two-tailed) test. RESULTS Novel mutations Valine27Alanine (V27A), Aspartate29Asparagine (D29N) and Histidine105Arginine (H105R) mutation corresponding to 107T>C, 115G>A and 341 A>G substitutions respectively were significantly (p<0.0001) obtained in 16/16(100%) ALF patients compared to none (0/16) of the AVH patients. HEV viral load and disease severity parameters corresponding to the samples with D29N and V27A mutations were significantly higher compared to the isolates lacking these mutations while the H105R mutation was associated with decreased viremia. CONCLUSION The D29N and V27A mutations had significant association with the poor outcome in ALF patients suggesting key role in enhancing HEV replication while the association of H105R mutation with decreased viremia creates interest on its antiviral aspects.
Collapse
Affiliation(s)
- Jayanta Borkakoti
- PCR Hepatitis Laboratory, Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India; Department of Biotechnology, Gauhati University, Assam, India
| | - Giasuddin Ahmed
- Department of Biotechnology, Gauhati University, Assam, India
| | - Arvind Rai
- Department of Biochemistry, National Centre for Disease Control, New Delhi, India
| | - Premashis Kar
- PCR Hepatitis Laboratory, Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India.
| |
Collapse
|
|
50
|
Parvez MK. Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity. World J Gastroenterol 2017; 23:590-602. [PMID: 28216965 PMCID: PMC5292332 DOI: 10.3748/wjg.v23.i4.590] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Revised: 12/05/2016] [Accepted: 01/04/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.
METHODS Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.
RESULTS In silico analysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.
CONCLUSION This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.
Collapse
|