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Brüggemann Y, Frericks N, Richter E, Kinast V, Steinmann E. How hepatitis E virus invades hepatocytes: the mystery of viral entry. Trends Microbiol 2025:S0966-842X(25)00111-8. [PMID: 40274493 DOI: 10.1016/j.tim.2025.03.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2025] [Revised: 03/28/2025] [Accepted: 03/31/2025] [Indexed: 04/26/2025]
Abstract
Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis globally. HEV infections can progress to chronic disease in immunocompromised individuals and may also cause extrahepatic complications. By contrast to other hepatitis viruses, HEV exhibits a broad tissue tropism, and certain genotypes have the ability to infect multiple species. The initial steps of surface attachment and host cell entry are critical steps in the viral infection cycle and serve as key determinants to establish an infection. This review summarizes the current understanding of HEV entry, focusing on molecular entry factors, such as viral receptors, and discusses differences between quasi-enveloped and non-enveloped HEV. We further cover recent developments of assay systems to study HEV entry and highlight experimental strategies to identify novel host components required for HEV entry. Advancing our understanding in these areas could help to guide the development of targeted antiviral strategies to block the early stages of HEV infection.
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Affiliation(s)
- Yannick Brüggemann
- Department of Molecular and Medical Virology, Faculty of Medicine, Ruhr University Bochum, Bochum, Germany
| | - Nicola Frericks
- Department of Molecular and Medical Virology, Faculty of Medicine, Ruhr University Bochum, Bochum, Germany
| | - Emely Richter
- Department of Molecular and Medical Virology, Faculty of Medicine, Ruhr University Bochum, Bochum, Germany
| | - Volker Kinast
- Department of Medical Microbiology and Virology, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Faculty of Medicine, Ruhr University Bochum, Bochum, Germany; German Centre for Infection Research (DZIF), External Partner Site, Bochum, Germany.
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2
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Ankavay M, Da Silva N, Pollán A, Oechslin N, Dinkelborg K, Behrendt P, Moradpour D, Gouttenoire J. Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein. JHEP Rep 2025; 7:101293. [PMID: 39991067 PMCID: PMC11847060 DOI: 10.1016/j.jhepr.2024.101293] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Revised: 11/25/2024] [Accepted: 11/28/2024] [Indexed: 02/25/2025] Open
Abstract
Background and Aims Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. Methods We exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV open reading frame 2 (ORF2) protein, corresponding to the viral capsid, allowing for the insertion of reporter sequences in a functional context. Results Short sequence insertions (5 amino acids) were tolerated at four distinct sites in the C-terminal region of the ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, with about a 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3 clones. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies. Conclusions We describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact and implications Hepatitis E virus infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing for the evaluation of antiviral drugs and neutralizing antibodies.
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Affiliation(s)
- Maliki Ankavay
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Nathalie Da Silva
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Angela Pollán
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Noémie Oechslin
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Katja Dinkelborg
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Institute for Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Patrick Behrendt
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Institute for Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Darius Moradpour
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Jérôme Gouttenoire
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
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Kim DH, Kim DY, Kim JH, Lim KB, Cho AY, Lee JB, Park SY, Song CS, Lee SW, Lee DH, Kim DG, Choi IS. Utility of hypervariable region in hepatitis E virus for genetic evolution analysis and epidemiological studies. J Gen Virol 2025; 106. [PMID: 39937581 DOI: 10.1099/jgv.0.002080] [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] [Indexed: 02/13/2025] Open
Abstract
Clinical and experimental studies have advanced our understanding of hepatitis E virus (HEV) infection; however, translating the findings to improve prevention and clinical outcomes remains challenging. Phylogenetic analyses of HEV show inconsistencies due to variations in the nucleotide regions studied. This study examined specific HEV regions to facilitate comprehensive molecular and phylogenetic analyses by examining the complete genome and commonly studied partial genome regions. We compared topological similarities between phylogenetic trees and evaluated evolutionary divergence using base substitutions and pairwise distances. The hypervariable region (HVR) showed the closest topology (Robinson-Foulds, Jaccard Robinson-Foulds and clustering information) to the complete genome and a higher mutation rate, resulting in longer branch lengths and clearer genotypic distinctions. Pairwise analysis revealed greater intra- and intergenotypic diversity in the HVR than in other regions. The higher base substitution rate and longer branch lengths of HVR suggest its key role in genotype evolution. Classifying HEV using HVR instead of the other partial genomic regions can reveal subtypes that more accurately reflect the genetic characteristics of HEV. Future research could focus on HVRs to better compare clinical symptoms and genetic features of HEV.
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Affiliation(s)
- Dong-Hwi Kim
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Da-Yoon Kim
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Jae-Hyeong Kim
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Kyu-Beom Lim
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Andrew Y Cho
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Joong-Bok Lee
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Seung-Yong Park
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Chang-Seon Song
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Sang-Won Lee
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Dong-Hun Lee
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
| | - Do-Geun Kim
- Korea Brain Research Institute (KBRI), Daegu, Republic of Korea
- Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Republic of Korea
| | - In-Soo Choi
- Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- Konkuk University Zoonotic Diseases Research Center, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
- KU Center for Animal Blood Medical Science, Konkuk University, 120 Neungdong-ro, Seoul 05029, Gwangjin-gu, Republic of Korea
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4
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Guan X, Wang T, Gao Y, Zhai H, Jiang F, Hou Q, Yang X, Wu H, Li LF, Luo Y, Li S, Sun Y, Qiu HJ, Li Y. The CP123L protein of African swine fever virus is a membrane-associated, palmitoylated protein required for viral replication. J Virol 2025; 99:e0144524. [PMID: 39714165 PMCID: PMC11784412 DOI: 10.1128/jvi.01445-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Accepted: 10/31/2024] [Indexed: 12/24/2024] Open
Abstract
African swine fever (ASF) is a highly contagious and often lethal disease caused by African swine fever virus (ASFV) in pigs. Protein palmitoylation is a prevalent posttranslational lipid modification that can modulate viral replication. In this study, we investigated the palmitoylation of ASFV proteins. The results revealed that the CP123L protein (pCP123L) of ASFV was palmitoylated at the cysteine residue at position 18 (C18). To further elucidate the functional significance of this posttranslational modification, abolishing palmitoylation through a cysteine-to-serine mutation at C18 (C18S) of pCP123L (pCP123L/C18S) or treatment with 2-bromopalmitate (2-BP), a palmitoylation inhibitor, led to altered cytomembrane localization and migration rate of pCP123L. Furthermore, depalmitoylation achieved through 2-BP treatment significantly suppressed ASFV replication and exerted a profound impact on virus budding. Remarkably, blocking pCP123L palmitoylation via the C18S mutation resulted in decreased replication of ASFV. Our study represents the first evidence for the presence of palmitoylation in ASFV proteins and underscores its crucial role in viral replication. IMPORTANCE African swine fever (ASF) poses a significant threat to the global pig industry. The causative agent of ASF is African swine fever virus (ASFV), which encodes more than 165 proteins. Protein palmitoylation, a common posttranslational lipid modification, can modulate viral infection. To date, the ASFV proteins that undergo palmitoylation and their impacts on viral replication remain elusive. In this study, the CP123L protein (pCP123L) of ASFV was identified as a palmitoylated protein, and the cysteine residue at position 18 of pCP123L is responsible for its palmitoylation. Notably, our findings demonstrate that palmitoylation plays significant roles in ASFV protein functions and facilitates viral replication.
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Affiliation(s)
- Xiangyu Guan
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Tao Wang
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Yuxuan Gao
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Huanjie Zhai
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Fengwei Jiang
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Qinghe Hou
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Xiaoke Yang
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Hongxia Wu
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Lian-Feng Li
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Yuzi Luo
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Su Li
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Yuan Sun
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Hua-Ji Qiu
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
| | - Yongfeng Li
- State Key Laboratory for Animal Disease Control and Prevention, CAAS Harbin Veterinary Research Institute, Harbin, Heilongjiang, China
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Liu X, Liu T, Shao Z, Xiong X, Qi S, Guan J, Wang M, Tang YD, Feng Z, Wang L, Yin X. Palmitoylation-dependent association with Annexin II directs hepatitis E virus ORF3 sorting into vesicles and quasi-enveloped virions. Proc Natl Acad Sci U S A 2025; 122:e2418751122. [PMID: 39793027 PMCID: PMC11725905 DOI: 10.1073/pnas.2418751122] [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: 09/19/2024] [Accepted: 11/25/2024] [Indexed: 01/12/2025] Open
Abstract
Historically considered to be nonenveloped, hepatitis E virus (HEV), an important zoonotic pathogen, has recently been discovered to egress from infected cells as quasi-enveloped virions. These quasi-enveloped virions circulating in the blood are resistant to neutralizing antibodies, thereby facilitating the stealthy spread of infection. Despite abundant evidence of the essential role of the HEV-encoded ORF3 protein in quasi-enveloped virus formation, the underlying mechanism remains unclear. Here, we demonstrate that the HEV ORF3 protein possesses an inherent capacity for self-secretion and that palmitoylation at two cysteine residues within the ORF3 N-terminal region is essential for its secretion and quasi-enveloped virus formation. We further found that only palmitoylated ORF3 proteins hijacked Annexin II for transport to the cytoskeleton and are then directed into multivesicular bodies through the nSMase-endosomal sorting complexes required for transport-III pathway for secretion. Finally, we show that infection of gerbils with HEV mutants harboring mutations at palmitoylation sites within ORF3 showed no fecal viral shedding but competent replication in the liver. Our study fills a gap in the understanding of the assembly and release of quasi-enveloped virions mediated by ORF3 and offers the potential for designing therapeutic strategies to control HEV infection.
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Affiliation(s)
- Xing Liu
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Tianxu Liu
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing100191, China
| | - Zhen Shao
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Xiaoyan Xiong
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
- Department of Animal Sciences, Quantitative Veterinary Epidemiology Group, Wageningen University, Wageningen6700 AH, The Netherlands
| | - Shuhui Qi
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Junyong Guan
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Menghang Wang
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Yan-Dong Tang
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
| | - Zongdi Feng
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, OH43205
- Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH43205
| | - Lin Wang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing100191, China
| | - Xin Yin
- Division of Livestock Infectious Diseases, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, China
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Wang Y, Ma H, Zhang B, Li S, Lu B, Qi Y, Liu T, Wang H, Kang X, Liang Y, Kong E, Cao L, Zhou B. Protein palmitoylation in hepatic diseases: Functional insights and therapeutic strategies. J Adv Res 2024:S2090-1232(24)00619-2. [PMID: 39732335 DOI: 10.1016/j.jare.2024.12.041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2024] [Revised: 12/24/2024] [Accepted: 12/25/2024] [Indexed: 12/30/2024] Open
Abstract
BACKGROUND Liver pathologies represent a spectrum of conditions ranging from fatty liver to the aggressive hepatocellular carcinoma (HCC), as well as parasitic infections, which collectively pose substantial global health challenges. S-palmitoylation (commonly referred to as palmitoylation), a post-translational modification (PTM) characterized by the covalent linkage of a 16-carbon palmitic acid (PA) chain to specific cysteine residues on target proteins, plays a pivotal role in diverse cellular functions and is intimately associated with the liver's physiological and pathological states. AIM OF REVIEW This study aims to elucidate how protein palmitoylation affects liver disease pathophysiology and evaluates its potential as a target for diagnostic and therapeutic interventions. KEY SCIENTIFIC CONCEPTS OF REVIEW Recent studies have identified the key role of protein palmitoylation in regulating the development and progression of liver diseases. This review summarizes the intricate mechanisms by which protein palmitoylation modulates the pathophysiological processes of liver diseases and explores the potential of targeting protein palmitoylation modifications or the enzymes regulating this modification as prospective diagnostic biomarkers and therapeutic targets.
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Affiliation(s)
- Ying Wang
- The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, PR China; Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Haoyuan Ma
- Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Bowen Zhang
- Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Sainan Li
- Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Beijia Lu
- Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Yingcheng Qi
- Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Tingting Liu
- Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Hua Wang
- Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Medical University, Hefei, Anhui, PR China.
| | - Xiaohong Kang
- The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, PR China.
| | - Yinming Liang
- Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China.
| | - Eryan Kong
- The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, PR China; Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China.
| | - Liu Cao
- Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China.
| | - Binhui Zhou
- The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, PR China; Institute of Psychiatry and Neuroscience of Xinxiang Medical University, Xinxiang, Henan, PR China; Laboratory of Genetic Regulators in the Immune System, School of Medical Technology, Xinxiang Medical University, Xinxiang, Henan, PR China.
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Klöhn M, Burkard T, Janzen J, Haase JA, Gömer A, Fu R, Ssebyatika G, Nocke MK, Brown RJP, Krey T, Dao Thi VL, Kinast V, Brüggemann Y, Todt D, Steinmann E. Targeting cellular cathepsins inhibits hepatitis E virus entry. Hepatology 2024; 80:1239-1251. [PMID: 38728662 PMCID: PMC11486972 DOI: 10.1097/hep.0000000000000912] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Accepted: 04/02/2024] [Indexed: 05/12/2024]
Abstract
BACKGROUND AND AIMS HEV is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potential of cellular protease during HEV infection. APPROACH AND RESULTS Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC 50 of ~0.02 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.
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Affiliation(s)
- Mara Klöhn
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Thomas Burkard
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Juliana Janzen
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Jil A. Haase
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - André Gömer
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Rebecca Fu
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany
- Heidelberg Biosciences International Graduate School (HBIGS), Heidelberg, Germany
| | - George Ssebyatika
- Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, Luebeck, Germany
| | - Maximilian K. Nocke
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Richard J. P. Brown
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Thomas Krey
- Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, Luebeck, Germany
- German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems
- Institute of Virology, Hannover Medical School, Hannover, Germany
- Excellence Cluster 2155 RESIST, Hannover Medical School, Hannover, Germany
- Centre for Structural Systems Biology (CSSB), Hamburg, Germany
| | - Viet Loan Dao Thi
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
| | - Volker Kinast
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
- Department of Medical Microbiology and Virology, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Yannick Brüggemann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Daniel Todt
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
- European Virus Bioinformatics Center (EVBC), Jena, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
- German Centre for Infection Research (DZIF), External Partner Site, Bochum, Germany
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8
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Brüggemann Y, Klöhn M, Wedemeyer H, Steinmann E. Hepatitis E virus: from innate sensing to adaptive immune responses. Nat Rev Gastroenterol Hepatol 2024; 21:710-725. [PMID: 39039260 DOI: 10.1038/s41575-024-00950-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 05/29/2024] [Indexed: 07/24/2024]
Abstract
Hepatitis E virus (HEV) infections are a major cause of acute viral hepatitis in humans worldwide. In immunocompetent individuals, the majority of HEV infections remain asymptomatic and lead to spontaneous clearance of the virus, and only a minority of individuals with infection (5-16%) experience symptoms of acute viral hepatitis. However, HEV infections can cause up to 30% mortality in pregnant women, become chronic in immunocompromised patients and cause extrahepatic manifestations. A growing body of evidence suggests that the host immune response to infection with different HEV genotypes is a critical determinant of distinct HEV infection outcomes. In this Review, we summarize key components of the innate and adaptive immune responses to HEV, including the underlying immunological mechanisms of HEV associated with acute and chronic liver failure and interactions between T cell and B cell responses. In addition, we discuss the current status of vaccines against HEV and raise outstanding questions regarding the immune responses induced by HEV and treatment of the disease, highlighting areas for future investigation.
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Affiliation(s)
- Yannick Brüggemann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Mara Klöhn
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Heiner Wedemeyer
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
- German Center for Infection Research (DZIF), Partner Sites Hannover-Braunschweig, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany.
- German Center for Infection Research (DZIF), External Partner Site, Bochum, Germany.
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9
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Quirino A, Marascio N, Branda F, Ciccozzi A, Romano C, Locci C, Azzena I, Pascale N, Pavia G, Matera G, Casu M, Sanna D, Giovanetti M, Ceccarelli G, Alaimo di Loro P, Ciccozzi M, Scarpa F, Maruotti A. Viral Hepatitis: Host Immune Interaction, Pathogenesis and New Therapeutic Strategies. Pathogens 2024; 13:766. [PMID: 39338957 PMCID: PMC11435051 DOI: 10.3390/pathogens13090766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2024] [Revised: 08/30/2024] [Accepted: 09/02/2024] [Indexed: 09/30/2024] Open
Abstract
Viral hepatitis is a major cause of liver illness worldwide. Despite advances in the understanding of these infections, the pathogenesis of hepatitis remains a complex process driven by intricate interactions between hepatitis viruses and host cells at the molecular level. This paper will examine in detail the dynamics of these host-pathogen interactions, highlighting the key mechanisms that regulate virus entry into the hepatocyte, their replication, evasion of immune responses, and induction of hepatocellular damage. The unique strategies employed by different hepatitis viruses, such as hepatitis B, C, D, and E viruses, to exploit metabolic and cell signaling pathways to their advantage will be discussed. At the same time, the innate and adaptive immune responses put in place by the host to counter viral infection will be analyzed. Special attention will be paid to genetic, epigenetic, and environmental factors that modulate individual susceptibility to different forms of viral hepatitis. In addition, this work will highlight the latest findings on the mechanisms of viral persistence leading to the chronic hepatitis state and the potential implications for the development of new therapeutic strategies. Fully understanding the complex host-pathogen interactions in viral hepatitis is crucial to identifying new therapeutic targets, developing more effective approaches for treatment, and shedding light on the mechanisms underlying progression to more advanced stages of liver damage.
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Affiliation(s)
- Angela Quirino
- Unit of Clinical Microbiology, Department of Health Sciences, “Magna Græcia” University of Catanzaro “Renato Dulbecco” Teaching Hospital, 88100 Catanzaro, Italy; (A.Q.); (N.M.); (G.P.); (G.M.)
| | - Nadia Marascio
- Unit of Clinical Microbiology, Department of Health Sciences, “Magna Græcia” University of Catanzaro “Renato Dulbecco” Teaching Hospital, 88100 Catanzaro, Italy; (A.Q.); (N.M.); (G.P.); (G.M.)
| | - Francesco Branda
- Unit of Medical Statistics and Molecular Epidemiology, Università Campus Bio-Medico di Roma, 00128 Rome, Italy; (C.R.); (M.C.)
| | - Alessandra Ciccozzi
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (A.C.); (C.L.); (D.S.); (F.S.)
| | - Chiara Romano
- Unit of Medical Statistics and Molecular Epidemiology, Università Campus Bio-Medico di Roma, 00128 Rome, Italy; (C.R.); (M.C.)
| | - Chiara Locci
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (A.C.); (C.L.); (D.S.); (F.S.)
- Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; (I.A.); (N.P.); (M.C.)
| | - Ilenia Azzena
- Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; (I.A.); (N.P.); (M.C.)
| | - Noemi Pascale
- Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; (I.A.); (N.P.); (M.C.)
- Department of Chemical Physical Mathematical and Natural Sciences, University of Sassari, 07100 Sassari, Italy
| | - Grazia Pavia
- Unit of Clinical Microbiology, Department of Health Sciences, “Magna Græcia” University of Catanzaro “Renato Dulbecco” Teaching Hospital, 88100 Catanzaro, Italy; (A.Q.); (N.M.); (G.P.); (G.M.)
| | - Giovanni Matera
- Unit of Clinical Microbiology, Department of Health Sciences, “Magna Græcia” University of Catanzaro “Renato Dulbecco” Teaching Hospital, 88100 Catanzaro, Italy; (A.Q.); (N.M.); (G.P.); (G.M.)
| | - Marco Casu
- Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; (I.A.); (N.P.); (M.C.)
| | - Daria Sanna
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (A.C.); (C.L.); (D.S.); (F.S.)
| | - Marta Giovanetti
- Department of Sciences and Technologies for Sustainable Development and One Health, Università Campus Bio-Medico di Roma, 00128 Rome, Italy;
- Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte 30190-002, MG, Brazil
- Climate Amplified Diseases and Epidemics (CLIMADE), Brasilia 70070-130, GO, Brazil
| | - Giancarlo Ceccarelli
- Department of Public Health and Infectious Diseases, University Hospital Policlinico Umberto I, Sapienza University of Rome, 00161 Rome, Italy;
| | | | - Massimo Ciccozzi
- Unit of Medical Statistics and Molecular Epidemiology, Università Campus Bio-Medico di Roma, 00128 Rome, Italy; (C.R.); (M.C.)
| | - Fabio Scarpa
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (A.C.); (C.L.); (D.S.); (F.S.)
| | - Antonello Maruotti
- Department GEPLI, Libera Università Maria Ss Assunta, 00193 Rome, Italy;
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10
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Ferrié M, Alexandre V, Montpellier C, Bouquet P, Tubiana T, Mézière L, Ankavay M, Bentaleb C, Dubuisson J, Bressanelli S, Aliouat-Denis CM, Rouillé Y, Cocquerel L. The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus. Cell Mol Life Sci 2024; 81:335. [PMID: 39117755 PMCID: PMC11335258 DOI: 10.1007/s00018-024-05367-0] [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: 10/13/2023] [Revised: 07/03/2024] [Accepted: 07/15/2024] [Indexed: 08/10/2024]
Abstract
Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.
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Affiliation(s)
- Martin Ferrié
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Virginie Alexandre
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Claire Montpellier
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Peggy Bouquet
- Unit of Clinical Microbiology, Institut Pasteur de Lille, Lille, F-59000, France
| | - Thibault Tubiana
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Léa Mézière
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Maliki Ankavay
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
- Division of Gastroenterology and Hepatology, Institute of Microbiology, Lausanne, Switzerland
| | - Cyrine Bentaleb
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Jean Dubuisson
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Stéphane Bressanelli
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Cécile-Marie Aliouat-Denis
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Yves Rouillé
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France
| | - Laurence Cocquerel
- Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL- Center for Infection and Immunity of Lille, Lille, F-59000, France.
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11
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Jiao F, Zhao Y, Zhou G, Meng C, Wang L, Wu S, Li J, Cao L, Zhou B, Luo Y, Jiao H. Multiple Functions of Hepatitis E Virus ORF3. Microorganisms 2024; 12:1405. [PMID: 39065173 PMCID: PMC11278674 DOI: 10.3390/microorganisms12071405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Revised: 07/05/2024] [Accepted: 07/08/2024] [Indexed: 07/28/2024] Open
Abstract
Hepatitis E (Hepatitis E, HE) is an acute and chronic infectious hepatitis caused by hepatitis E virus (Hepatitis E Virus, HEV) infection, which is responsible for most acute hepatitis in the world and is a significant public health problem. The pathogen, HEV, has three Open Reading Frames (ORFs) ORF1, ORF2, and ORF3, each of which has a different function. Most of the current research is focused on ORF1 and ORF2, while the research on ORF3 is still relatively small. To provide more ideas for the study of HEV pathogenesis and the prevention and treatment of HE, this paper reviews the effects of ORF3 on the ERK pathway, growth factors, immune response, and virus release.
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Affiliation(s)
- Fengyuan Jiao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Yu Zhao
- Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Genitic Resources and Germplasm Innovation in Karst Region, Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Science, Guiyang 550005, China;
| | - Gengxu Zhou
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Chi Meng
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Lingjie Wang
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Shengping Wu
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Jixiang Li
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Liting Cao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Bo Zhou
- Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Yujinxiang Street 573, Changchun 130102, China;
| | - Yichen Luo
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Hanwei Jiao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
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12
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Ding Q, Hu B, Yao X, Gan M, Chen D, Zhang N, Wei J, Cai K, Zheng Z. Prevalence and molecular characterization of hepatitis E virus (HEV) from wild rodents in Hubei Province, China. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2024; 121:105602. [PMID: 38734397 DOI: 10.1016/j.meegid.2024.105602] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 04/28/2024] [Accepted: 05/05/2024] [Indexed: 05/13/2024]
Abstract
Hepatitis E, caused by the hepatitis E virus (HEV), is a global public health issue. Low similarity between the gene sequences of mouse and human HEV led to the belief that the risk of human infection was low. Recent reports of chronic and acute hepatitis E caused by murine HEV infection in humans in Hong Kong have raised global concerns. Therefore, it is crucial to investigate the epidemiology and prevalence of HEV in China. We comprehensively analyzed different rodent HEV strains to understand rocahepevirus occurrence in Hubei Province, China. The HEV positivity rate for was 6.43% (73/1136). We identified seven near-full-length rocahepevirus strains and detected rat HEV antigens in tissues from different mouse species. HEV has extensive tissue tropism and a high viral load in the liver. We highlight the genetic diversity of HEVs in rodents and underscore the importance of paying attention to their variation and evolution.
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Affiliation(s)
- Qingwen Ding
- Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Bing Hu
- Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei 430079, China
| | - Xuan Yao
- Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei 430079, China
| | - Min Gan
- Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Dan Chen
- College of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430024, China
| | - Nailou Zhang
- Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Jinbo Wei
- Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Kun Cai
- Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei 430079, China.
| | - Zhenhua Zheng
- Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China.
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Shahini E, Argentiero A, Andriano A, Losito F, Maida M, Facciorusso A, Cozzolongo R, Villa E. Hepatitis E Virus: What More Do We Need to Know? MEDICINA (KAUNAS, LITHUANIA) 2024; 60:998. [PMID: 38929615 PMCID: PMC11205503 DOI: 10.3390/medicina60060998] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Revised: 06/14/2024] [Accepted: 06/17/2024] [Indexed: 06/28/2024]
Abstract
Hepatitis E virus (HEV) infection is typically a self-limiting, acute illness that spreads through the gastrointestinal tract but replicates in the liver. However, chronic infections are possible in immunocompromised individuals. The HEV virion has two shapes: exosome-like membrane-associated quasi-enveloped virions (eHEV) found in circulating blood or in the supernatant of infected cell cultures and non-enveloped virions ("naked") found in infected hosts' feces and bile to mediate inter-host transmission. Although HEV is mainly spread via enteric routes, it is unclear how it penetrates the gut wall to reach the portal bloodstream. Both virion types are infectious, but they infect cells in different ways. To develop personalized treatment/prevention strategies and reduce HEV impact on public health, it is necessary to decipher the entry mechanism for both virion types using robust cell culture and animal models. The contemporary knowledge of the cell entry mechanism for these two HEV virions as possible therapeutic target candidates is summarized in this narrative review.
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Affiliation(s)
- Endrit Shahini
- Gastroenterology Unit, National Institute of Gastroenterology-IRCCS “Saverio de Bellis”, Castellana Grotte, 70013 Bari, Italy; (F.L.); (R.C.)
| | | | - Alessandro Andriano
- Department of Precision and Regenerative Medicine and Ionian Area, University of Bari Aldo Moro Medical School, 70124 Bari, Italy;
| | - Francesco Losito
- Gastroenterology Unit, National Institute of Gastroenterology-IRCCS “Saverio de Bellis”, Castellana Grotte, 70013 Bari, Italy; (F.L.); (R.C.)
| | - Marcello Maida
- Gastroenterology and Endoscopy Unit, S. Elia-Raimondi Hospital, 93100 Caltanissetta, Italy;
| | - Antonio Facciorusso
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy;
| | - Raffaele Cozzolongo
- Gastroenterology Unit, National Institute of Gastroenterology-IRCCS “Saverio de Bellis”, Castellana Grotte, 70013 Bari, Italy; (F.L.); (R.C.)
| | - Erica Villa
- Gastroenterology Unit, CHIMOMO Department, University of Modena & Reggio Emilia, Via del Pozzo 71, 41121 Modena, Italy
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14
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Paronetto O, Allioux C, Diméglio C, Lobjois L, Jeanne N, Ranger N, Boineau J, Pucelle M, Demmou S, Abravanel F, Chapuy-Regaud S, Izopet J, Lhomme S. Characterization of virus‒host recombinant variants of the hepatitis E virus. J Virol 2024; 98:e0029524. [PMID: 38712945 PMCID: PMC11237545 DOI: 10.1128/jvi.00295-24] [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: 02/13/2024] [Accepted: 04/09/2024] [Indexed: 05/08/2024] Open
Abstract
Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.
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Affiliation(s)
- Olivia Paronetto
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
| | - Claire Allioux
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
| | - Chloé Diméglio
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Lhorane Lobjois
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
| | - Nicolas Jeanne
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Noémie Ranger
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Jérôme Boineau
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Mélanie Pucelle
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Sofia Demmou
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Florence Abravanel
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Sabine Chapuy-Regaud
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Jacques Izopet
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
| | - Sébastien Lhomme
- Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), UMR 5051 (CNRS), UMR 1291 (INSERM), Université Toulouse III-Paul Sabatier, Toulouse, France
- Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, Toulouse, France
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15
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Li X, Sun X, Pinpin J, Zhao Q, Sun Y. Multifunctional ORF3 protein of hepatitis E virus. J Med Virol 2024; 96:e29691. [PMID: 38783788 DOI: 10.1002/jmv.29691] [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: 02/02/2024] [Revised: 04/23/2024] [Accepted: 05/14/2024] [Indexed: 05/25/2024]
Abstract
Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is transmitted primarily through the fecal-oral route and can cause acute hepatitis in humans. Since HEV was identified as a zoonotic pathogen, different species of HEV strains have been globally identified from various hosts, leading to an expanding range of hosts. The HEV genome consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. The ORF3 protein is the smallest but has many functions in HEV release and pathogenesis. In this review, we systematically summarize recent progress in understanding the functions of the HEV ORF3 protein in virion release, biogenesis of quasi-enveloped viruses, antigenicity, and host environmental regulation. This review will help us to understand HEV replication and pathogenesis mechanisms better.
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Affiliation(s)
- Xiaoxuan Li
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
| | - Xuwen Sun
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
| | - Ji Pinpin
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
| | - Qin Zhao
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
| | - Yani Sun
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
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16
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Sheng Y, Deng Y, Li X, Ji P, Sun X, Liu B, Zhu J, Zhao J, Nan Y, Zhou EM, Hiscox JA, Stewart JP, Sun Y, Zhao Q. Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release. J Virol 2024; 98:e0164923. [PMID: 38548704 PMCID: PMC11019958 DOI: 10.1128/jvi.01649-23] [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: 01/10/2024] [Accepted: 03/06/2024] [Indexed: 04/17/2024] Open
Abstract
Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.
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Affiliation(s)
- Yamin Sheng
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Yingying Deng
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Xiaoxuan Li
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Pinpin Ji
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Xuwen Sun
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Baoyuan Liu
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jiahong Zhu
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jiakai Zhao
- 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
| | - En-Min Zhou
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Julian A. Hiscox
- Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom
| | - James P. Stewart
- Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom
| | - Yani Sun
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Qin Zhao
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
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17
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Bai M, Gallen E, Memarzadeh S, Howie J, Gao X, Kuo CWS, Brown E, Swingler S, Wilson SJ, Shattock MJ, France DJ, Fuller W. Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation. PLoS One 2024; 19:e0299665. [PMID: 38512906 PMCID: PMC10956751 DOI: 10.1371/journal.pone.0299665] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Accepted: 02/14/2024] [Indexed: 03/23/2024] Open
Abstract
Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.
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Affiliation(s)
- Mingjie Bai
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Emily Gallen
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Sarah Memarzadeh
- School of Chemistry, University of Glasgow, Glasgow, United Kingdom
| | - Jacqueline Howie
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Xing Gao
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Chien-Wen S. Kuo
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Elaine Brown
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Simon Swingler
- Medical Research Council–University of Glasgow Centre for Virus Research, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Sam J. Wilson
- Medical Research Council–University of Glasgow Centre for Virus Research, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Michael J. Shattock
- School of Cardiovascular and Metabolic Medicine & Sciences, King’s College London, London, United Kingdom
| | - David J. France
- School of Chemistry, University of Glasgow, Glasgow, United Kingdom
| | - William Fuller
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
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18
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Glitscher M, Spannaus IM, Behr F, Murra RO, Woytinek K, Bender D, Hildt E. The Protease Domain in HEV pORF1 Mediates the Replicase's Localization to Multivesicular Bodies and Its Exosomal Release. Cell Mol Gastroenterol Hepatol 2024; 17:589-605. [PMID: 38190941 PMCID: PMC10900777 DOI: 10.1016/j.jcmgh.2024.01.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2023] [Revised: 01/03/2024] [Accepted: 01/03/2024] [Indexed: 01/10/2024]
Abstract
BACKGROUND A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. METHODS Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase-polymerase chain reaction to analyze separated particles in more detail. RESULTS We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein's 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. CONCLUSIONS Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.
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Affiliation(s)
- Mirco Glitscher
- Department of Virology, Paul-Ehrlich-Institute, Langen, Germany
| | | | - Fabiane Behr
- Department of Virology, Paul-Ehrlich-Institute, Langen, Germany
| | | | | | - Daniela Bender
- Department of Virology, Paul-Ehrlich-Institute, Langen, Germany
| | - Eberhard Hildt
- Department of Virology, Paul-Ehrlich-Institute, Langen, Germany.
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19
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Oechslin N, Da Silva N, Ankavay M, Moradpour D, Gouttenoire J. A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication. Proc Natl Acad Sci U S A 2023; 120:e2307423120. [PMID: 38109552 PMCID: PMC10756275 DOI: 10.1073/pnas.2307423120] [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: 05/08/2023] [Accepted: 11/17/2023] [Indexed: 12/20/2023] Open
Abstract
Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.
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Affiliation(s)
- Noémie Oechslin
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne1011, Switzerland
| | - Nathalie Da Silva
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne1011, Switzerland
| | - Maliki Ankavay
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne1011, Switzerland
| | - Darius Moradpour
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne1011, Switzerland
| | - Jérôme Gouttenoire
- Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne1011, Switzerland
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20
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Das A, Rivera-Serrano EE, Yin X, Walker CM, Feng Z, Lemon SM. Cell entry and release of quasi-enveloped human hepatitis viruses. Nat Rev Microbiol 2023; 21:573-589. [PMID: 37185947 PMCID: PMC10127183 DOI: 10.1038/s41579-023-00889-z] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/22/2023] [Indexed: 05/17/2023]
Abstract
Infectious hepatitis type A and type E are caused by phylogenetically distinct single-stranded, positive-sense RNA viruses that were once considered to be non-enveloped. However, studies show that both are released nonlytically from hepatocytes as 'quasi-enveloped' virions cloaked in host membranes. These virion types predominate in the blood of infected individuals and mediate virus spread within the liver. They lack virally encoded proteins on their surface and are resistant to neutralizing anti-capsid antibodies induced by infection, yet they efficiently enter cells and initiate new rounds of virus replication. In this Review, we discuss the mechanisms by which specific peptide sequences in the capsids of these quasi-enveloped virions mediate their endosomal sorting complexes required for transport (ESCRT)-dependent release from hepatocytes through multivesicular endosomes, what is known about how they enter cells, and the impact of capsid quasi-envelopment on host immunity and pathogenesis.
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Affiliation(s)
- Anshuman Das
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Lentigen Technology, Inc., Gaithersburg, MD, USA
| | - Efraín E Rivera-Serrano
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Biology, Elon University, Elon, NC, USA
| | - Xin Yin
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China
| | - Christopher M Walker
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA
- Department of Paediatrics, The Ohio State University College of Medicine, Columbus, OH, USA
| | - Zongdi Feng
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
- Department of Paediatrics, The Ohio State University College of Medicine, Columbus, OH, USA.
| | - Stanley M Lemon
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
- Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
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21
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Takahashi M, Kunita S, Nishizawa T, Ohnishi H, Primadharsini PP, Nagashima S, Murata K, Okamoto H. Infection Dynamics and Genomic Mutations of Hepatitis E Virus in Naturally Infected Pigs on a Farrow-to-Finish Farm in Japan: A Survey from 2012 to 2021. Viruses 2023; 15:1516. [PMID: 37515202 PMCID: PMC10385168 DOI: 10.3390/v15071516] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2023] [Revised: 07/05/2023] [Accepted: 07/06/2023] [Indexed: 07/30/2023] Open
Abstract
Hepatitis E virus (HEV) causes acute or chronic hepatitis in humans. Pigs are the primary reservoir for zoonotic HEV genotypes 3 and 4 worldwide. This study investigated the infection dynamics and genomic mutations of HEV in domestic pigs on a farrow-to-finish pig farm in Japan between 2012 and 2021. A high prevalence of anti-HEV IgG antibodies was noted among pigs on this farm in 2012, when the survey started, and persisted for at least nine years. During 2012-2021, HEV RNA was detected in both serum and fecal samples, indicating active viral replication. Environmental samples, including slurry samples in manure pits, feces on the floor, floor and wall swabs in pens, and dust samples, also tested positive for HEV RNA, suggesting potential sources of infection within the farm environment. Indeed, pigs raised in HEV-contaminated houses had a higher rate of HEV infection than those in an HEV-free house. All 104 HEV strains belonged to subgenotype 3b, showing a gradual decrease in nucleotide identities over time. The 2012 (swEJM1201802S) and 2021 (swEJM2100729F) HEV strains shared 97.9% sequence identity over the entire genome. Importantly, the swEJM2100729F strain efficiently propagated in human hepatoma cells, demonstrating its infectivity. These findings contribute to our understanding of the prevalence, transmission dynamics, and genetic characteristics of HEV in domestic pigs, emphasizing the potential risks associated with HEV infections and are crucial for developing effective strategies to mitigate the risk of HEV infection in both animals and humans.
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Affiliation(s)
- Masaharu Takahashi
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Satoshi Kunita
- Center for Experimental Medicine, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Tsutomu Nishizawa
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Hiroshi Ohnishi
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Putu Prathiwi Primadharsini
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Shigeo Nagashima
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Kazumoto Murata
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Hiroaki Okamoto
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
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22
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Ma X, Xia Q, Liu K, Wu Z, Li C, Xiao C, Dong N, Hameed M, Anwar MN, Li Z, Shao D, Li B, Qiu Y, Wei J, Ma Z. Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence. J Virol 2023; 97:e0038223. [PMID: 37289075 PMCID: PMC10308905 DOI: 10.1128/jvi.00382-23] [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: 03/14/2023] [Accepted: 05/13/2023] [Indexed: 06/09/2023] Open
Abstract
Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.
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Affiliation(s)
- Xiaochun Ma
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
- College of Veterinary Medicine, Shandong Vocational Animal Science and Veterinary College, Weifang, People’s Republic of China
| | - Qiqi Xia
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Ke Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Zhuanchang Wu
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, School of Basic Medical Science, Shandong University, Jinan, People’s Republic of China
| | - Chenxi Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Changguang Xiao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Nihua Dong
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Muddassar Hameed
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Muhammad Naveed Anwar
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
- Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
| | - Zongjie Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Donghua Shao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Beibei Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Yafeng Qiu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Jianchao Wei
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Zhiyong Ma
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
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23
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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: 12] [Impact Index Per Article: 6.0] [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.
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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
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24
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Bhise N, Agarwal M, Thakur N, Akshay PS, Cherian S, Lole K. Repurposing of artesunate, an antimalarial drug, as a potential inhibitor of hepatitis E virus. Arch Virol 2023; 168:147. [PMID: 37115342 PMCID: PMC10141844 DOI: 10.1007/s00705-023-05770-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2022] [Accepted: 03/29/2023] [Indexed: 04/29/2023]
Abstract
Hepatitis E virus (HEV) is endemic in several developing countries of Africa and Asia. It mainly causes self-limiting waterborne infections, in either sporadic or outbreak form. Recently, HEV was shown to cause chronic infections in immunosuppressed individuals. Ribavirin and interferon, the current off-label treatment options for hepatitis E, have several side effects. Hence, there is a need for new drugs. We evaluated the antimalarial drug artesunate (ART) against genotype 1 HEV (HEV-1) and HEV-3 using a virus-replicon-based cell culture system. ART exhibited 59% and 43% inhibition of HEV-1 and HEV-3, respectively, at the highest nontoxic concentration. Computational molecular docking analysis showed that ART can bind to the helicase active site (affinity score, -7.4 kcal/mol), indicating its potential to affect ATP hydrolysis activity. An in vitro ATPase activity assay of the helicase indeed showed 24% and 55% inhibition at 19.5 µM (EC50) and 78 µM concentrations of ART, respectively. Since ATP is a substrate of RNA-dependent RNA polymerase (RdRp) as well, we evaluated the effect of ART on the enzymatic activity of the viral polymerase. Interestingly, ART showed 26% and 40% inhibition of the RdRp polymerase activity at 19.5 µM and 78 µM concentrations of ART, respectively. It could be concluded from these findings that ART inhibited replication of both HEV-1 and HEV-3 by directly targeting the activities of the viral enzymes helicase and RdRp. Considering that ART is known to be safe in pregnant women, we think this antimalarial drug deserves further evaluation in animal models.
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Affiliation(s)
- Neha Bhise
- Hepatitis Group, Indian Council of Medical Research-National Institute of Virology, Microbial Containment Complex, Pune, India
| | - Megha Agarwal
- Bioinformatics and Data Management Group, Indian Council of Medical Research-National Institute of Virology, Dr. Ambedkar Road, Pune, India
| | - Nidhi Thakur
- Hepatitis Group, Indian Council of Medical Research-National Institute of Virology, Microbial Containment Complex, Pune, India
| | - P S Akshay
- Hepatitis Group, Indian Council of Medical Research-National Institute of Virology, Microbial Containment Complex, Pune, India
| | - Sarah Cherian
- Bioinformatics and Data Management Group, ICMR-National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune, 411001, India.
| | - Kavita Lole
- Hepatitis Group, ICMR-National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune, 411021, India.
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25
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Oechslin N, Ankavay M, Moradpour D, Gouttenoire J. Expanding the Hepatitis E Virus Toolbox: Selectable Replicons and Recombinant Reporter Genomes. Viruses 2023; 15:v15040869. [PMID: 37112849 PMCID: PMC10147066 DOI: 10.3390/v15040869] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 03/27/2023] [Accepted: 03/28/2023] [Indexed: 03/31/2023] Open
Abstract
Hepatitis E virus (HEV) has received relatively little attention for decades although it is now considered as one of the most frequent causes of acute hepatitis worldwide. Our knowledge of this enterically-transmitted, positive-strand RNA virus and its life cycle remains scarce but research on HEV has gained momentum more recently. Indeed, advances in the molecular virology of hepatitis E, including the establishment of subgenomic replicons and infectious molecular clones, now allow study of the entire viral life cycle and to explore host factors required for productive infection. Here, we provide an overview on currently available systems, with an emphasis on selectable replicons and recombinant reporter genomes. Furthermore, we discuss the challenges in developing new systems which should enable to further investigate this widely distributed and important pathogen.
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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.
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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.
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27
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Wang L, Wang Y, Zhuang H. Puzzles for Hepatitis E Virus. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1417:247-256. [PMID: 37223871 DOI: 10.1007/978-981-99-1304-6_17] [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 an important but understudied virus that has been the major cause of acute viral hepatitis worldwide. In recent decades, our understanding of this neglected virus has changed greatly: novel forms of viral proteins and their functions have been discovered; HEV can transmit via blood transfusion and organ transplantation; HEV can infect many animal species and the number is still increasing; HEV can induce chronic hepatitis and extra-hepatic manifestations. However, we are short of effective treatment measures to counter the virus. In this chapter we tend to briefly introduce the puzzles and major knowledge gaps existed in the field of HEV research.
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Affiliation(s)
- Lin Wang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
| | - Youchun Wang
- Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Kunming, China
| | - Hui Zhuang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
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28
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Liu X, Qi S, Yin X. Morphogenesis of Hepatitis E Virus. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1417:159-169. [PMID: 37223865 DOI: 10.1007/978-981-99-1304-6_11] [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, a leading cause of acute hepatitis worldwide, has been recognized as non-enveloped virus since its discovery in the 1980s. However, the recent identification of lipid membrane-associated form termed as "quasi-enveloped" HEV has changed this long-held notion. Both naked HEV and quasi-enveloped HEV play important roles in the pathogenesis of hepatitis E. However, the biogenesis and the mechanisms underlying the composition, biogenesis regulation, and functions of the novel quasi-enveloped virions remain enigmatic. In this chapter, we highlight the most recent discoveries on the dual life cycle of these two different types of virions, and further discuss the implication of the quasi-envelopment in our understanding of the molecular biology of HEV.
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Affiliation(s)
- Xing Liu
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Shuhui Qi
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Xin Yin
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
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29
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Kinast V, Klöhn M, Nocke MK, Todt D, Steinmann E. Hepatitis E virus species barriers: seeking viral and host determinants. Curr Opin Virol 2022; 56:101274. [PMID: 36283248 DOI: 10.1016/j.coviro.2022.101274] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 09/19/2022] [Accepted: 09/21/2022] [Indexed: 11/03/2022]
Abstract
The intimate relationship between virus and host cell can result in highly adapted viruses that are restricted to a single host. However, some viruses have the ability to infect multiple host species. Remarkably, hepatitis E viruses (HEV) comprise genotypes that are either 'single-host' or 'multi-host' genotypes, a trait that raises fundamental questions: Why do different genotypes differ in their host range, despite a high degree of genomic similarity? What are the underlying molecular determinants that shape species barriers? Here, we review the current knowledge of viral and host determinants that may affect the evolutionary trajectories of HEV. We also provide a perspective on techniques and methods that address open questions of HEV host range and adaptation.
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Affiliation(s)
- Volker Kinast
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany; Department of Medical Microbiology and Virology, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Mara Klöhn
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Maximilian K Nocke
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Daniel Todt
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany; European Virus Bioinformatics Center (EVBC), 07743 Jena, Germany.
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany; German Centre for Infection Research (DZIF), External Partner Site, Bochum, Germany.
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30
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Gremmel N, Keuling O, Becher P, Baechlein C. Isolation of 15 hepatitis E virus strains lacking ORF1 rearrangements from wild boar and pig organ samples and efficient replication in cell culture. Transbound Emerg Dis 2022; 69:e2617-e2628. [PMID: 35678772 DOI: 10.1111/tbed.14608] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 05/03/2022] [Accepted: 05/30/2022] [Indexed: 11/29/2022]
Abstract
As a zoonotic pathogen, the hepatitis E virus (HEV) leads to numerous infections in humans with different clinical manifestations. Especially genotype 3, as causative agent of a foodborne zoonosis, is transmitted to humans by ingestion of undercooked or raw meat containing liver from HEV-infected animals. Although the virus' prevalence and dissemination in hosts like wild boar and pig have been well characterized, HEV is greatly understudied on a molecular level and reliable cell culture models are lacking. For this reason, the present study concentrated on the isolation and subsequent characterization of porcine HEV from tissue samples derived from wild boar and domestic pigs: 222 wild boars hunted in Northern Germany were investigated for the presence of HEV RNA with a detection rate of 5.9%. Three additional HEV-positive wild boar liver samples as well as an HEV-positive spleen and a positive kidney from domestic pigs were included. After inoculation of positive samples onto the human hepatoma cell line PLC/PRF/5, cells were grown for several weeks. Successful isolation was confirmed by RT-qPCR, virus passage, immunofluorescence staining and titration. Overall, 15 strains from a total of 18 RNA-positive organ samples could be obtained and viral loads >109 RNA copies/ml were measured in cell culture supernatants. Accordingly, 83.3% of the HEV RNA-positive samples contained infectious hepatitis E viral particles and therefore must be considered as a potential source for human infections. Phylogenetic analyses revealed that all isolated strains belong to genotype 3. Further genetic characterization showed a high degree of sequence variability, but no sequence insertions, in the hypervariable region within the open reading frame 1.
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Affiliation(s)
- Nele Gremmel
- Department of Infectious Diseases, Institute of Virology, University of Veterinary Medicine, Hannover, Germany
| | - Oliver Keuling
- Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine, Hannover, Germany
| | - Paul Becher
- Department of Infectious Diseases, Institute of Virology, University of Veterinary Medicine, Hannover, Germany
| | - Christine Baechlein
- Department of Infectious Diseases, Institute of Virology, University of Veterinary Medicine, Hannover, Germany.,Present address: Lower Saxony State Office for Consumer Protection and Food Safety, Food and Veterinary Institute Braunschweig/Hannover, Hannover, Germany
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31
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The Evolutionary Dance between Innate Host Antiviral Pathways and SARS-CoV-2. Pathogens 2022; 11:pathogens11050538. [PMID: 35631059 PMCID: PMC9147806 DOI: 10.3390/pathogens11050538] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Revised: 04/29/2022] [Accepted: 04/30/2022] [Indexed: 02/04/2023] Open
Abstract
Compared to what we knew at the start of the SARS-CoV-2 global pandemic, our understanding of the interplay between the interferon signaling pathway and SARS-CoV-2 infection has dramatically increased. Innate antiviral strategies range from the direct inhibition of viral components to reprograming the host’s own metabolic pathways to block viral infection. SARS-CoV-2 has also evolved to exploit diverse tactics to overcome immune barriers and successfully infect host cells. Herein, we review the current knowledge of the innate immune signaling pathways triggered by SARS-CoV-2 with a focus on the type I interferon response, as well as the mechanisms by which SARS-CoV-2 impairs those defenses.
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32
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Aliyari SR, Ghaffari AA, Pernet O, Parvatiyar K, Wang Y, Gerami H, Tong AJ, Vergnes L, Takallou A, Zhang A, Wei X, Chilin LD, Wu Y, Semenkovich CF, Reue K, Smale ST, Lee B, Cheng G. Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2. Acta Pharm Sin B 2022; 12:1624-1635. [PMID: 35251918 PMCID: PMC8883762 DOI: 10.1016/j.apsb.2022.02.019] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2021] [Revised: 01/27/2022] [Accepted: 02/08/2022] [Indexed: 12/15/2022] Open
Abstract
SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.
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Affiliation(s)
- Saba R. Aliyari
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Amir Ali Ghaffari
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Olivier Pernet
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
- EnViro International Laboratories, Los Angeles, CA 90077, USA
| | - Kislay Parvatiyar
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Yao Wang
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Hoda Gerami
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Ann-Jay Tong
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Laurent Vergnes
- Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Armin Takallou
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Adel Zhang
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Xiaochao Wei
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Linda D. Chilin
- Center for Infectious Disease Research, School of Systems Biology, George Mason University Manassas, VA 20110, USA
| | - Yuntao Wu
- Center for Infectious Disease Research, School of Systems Biology, George Mason University Manassas, VA 20110, USA
| | - Clay F. Semenkovich
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO 63110, USA
- Diabetic Cardiovascular Disease Center, Washington, University School of Medicine, St. Louis, MO 63110, USA
| | - Karen Reue
- Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Stephen T. Smale
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Benhur Lee
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Genhong Cheng
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
- Corresponding author. Tel.:+1 310 825 8896; fax: +1 310 206 5553.
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33
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Li X, Shen L, Xu Z, Liu W, Li A, Xu J. Protein Palmitoylation Modification During Viral Infection and Detection Methods of Palmitoylated Proteins. Front Cell Infect Microbiol 2022; 12:821596. [PMID: 35155279 PMCID: PMC8829041 DOI: 10.3389/fcimb.2022.821596] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Accepted: 01/12/2022] [Indexed: 01/31/2023] Open
Abstract
Protein palmitoylation—a lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group—is a significant post-translational biological process. This process regulates the trafficking, subcellular localization, and stability of different proteins in cells. Since palmitoylation participates in various biological processes, it is related to the occurrence and development of multiple diseases. It has been well evidenced that the proteins whose functions are palmitoylation-dependent or directly involved in key proteins’ palmitoylation/depalmitoylation cycle may be a potential source of novel therapeutic drugs for the related diseases. Many researchers have reported palmitoylation of proteins, which are crucial for host-virus interactions during viral infection. Quite a few explorations have focused on figuring out whether targeting the acylation of viral or host proteins might be a strategy to combat viral diseases. All these remarkable achievements in protein palmitoylation have been made to technological advances. This paper gives an overview of protein palmitoylation modification during viral infection and the methods for palmitoylated protein detection. Future challenges and potential developments are proposed.
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Affiliation(s)
- Xiaoling Li
- College of Life Sciences, Henan Agricultural University, Zhengzhou, China
| | - Lingyi Shen
- College of Life Sciences, Henan Agricultural University, Zhengzhou, China
| | - Zhao Xu
- College of Life Sciences, Henan Agricultural University, Zhengzhou, China
| | - Wei Liu
- College of Life Sciences, Henan Agricultural University, Zhengzhou, China
| | - Aihua Li
- Clinical Lab, Henan Provincial Chest Hospital, Zhengzhou, China
| | - Jun Xu
- College of Life Sciences, Henan Agricultural University, Zhengzhou, China
- *Correspondence: Jun Xu, ;
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34
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Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector. Viruses 2022; 14:v14020266. [PMID: 35215859 PMCID: PMC8878420 DOI: 10.3390/v14020266] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 01/23/2022] [Accepted: 01/24/2022] [Indexed: 01/25/2023] Open
Abstract
The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.
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Chapuy-Regaud S, Allioux C, Capelli N, Migueres M, Lhomme S, Izopet J. Vectorial Release of Human RNA Viruses from Epithelial Cells. Viruses 2022; 14:231. [PMID: 35215825 PMCID: PMC8875463 DOI: 10.3390/v14020231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 01/07/2022] [Accepted: 01/21/2022] [Indexed: 02/04/2023] Open
Abstract
Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.
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Affiliation(s)
- Sabine Chapuy-Regaud
- Department of Virology, CHU Purpan, F-31059 Toulouse, France; (N.C.); (M.M.); (S.L.); (J.I.)
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
| | - Claire Allioux
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
| | - Nicolas Capelli
- Department of Virology, CHU Purpan, F-31059 Toulouse, France; (N.C.); (M.M.); (S.L.); (J.I.)
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
| | - Marion Migueres
- Department of Virology, CHU Purpan, F-31059 Toulouse, France; (N.C.); (M.M.); (S.L.); (J.I.)
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
| | - Sébastien Lhomme
- Department of Virology, CHU Purpan, F-31059 Toulouse, France; (N.C.); (M.M.); (S.L.); (J.I.)
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
| | - Jacques Izopet
- Department of Virology, CHU Purpan, F-31059 Toulouse, France; (N.C.); (M.M.); (S.L.); (J.I.)
- INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), INSERM UMR1291, CNRS UMR5051, Université Toulouse III, CHU Purpan, F-31024 Toulouse, France;
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36
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Oechslin N, Da Silva N, Szkolnicka D, Cantrelle F, Hanoulle X, Moradpour D, Gouttenoire J. Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production. Hepatology 2022; 75:170-181. [PMID: 34387882 PMCID: PMC9300124 DOI: 10.1002/hep.32100] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 07/09/2021] [Accepted: 07/31/2021] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIMS Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. APPROACH AND RESULTS Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. CONCLUSIONS Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.
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Affiliation(s)
- Noémie Oechslin
- Division of Gastroenterology and HepatologyLausanne University Hospital and University of LausanneLausanneSwitzerland
| | - Nathalie Da Silva
- Division of Gastroenterology and HepatologyLausanne University Hospital and University of LausanneLausanneSwitzerland
| | - Dagmara Szkolnicka
- Division of Gastroenterology and HepatologyLausanne University Hospital and University of LausanneLausanneSwitzerland
| | - François‐Xavier Cantrelle
- UMR1167University of LilleINSERMLille University HospitalInstitut Pasteur de LilleLilleFrance,CNRS, ERL9002 ‐ Integrative Structural BiologyLilleFrance
| | - Xavier Hanoulle
- UMR1167University of LilleINSERMLille University HospitalInstitut Pasteur de LilleLilleFrance,CNRS, ERL9002 ‐ Integrative Structural BiologyLilleFrance
| | - Darius Moradpour
- Division of Gastroenterology and HepatologyLausanne University Hospital and University of LausanneLausanneSwitzerland
| | - Jérôme Gouttenoire
- Division of Gastroenterology and HepatologyLausanne University Hospital and University of LausanneLausanneSwitzerland
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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.
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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
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38
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Bentaleb C, Hervouet K, Montpellier C, Camuzet C, Ferrié M, Burlaud-Gaillard J, Bressanelli S, Metzger K, Werkmeister E, Ankavay M, Janampa NL, Marlet J, Roux J, Deffaud C, Goffard A, Rouillé Y, Dubuisson J, Roingeard P, Aliouat-Denis CM, Cocquerel L. The endocytic recycling compartment serves as a viral factory for hepatitis E virus. Cell Mol Life Sci 2022; 79:615. [PMID: 36460928 PMCID: PMC9718719 DOI: 10.1007/s00018-022-04646-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 11/04/2022] [Accepted: 11/23/2022] [Indexed: 12/04/2022]
Abstract
Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.
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Affiliation(s)
- Cyrine Bentaleb
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Kévin Hervouet
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Claire Montpellier
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Charline Camuzet
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Martin Ferrié
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Julien Burlaud-Gaillard
- grid.411167.40000 0004 1765 1600Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France ,Université de Tours et CHRU de Tours, Plateforme IBiSA de Microscopie Electronique, Tours, France
| | - Stéphane Bressanelli
- grid.457334.20000 0001 0667 2738Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-Sur-Yvette, France
| | - Karoline Metzger
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Elisabeth Werkmeister
- grid.503422.20000 0001 2242 6780Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR2014-US41-PLBS-Plateformes Lilloises de Biologie and Santé, Lille, France
| | - Maliki Ankavay
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France ,Present Address: Division of Gastroenterology and Hepatology, Institute of Microbiology, Lausanne, Switzerland
| | - Nancy Leon Janampa
- grid.411167.40000 0004 1765 1600Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France
| | - Julien Marlet
- grid.411167.40000 0004 1765 1600Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France
| | | | | | - Anne Goffard
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Yves Rouillé
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Jean Dubuisson
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Philippe Roingeard
- grid.411167.40000 0004 1765 1600Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France ,Université de Tours et CHRU de Tours, Plateforme IBiSA de Microscopie Electronique, Tours, France
| | - Cécile-Marie Aliouat-Denis
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Laurence Cocquerel
- grid.503422.20000 0001 2242 6780University of Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
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Klöhn M, Schrader JA, Brüggemann Y, Todt D, Steinmann E. Beyond the Usual Suspects: Hepatitis E Virus and Its Implications in Hepatocellular Carcinoma. Cancers (Basel) 2021; 13:5867. [PMID: 34831021 PMCID: PMC8616277 DOI: 10.3390/cancers13225867] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 11/16/2021] [Accepted: 11/19/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis E virus infections are the leading cause of viral hepatitis in humans, contributing to an estimated 3.3 million symptomatic cases and almost 44,000 deaths annually. Recently, HEV infections have been found to result in chronic liver infection and cirrhosis in severely immunocompromised patients, suggesting the possibility of HEV-induced hepatocarcinogenesis. While HEV-associated formation of HCC has rarely been reported, the expansion of HEV's clinical spectrum and the increasing evidence of chronic HEV infections raise questions about the connection between HEV and HCC. The present review summarizes current clinical evidence of the relationship between HEV and HCC and discusses mechanisms of virus-induced HCC development with regard to HEV pathogenesis. We further elucidate why the development of HEV-induced hepatocellular carcinoma has so rarely been observed and provide an outlook on possible experimental set-ups to study the relationship between HEV and HCC formation.
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Affiliation(s)
- Mara Klöhn
- Department of Molecular and Medical Virology, Ruhr-Universität Bochum, 44801 Bochum, Germany; (M.K.); (J.A.S.); (Y.B.); (D.T.)
| | - Jil Alexandra Schrader
- Department of Molecular and Medical Virology, Ruhr-Universität Bochum, 44801 Bochum, Germany; (M.K.); (J.A.S.); (Y.B.); (D.T.)
| | - Yannick Brüggemann
- Department of Molecular and Medical Virology, Ruhr-Universität Bochum, 44801 Bochum, Germany; (M.K.); (J.A.S.); (Y.B.); (D.T.)
| | - Daniel Todt
- Department of Molecular and Medical Virology, Ruhr-Universität Bochum, 44801 Bochum, Germany; (M.K.); (J.A.S.); (Y.B.); (D.T.)
- European Virus Bioinformatics Center (EVBC), 07743 Jena, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr-Universität Bochum, 44801 Bochum, Germany; (M.K.); (J.A.S.); (Y.B.); (D.T.)
- German Centre for Infection Research (DZIF), External Partner Site, 44801 Bochum, Germany
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The Viral ORF3 Protein Is Required for Hepatitis E Virus Apical Release and Efficient Growth in Polarized Hepatocytes and Humanized Mice. J Virol 2021; 95:e0058521. [PMID: 34523963 DOI: 10.1128/jvi.00585-21] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Hepatitis E virus (HEV), an enterically transmitted RNA virus, is a major cause of acute hepatitis worldwide. Additionally, HEV genotype 3 (gt3) can frequently persist in immunocompromised individuals with an increased risk for developing severe liver disease. Currently, no HEV-specific treatment is available. The viral open reading frame 3 (ORF3) protein facilitates HEV egress in vitro and is essential for establishing productive infection in macaques. Thus, ORF3, which is unique to HEV, has the potential to be explored as a target for antiviral therapy. However, significant gaps exist in our understanding of the critical functions of ORF3 in HEV infection in vivo. Here, we utilized a polarized hepatocyte culture model and a human liver chimeric mouse model to dissect the roles of ORF3 in gt3 HEV release and persistent infection. We show that ORF3's absence substantially decreased HEV replication and virion release from the apical surface but not the basolateral surface of polarized hepatocytes. While wild-type HEV established a persistent infection in humanized mice, mutant HEV lacking ORF3 (ORF3null) failed to sustain the infection despite transient replication in the liver and was ultimately cleared. Strikingly, mice inoculated with the ORF3null virus displayed no fecal shedding throughout the 6-week experiment. Overall, our results demonstrate that ORF3 is required for HEV fecal shedding and persistent infection, providing a rationale for targeting ORF3 as a treatment strategy for HEV infection. IMPORTANCE HEV infections are associated with significant morbidity and mortality. HEV gt3 additionally can cause persistent infection, which can rapidly progress to liver cirrhosis. Currently, no HEV-specific treatments are available. The poorly understood HEV life cycle hampers the development of antivirals for HEV. Here, we investigated the role of the viral ORF3 protein in HEV infection in polarized hepatocyte cultures and human liver chimeric mice. We found that two major aspects of the HEV life cycle require ORF3: fecal virus shedding and persistent infection. These results provide a rationale for targeting ORF3 to treat HEV infection.
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Jiao H, Zhao Y, Zhou Z, Li W, Li B, Gu G, Luo Y, Shuai X, Fan C, Wu L, Chen J, Huang Q, Wang F, Liu J. Identifying Circular RNAs in HepG2 Expressing Genotype IV Swine Hepatitis E Virus ORF3 Via Whole Genome Sequencing. Cell Transplant 2021; 30:9636897211055042. [PMID: 34699255 PMCID: PMC8552397 DOI: 10.1177/09636897211055042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Swine hepatitis E (SHE) is a new type of zoonotic infectious disease caused by swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key regulatory and virulent protein of SHEV. Circular RNAs (circRNAs) are a special kind of non-coding RNA molecule, which has a closed ring structure. In this study, to identify the circRNA profile in host cells affected by SHEV ORF3, adenovirus ADV4-ORF3 mediated the overexpression of ORF3 in HepG2 cells, whole genome sequencing was used to investigate the differentially expressed circRNAs, GO and KEGG were performed to enrichment analyze of differentially expressed circRNA-hosting gene, and Targetscan and miRanda softwares were used to analyze the interaction between circRNA and miRNA. The results showed adenovirus successfully mediated the overexpression of ORF3 in HepG2 cells, 1,105 up-regulation circRNAs and 1,556 down-regulation circRNAs were identified in ADV4-ORF3 infection group compared with the control. GO function enrichment analysis of differentially expressed circRNAs-hosting genes classified three main categories (cellular component, biological process and molecular function). KEGG pathway enrichment analysis scatter plot showed the pathway term of top20. The circRNAs with top10 number of BS sites for qRT-PCR validation were selected to confirmed, the results indicated that the up-regulated hsa_circ_0001423 and hsa_circ_0006404, and down-regulated of hsa_circ_0004833 and hsa_circ_0007444 were consistent with the sequencing data. Our findings first preliminarily found that ORF3 protein may affect triglyceride activation (GO:0006642) and riboflavin metabolism (ko00740) in HepG2 cells, which provides a scientific basis for further elucidating the effect of ORF3 on host lipid metabolism and the mechanism of SHEV infection.
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Affiliation(s)
- Hanwei Jiao
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Yu Zhao
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Science, Guiyang, China
| | - Zhixiong Zhou
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Wenjie Li
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Bowen Li
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Guojing Gu
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Yichen Luo
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Xuehong Shuai
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Cailiang Fan
- Rongchang Animal Epidemic Prevention and Control Center, Chongqing, Rongchang, China
| | - Li Wu
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Jixuan Chen
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Qingzhou Huang
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Fengyang Wang
- Hainan Key Lab of Tropical Animal Reproduction and Breeding and Epidemic Disease Research, College of Animal Science and Technology, Hainan University, Haikou, China
| | - Juan Liu
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
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42
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Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus. Appl Microbiol Biotechnol 2021; 105:8505-8516. [PMID: 34633486 DOI: 10.1007/s00253-021-11621-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2021] [Revised: 09/19/2021] [Accepted: 09/26/2021] [Indexed: 10/20/2022]
Abstract
Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3SAR-55 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 μg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.
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Cellular Organelles Involved in Hepatitis E Virus Infection. Pathogens 2021; 10:pathogens10091206. [PMID: 34578238 PMCID: PMC8469867 DOI: 10.3390/pathogens10091206] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2021] [Revised: 09/16/2021] [Accepted: 09/16/2021] [Indexed: 12/24/2022] Open
Abstract
Hepatitis E virus (HEV), a major cause of acute hepatitis worldwide, infects approximately 20 million individuals annually. HEV can infect a wide range of mammalian and avian species, and cause frequent zoonotic spillover, increasingly raising public health concerns. To establish a successful infection, HEV needs to usurp host machineries to accomplish its life cycle from initial attachment to egress. However, relatively little is known about the HEV life cycle, especially the functional role(s) of cellular organelles and their associated proteins at different stages of HEV infection. Here, we summarize current knowledge regarding the relation of HEV with the different cell organelles during HEV infection. Furthermore, we discuss the underlying mechanisms by which HEV infection is precisely regulated in infected cells and the modification of host cell organelles and their associated proteins upon HEV infection.
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44
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Coronel Arrechea C, Giolito ML, García IA, Soria G, Valdez Taubas J. A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors. Open Biol 2021; 11:200415. [PMID: 34343464 PMCID: PMC8331233 DOI: 10.1098/rsob.200415] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia. As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.
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Affiliation(s)
- Consuelo Coronel Arrechea
- Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC) CONICET, Córdoba, Argentina.,Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Córdoba, Argentina
| | - María Luz Giolito
- Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC) CONICET, Córdoba, Argentina.,Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Córdoba, Argentina
| | - Iris Alejandra García
- Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Córdoba, Argentina
| | - Gastón Soria
- Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.,Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Córdoba, Argentina
| | - Javier Valdez Taubas
- Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC) CONICET, Córdoba, Argentina.,Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Córdoba, Argentina
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45
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Kerviel A, Zhang M, Altan-Bonnet N. A New Infectious Unit: Extracellular Vesicles Carrying Virus Populations. Annu Rev Cell Dev Biol 2021; 37:171-197. [PMID: 34270326 DOI: 10.1146/annurev-cellbio-040621-032416] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Viral egress and transmission have long been described to take place through single free virus particles. However, viruses can also shed into the environment and transmit as populations clustered inside extracellular vesicles (EVs), a process we had first called vesicle-mediated en bloc transmission. These membrane-cloaked virus clusters can originate from a variety of cellular organelles including autophagosomes, plasma membrane, and multivesicular bodies. Their viral cargo can be multiples of nonenveloped or enveloped virus particles or even naked infectious genomes, but egress is always nonlytic, with the cell remaining intact. Here we put forth the thesis that EV-cloaked viral clusters are a distinct form of infectious unit as compared to free single viruses (nonenveloped or enveloped) or even free virus aggregates. We discuss how efficient and prevalent these infectious EVs are in the context of virus-associated diseases and highlight the importance of their proper detection and disinfection for public health. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Adeline Kerviel
- Laboratory of Host-Pathogen Dynamics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;
| | - Mengyang Zhang
- Laboratory of Host-Pathogen Dynamics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; .,Department of Civil and Environmental Engineering, The George Washington University, Washington, DC 20052, USA
| | - Nihal Altan-Bonnet
- Laboratory of Host-Pathogen Dynamics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;
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46
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Glitscher M, Hildt E. Hepatitis E virus egress and beyond - the manifold roles of the viral ORF3 protein. Cell Microbiol 2021; 23:e13379. [PMID: 34272798 DOI: 10.1111/cmi.13379] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 06/18/2021] [Accepted: 07/06/2021] [Indexed: 11/30/2022]
Abstract
Although the hepatitis E virus represents an uprising threat to the global community by representing the commonest cause of an acute viral hepatitis worldwide, its life cycle is grossly understudied. Albeit HEV is a non-enveloped virus, its progeny is released as quasi-enveloped virions. Thus, the responsible accessory protein pORF3 gained rising attention in the past years. It mediates viral release via the exosomal route by targeting the viral capsid to the endosomal system, more precisely to multivesicular bodies. As this is followed by quasi-envelopment, pORF3 may in terms represent a substitute to a conventional envelope protein. This feature proofs to be rather unique with respect to other enteric viruses, although the protein's role in the viral life cycle seems to reach far beyond simply maintaining release of progeny viruses. How pORF3 affects viral morphogenesis, how it mediates efficient viral release and how it supports viral spread is summarised in this microreview. With this, we aim to shed light on functions of pORF3 to gain further insights in still enigmatic aspects of the HEV life cycle. TAKE AWAYS: HEV is released as exosome via multivesicular bodies Viral pORF3 mediates release via endosomal complexes required for transport pORF3 modulates various cellular processes in infected cells Elucidation of pORF3-related processes imply novel clinical strategies.
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Affiliation(s)
| | - Eberhard Hildt
- Department Virology, Paul-Ehrlich-Institut, Langen, Germany
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47
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Yang YL, Nan YC. Open reading frame 3 protein of hepatitis E virus: Multi-function protein with endless potential. World J Gastroenterol 2021; 27:2458-2473. [PMID: 34092969 PMCID: PMC8160619 DOI: 10.3748/wjg.v27.i20.2458] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 03/10/2021] [Accepted: 04/12/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen, causes acute hepatitis in humans and is responsible for hepatitis E outbreaks worldwide. Since the identification of HEV as a zoonotic agent, this virus has been isolated from a variety of hosts with an ever-expanding host range. HEV-open reading frame (ORF) 3, the smallest ORF in HEV genomes, initially had been perceived as an unremarkable HEV accessory protein. However, as novel HEV-ORF3 function has been discovered that is related to the existence of a putative third virion structural form, referred to as “quasi-enveloped” HEV particles, HEV is challenging the conventional virion structure-based classification scheme, which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In this review, we systematically describe recent progress that has identified multiple pathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesis of quasi-enveloped virus, regulation of the host innate immune response, and interference with host signaling pathways. In addition, implications of HEV-ORF3-associated quasi-enveloped virions are discussed to guide future development of improved vaccines against zoonotic HEV infection.
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Affiliation(s)
- Yong-Lin Yang
- Department of Infectious Diseases, Taizhou People's Hospital, The Fifth Affiliated Hospital of Nantong University, Taizhou 225300, Jiangsu Province, China
- Department of General Practice, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu Province, China
| | - Yu-Chen Nan
- Department of Preventive Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi Province, China
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Tietcheu Galani BR, Ayissi Owona VB, Guemmogne Temdie RJ, Metzger K, Atsama Amougou M, Djamen Chuisseu PD, Fondjo Kouam A, Ngounoue Djuidje M, Aliouat-Denis CM, Cocquerel L, Fewou Moundipa P. In silico and in vitro screening of licensed antimalarial drugs for repurposing as inhibitors of hepatitis E virus. In Silico Pharmacol 2021; 9:35. [PMID: 33959472 PMCID: PMC8093904 DOI: 10.1007/s40203-021-00093-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Accepted: 04/16/2021] [Indexed: 01/15/2023] Open
Abstract
ABSTRACT Hepatitis E virus (HEV) infection is emerging in Cameroon and represents one of the most common causes of acute hepatitis and jaundice. Moreover, earlier reports showed evidence of falciparum malaria/HEVcoexistence. Although the Sofosbuvir/Ribavirin combination was recently proposed in the treatment of HEV-infected patients, no specific antiviral drug has been approved so far, thereby urging the search for new therapies. Fortunately, drug repurposing offers a good alternative to this end. In this study, we report the in silico and in vitro activities of 8 licensed antimalarial drugs and two anti-hepatitis C virus agents used as references (Sofosbuvir, and Ribavirin), for repurposing as antiviral inhibitors against HEV. Compounds were docked against five HEV-specific targets including the Zinc-binding non-structural protein (6NU9), RNA-dependent RNA polymerase (RdRp), cryoEM structure of HEV VLP, genotype 1 (6LAT), capsid protein ORF-2, genotype 3 (2ZTN), and the E2s domain of genotype 1 (3GGQ) using the iGEMDOCK software and their pharmacokinetic profiles and toxicities were predicted using ADMETlab2.0. Their in vitro effects were also assessed on a gt 3 p6Gluc replicon system using the luciferase reporter assay. The docking results showed that Sofosbuvir had the best binding affinities with 6NU9 (- 98.22 kcal/mol), RdRp (- 113.86 kcal/mol), 2ZTN (- 106.96 kcal/mol), while Ribavirin better collided with 6LAT (- 99.33 kcal/mol). Interestingly, Lumefantrine showed the best affinity with 3GGQ (-106.05 kcal/mol). N-desethylamodiaquine and Amodiaquine presented higher binding scores with 6NU9 (- 93.5 and - 89.9 kcal/mol respectively vs - 80.83 kcal/mol), while Lumefantrine had the greatest energies with RdRp (- 102 vs - 84.58), and Pyrimethamine and N-desethylamodiaquine had stronger affinities with 2ZTN compared to Ribavirin (- 105.17 and - 102.65 kcal/mol vs - 96.04 kcal/mol). The biological screening demonstrated a significant (P < 0.001) antiviral effect on replication with 1 µM N-desethylamodiaquine, the major metabolite of Amodiaquine. However, Lumefantrine showed no effect at the tested concentrations (1, 5, and 10 µM). The biocomputational analysis of the pharmacokinetic profile of both drugs revealed a low permeability of Lumefantrine and a specific inactivation by CYP3A2 which might partly contribute to the short half-time of this drug. In conclusion, Amodiaquine and Lumefantrine may be good antimalarial drug candidates for repurposing against HEV. Further in vitro and in vivo experiments are necessary to validate these predictions. GRAPHIC ABSTRACT SUPPLEMENTARY INFORMATION The online version contains supplementary material available at 10.1007/s40203-021-00093-y.
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Affiliation(s)
- Borris Rosnay Tietcheu Galani
- Laboratory of Applied Biochemistry, Department of Biological Sciences, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundere, Cameroon
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
| | - Vincent Brice Ayissi Owona
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
| | - Romeo Joel Guemmogne Temdie
- Laboratory of Medicinal Plants, Health, and Galenic Formulation, Department of Biological Sciences, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundere, Cameroon
| | - Karoline Metzger
- University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Marie Atsama Amougou
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
- Research Center for Emerging and Reemerging Infectious Diseases (CREMER-IMPM), Virology Unit, P.O. Box 906, Yaounde, Cameroon
| | - Pascal Dieudonné Djamen Chuisseu
- Department of Medicine, Medical and Biomedical Sciences, Higher Institute of Health Sciences, Université Des Montagnes, P.O. Box 208, Bangangte, Cameroon
| | - Arnaud Fondjo Kouam
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
- Department of Biomedical Sciences, Faculty of Health Sciences, University of Buea, P.O Box 63, Buea, South West Region Cameroon
| | - Marceline Ngounoue Djuidje
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
| | - Cécile-Marie Aliouat-Denis
- University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Laurence Cocquerel
- University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR9017-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France
| | - Paul Fewou Moundipa
- Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty ofScience, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
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Jiao H, Shuai X, Luo Y, Zhou Z, Zhao Y, Li B, Gu G, Li W, Li M, Zeng H, Guo X, Xiao Y, Song Z, Gan L, Huang Q. Deep Insight Into Long Non-coding RNA and mRNA Transcriptome Profiling in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3. Front Vet Sci 2021; 8:625609. [PMID: 33996960 PMCID: PMC8116512 DOI: 10.3389/fvets.2021.625609] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 03/10/2021] [Indexed: 12/14/2022] Open
Abstract
Swine hepatitis E (swine HE) is a new type of zoonotic infectious disease caused by the swine hepatitis E virus (swine HEV). Open reading frame 3 (ORF3) is an important virulent protein of swine HEV, but its function still is mainly unclear. In this study, we generated adenoviruses ADV4-ORF3 and ADV4 negative control (ADV4-NC), which successfully mediated overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP, respectively, in HepG2 cells. High-throughput sequencing was used to screen for differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The cis-target genes of lncRNAs were predicted, functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed, and 12 lncRNAs with statistically significant different expressions (p ≤ 0.05 and q ≤ 1) were selected for further quantitative real-time reverse transcription (qRT-PCR) validation. In HepG2 cells, we identified 62 significantly differentially expressed genes (DEGs) (6,564 transcripts) and 319 lncRNAs (124 known lncRNAs and 195 novel lncRNAs) that were affected by ORF3, which were involved in systemic lupus erythematosus, Staphylococcus aureus infection, signaling pathways pluripotency regulation of stem cells, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and platinum drug resistance pathways. Cis-target gene prediction identified 45 lncRNAs corresponding to candidate mRNAs, among which eight were validated by qRT-PCR: LINC02476 (two transcripts), RAP2C-AS1, AC016526, AL139099, and ZNF337-AS1 (3 transcripts). Our results revealed that the lncRNA profile in host cells affected by ORF3, swine HEV ORF3, might affect the pentose and glucuronate interconversions and mediate the formation of obstructive jaundice by influencing bile secretion, which will help to determine the function of ORF3 and the infection mechanism and treatment of swine HE.
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Affiliation(s)
- Hanwei Jiao
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Xuehong Shuai
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Yichen Luo
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Zhixiong Zhou
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Yu Zhao
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Science, Guiyang, China
| | - Bowen Li
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Guojing Gu
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Wenjie Li
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Mengjuan Li
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Hui Zeng
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Xiaoyi Guo
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Yu Xiao
- College of Veterinary Medicine, Southwest University, Chongqing, China
| | - Zhenhui Song
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Ling Gan
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
| | - Qingzhou Huang
- College of Veterinary Medicine, Southwest University, Chongqing, China.,Chongqing Veterinary Scientific Engineering Research Center, Southwest University, Chongqing, China
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50
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Scholz J, Falkenhagen A, Johne R. The Translated Amino Acid Sequence of an Insertion in the Hepatitis E Virus Strain 47832c Genome, But Not the RNA Sequence, Is Essential for Efficient Cell Culture Replication. Viruses 2021; 13:v13050762. [PMID: 33926134 PMCID: PMC8145396 DOI: 10.3390/v13050762] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Revised: 04/14/2021] [Accepted: 04/22/2021] [Indexed: 12/19/2022] Open
Abstract
The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.
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