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Woytinek K, Glitscher M, Hildt E. Antagonism of epidermal growth factor receptor signaling favors hepatitis E virus life cycle. J Virol 2024; 98:e0058024. [PMID: 38856640 PMCID: PMC11265270 DOI: 10.1128/jvi.00580-24] [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: 04/24/2024] [Accepted: 05/09/2024] [Indexed: 06/11/2024] Open
Abstract
Hepatitis E virus (HEV) poses a global threat, which currently remains understudied in terms of host interactions. Epidermal growth factor receptor (EGFR) plays multifaceted roles in viral pathogenesis, impacting host-cell entry, viral replication, and host-defense modulation. On the one hand, EGFR signaling emerged as a major driver in innate immunity; on the other hand, a crosstalk between HEV and EGFR requires deeper analysis. We therefore aimed to dissect the receptor's involvement in the HEV life cycle. In persistently HEV-infected cells, the EGFR amount is decreased alongside with enhanced receptor internalization. As compared with the control ligand-induced EGFR, activation revealed an early receptor internalization and degradation in HEV-replicating cells, resulting in a notable EGFR signaling delay. Interestingly, inhibition or silencing of EGFR increased viral replication, extracellular and intracellular viral transcripts, and released infectious particles. The pro-viral impact of EGFR inhibition was attributed to (i) impaired expression of interferon-stimulated genes, (ii) activation of the autophagosomal system, (iii) virus-induced inhibition of lysosomal acidification, and (iv) a decrease of the cellular cholesterol level. IMPORTANCE This study identifies epidermal growth factor receptor (EGFR) as a novel host factor affecting hepatitis E virus (HEV): EGFR downregulation promotes viral replication, release, and evasion from the innate immune response. The discovery that EGFR inhibition favors viral spread is particularly concerning for HEV patients undergoing EGFR inhibitor treatment.
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Affiliation(s)
| | - Mirco Glitscher
- Division of Virology, Paul Ehrlich Institute, Langen, Germany
| | - Eberhard Hildt
- Division of Virology, Paul Ehrlich Institute, Langen, Germany
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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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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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Dai J, Feng Y, Liao Y, Tan L, Sun Y, Song C, Qiu X, Ding C. ESCRT machinery and virus infection. Antiviral Res 2024; 221:105786. [PMID: 38147902 DOI: 10.1016/j.antiviral.2023.105786] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Revised: 12/18/2023] [Accepted: 12/19/2023] [Indexed: 12/28/2023]
Abstract
The endosomal sorting complex required for transport (ESCRT) machinery plays a significant role in the spread of human viruses. However, our understanding of how the host ESCRT machinery responds to viral infection remains limited. Emerging evidence suggests that the ESCRT machinery can be hijacked by viruses of different families to enhance their replication. Throughout their life cycle, these viruses can interfere with or exploit ESCRT-mediated physiological processes to increase their chances of infecting the host. In contrast, to counteract virus infection, the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) system within the infected cells is activated to degrade the ESCRT proteins. Many retroviral and RNA viral proteins have evolved "late (L) domain" motifs, which enable them to recruit host ESCRT subunit proteins to facilitate virus transport, replication, budding, mature, and even endocytosis, Therefore, the L domain motifs and ESCRT subunit proteins could serve as promising drug targets for antiviral therapy. This review investigated the composition and essential functions of the ESCRT, shedding light on the impact of ESCRT subunits and viral L domain motifs on the replication of viruses. Furthermore, the antiviral effects facilitated by the ESCRT machinery have been investigated, aiming to provide valuable insights to guide the development and utilization of antiviral drugs.
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Affiliation(s)
- Jun Dai
- Experimental Animal Center, Zunyi Medical University, Zunyi, 563099, China; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Yiyi Feng
- Laboratory of Veterinary Microbiology and Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, 530004, Guangxi, China.
| | - Ying Liao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Lei Tan
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Yingjie Sun
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Cuiping Song
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Xusheng Qiu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
| | - Chan Ding
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
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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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Zhang B, Fan M, Fan J, Luo Y, Wang J, Wang Y, Liu B, Sun Y, Zhao Q, Hiscox JA, Nan Y, Zhou EM. Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization. Microbiol Spectr 2022; 10:e0226521. [PMID: 35138149 PMCID: PMC8826821 DOI: 10.1128/spectrum.02265-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Accepted: 01/05/2022] [Indexed: 12/31/2022] Open
Abstract
Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/β1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/β1 integrins. The triggering of the signaling pathway-associated Integrin α5/β1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.
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Affiliation(s)
- Beibei Zhang
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Mengnan Fan
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jie Fan
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Yuhang Luo
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jie Wang
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Yajing Wang
- 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
| | - 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
| | - Julian A. Hiscox
- Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom
| | - 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
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Shirazi R, Pozzi P, Gozlan Y, Wax M, Lustig Y, Linial M, Mendelson E, Bardenstein S, Mor O. Identification of Hepatitis E Virus Genotypes 3 and 7 in Israel: A Public Health Concern? Viruses 2021; 13:v13112326. [PMID: 34835132 PMCID: PMC8625709 DOI: 10.3390/v13112326] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 11/16/2021] [Accepted: 11/20/2021] [Indexed: 12/12/2022] Open
Abstract
Background: Hepatitis E (HEV) is an emerging cause of viral hepatitis worldwide. Swine carrying hepatitis E genotype 3 (HEV-3) are responsible for the majority of chronic viral hepatitis cases in developed countries. Recently, genotype 7 (HEV-7), isolated from a dromedary camel in the United Arab Emirates, was also associated with chronic viral hepatitis in a transplant recipient. In Israel, chronic HEV infection has not yet been reported, although HEV seroprevalence in humans is ~10%. Camels and swine are >65% seropositive. Here we report on the isolation and characterization of HEV from local camels and swine. Methods: Sera from camels (n = 142), feces from swine (n = 18) and blood from patients suspected of hepatitis E (n = 101) were collected during 2017–2020 and used to detect and characterize HEV sequences. Results: HEV-3 isolated from local swine and the camel-derived HEV-7 sequence were highly similar to HEV-3f and HEV-7 sequences (88.2% and 86.4%, respectively) related to viral hepatitis. The deduced amino acid sequences of both isolates were also highly conserved (>98%). Two patients were HEV-RNA positive; acute HEV-1 infection could be confirmed in one of them. Discussion: The absence of any reported HEV-3 and HEV-7 infection in humans remains puzzling, especially considering the reported seroprevalence rates, the similarity between HEV sequences related to chronic hepatitis and the HEV genotypes identified in swine and camels in Israel.
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Affiliation(s)
- Rachel Shirazi
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
| | - Paolo Pozzi
- Department of Veterinary Sciences, University of Torino, 10095 Grugliasco, Italy;
| | - Yael Gozlan
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
| | - Marina Wax
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
| | - Yaniv Lustig
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
- Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Michal Linial
- Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel;
| | - Ella Mendelson
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
- Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | | | - Orna Mor
- Central Virology Laboratory, Ministry of Health, Public Health Services, The Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 52620, Israel; (R.S.); (Y.G.); (M.W.); (Y.L.); (E.M.)
- Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
- Correspondence: ; Tel.: +972-3-530-2458
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Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada. Viruses 2021; 13:v13091730. [PMID: 34578311 PMCID: PMC8472430 DOI: 10.3390/v13091730] [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: 06/25/2021] [Revised: 08/25/2021] [Accepted: 08/26/2021] [Indexed: 12/14/2022] Open
Abstract
The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.
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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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10
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Hartard C, Fenaux H, Gentilhomme A, Murray JM, Akand E, Laugel E, Berger S, Maul A, de Rougemont A, Jeulin H, Remen T, Bensenane M, Bronowicki JP, Gantzer C, Bertrand I, Schvoerer E. Variability in molecular characteristics of Hepatitis E virus quasispecies could modify viral surface properties and transmission. J Viral Hepat 2021; 28:1078-1090. [PMID: 33877740 DOI: 10.1111/jvh.13513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Revised: 03/10/2021] [Accepted: 04/04/2021] [Indexed: 12/09/2022]
Abstract
Hepatitis E virus (HEV) usually causes self-limited liver diseases but can also result in severe cases. Genotypes 1 (G1) and 2 circulate in developing countries are human-restricted and waterborne, while zoonotic G3 and G4 circulating in industrialized countries preferentially infect human through consumption of contaminated meat. Our aims were to identify amino acid patterns in HEV variants that could be involved in pathogenicity or in transmission modes, related to their impact on antigenicity and viral surface hydrophobicity. HEV sequences from human (n = 37) and environmental origins (wild boar [n = 3], pig slaughterhouse effluent [n = 6] and urban wastewater [n = 2]) were collected for the characterization of quasispecies using ultra-deep sequencing (ORF2/ORF3 overlap). Predictive and functional assays were carried out to investigate viral particle antigenicity and hydrophobicity. Most quasispecies showed a major variant while a mixture was observed in urban wastewater and in one chronically infected patient. Amino acid signatures were identified, as a rabbit-linked HEV pattern in two infected patients, or the S68L (ORF2) / H81C (ORF3) residue mostly identified in wild boars. By comparison with environmental strains, molecular patterns less likely represented in humans were identified. Patterns impacting viral hydrophobicity and/or antigenicity were also observed, and the higher hydrophobicity of HEV naked particles compared with the enveloped forms was demonstrated. HEV variants isolated from human and environment present molecular patterns that could impact their surface properties as well as their transmission. These molecular patterns may concern only one minor variant of a quasispecies and could emerge under selective pressure.
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Affiliation(s)
- Cédric Hartard
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France.,Université de Lorraine, CNRS, LCPME, Nancy, France
| | - Honorine Fenaux
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France.,Université de Lorraine, CNRS, LCPME, Nancy, France
| | - Alexis Gentilhomme
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France
| | - John M Murray
- School of Mathematics and Statistics, UNSW Sydney, Sydney, NSW, Australia
| | - Elma Akand
- School of Mathematics and Statistics, UNSW Sydney, Sydney, NSW, Australia
| | - Elodie Laugel
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France.,Université de Lorraine, CNRS, LCPME, Nancy, France
| | - Sibel Berger
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France
| | - Armand Maul
- LIEC (Laboratoire Interdisciplinaire des Environnements Continentaux), Université de Lorraine, CNRS, Metz, France
| | - Alexis de Rougemont
- CHU de Dijon, Centre national de référence des virus entériques, Dijon, France
| | - Hélène Jeulin
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France.,Université de Lorraine, CNRS, LCPME, Nancy, France
| | - Thomas Remen
- DRCI, Délégation à la Recherche Clinique et à l'Innovation, Unité de Méthodologie, Data Management et Statistique, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France
| | - Mouni Bensenane
- Service d'hépato-gastro-entérologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France
| | - Jean-Pierre Bronowicki
- Service d'hépato-gastro-entérologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France
| | | | | | - Evelyne Schvoerer
- Laboratoire de Virologie, CHRU de Nancy Brabois, Vandoeuvre-lès-Nancy, France.,Université de Lorraine, CNRS, LCPME, Nancy, France
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11
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Ji H, Chen S, He Q, Wang W, Gong S, Qian Z, Zhang Y, Wei D, Yu W, Huang F. The different replication between nonenveloped and quasi-enveloped hepatitis E virus. J Med Virol 2021; 93:6267-6277. [PMID: 34076903 DOI: 10.1002/jmv.27121] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2021] [Accepted: 05/31/2021] [Indexed: 12/15/2022]
Abstract
Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. However, the understanding of the HEV life cycle is limited. In the present study, cells were separately infected with nonenveloped HEV (derived from feces or bile) or quasi-enveloped HEV (derived from the cell culture after serial passages, eHEV) and observed by confocal fluorescence microscopy to investigate the life cycle of HEV. HEV finished its binding and entry into host cells at first 6 h postinoculation (hpi). Cells inoculated with eHEV showed less infectivity than cells inoculated with nonenveloped HEV. Newly synthesized progeny virions were released into the supernatant of cell cultures from 48 hpi. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis results showed that the supernatant's progeny viruses were infectious even after five serial passages. These results show the significant difference between nonenveloped HEV and eHEV, which will provide novel insights into the HEV replication cycle. The efficient cell culture of HEV will promote the development of anti-HEV drugs and vaccines.
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Affiliation(s)
- Hanbin Ji
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Shuangfeng Chen
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Qiuxia He
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Wenjing Wang
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Shilin Gong
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Zhongyao Qian
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Yike Zhang
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Daqiao Wei
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China
| | - Wenhai Yu
- Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, PR China
| | - Fen Huang
- Medical Faculty, Kunming University of Science and Technology, Kunming, PR China.,Yunnan Provincial Key Laboratory of Clinical Virology, Kunming, PR China
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12
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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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13
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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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14
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Wang B, Meng XJ. Structural and molecular biology of hepatitis E virus. Comput Struct Biotechnol J 2021; 19:1907-1916. [PMID: 33995894 PMCID: PMC8079827 DOI: 10.1016/j.csbj.2021.03.038] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 03/26/2021] [Accepted: 03/29/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is one of the most common causes of acute viral hepatitis, mainly transmitted by fecal-oral route but has also been linked to fulminant hepatic failure, chronic hepatitis, and extrahepatic neurological and renal diseases. HEV is an emerging zoonotic pathogen with a broad host range, and strains of HEV from numerous animal species are known to cross species barriers and infect humans. HEV is a single-stranded, positive-sense RNA virus in the family Hepeviridae. The genome typically contains three open reading frames (ORFs): ORF1 encodes a nonstructural polyprotein for virus replication and transcription, ORF2 encodes the capsid protein that elicits neutralizing antibodies, and ORF3, which partially overlaps ORF2, encodes a multifunctional protein involved in virion morphogenesis and pathogenesis. HEV virions are non-enveloped spherical particles in feces but exist as quasi-enveloped particles in circulating blood. Two types of HEV virus-like particles (VLPs), small T = 1 (270 Å) and native virion-sized T = 3 (320-340 Å) have been reported. There exist two distinct forms of capsid protein, the secreted form (ORF2S) inhibits antibody neutralization, whereas the capsid-associated form (ORF2C) self-assembles to VLPs. Four cis-reactive elements (CREs) containing stem-loops from secondary RNA structures have been identified in the non-coding regions and are critical for virus replication. This mini-review discusses the current knowledge and gaps regarding the structural and molecular biology of HEV with emphasis on the virion structure, genomic organization, secondary RNA structures, viral proteins and their functions, and life cycle of HEV.
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Affiliation(s)
- Bo Wang
- Center for Emerging, Zoonotic and Arthropod-borne Pathogens, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
| | - Xiang-Jin Meng
- Center for Emerging, Zoonotic and Arthropod-borne Pathogens, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
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15
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Advances in Hepatitis E Virus Biology and Pathogenesis. Viruses 2021; 13:v13020267. [PMID: 33572257 PMCID: PMC7915517 DOI: 10.3390/v13020267] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Revised: 01/21/2021] [Accepted: 02/02/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.
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16
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Multivesicular body sorting and the exosomal pathway are required for the release of rat hepatitis E virus from infected cells. Virus Res 2020; 278:197868. [PMID: 31962066 DOI: 10.1016/j.virusres.2020.197868] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2019] [Revised: 01/17/2020] [Accepted: 01/17/2020] [Indexed: 01/14/2023]
Abstract
Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.
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17
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Dao Thi VL, Wu X, Rice CM. Stem Cell-Derived Culture Models of Hepatitis E Virus Infection. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a031799. [PMID: 29686039 DOI: 10.1101/cshperspect.a031799] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Similar to other hepatotropic viruses, hepatitis E virus (HEV) has been notoriously difficult to propagate in cell culture, limiting studies to unravel its biology. Recently, major advances have been made by passaging primary HEV isolates and selecting variants that replicate efficiently in carcinoma cells. These adaptations, however, can alter HEV biology. We have explored human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) as an alternative to conventional hepatoma and hepatocyte cell culture systems for HEV studies. HLCs are permissive for nonadapted HEV isolate genotypes (gt)1-4 replication and can be readily genetically manipulated. HLCs, therefore, enable studies of pan-genotype HEV biology and will serve as a platform for testing anti-HEV treatments. Finally, we discuss how hepatocyte polarity is likely an important factor in the maturation and spread of infectious HEV particles.
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Affiliation(s)
- Viet Loan Dao Thi
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
| | - Xianfang Wu
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
| | - Charles M Rice
- Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10065
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18
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Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion. PLoS Pathog 2018; 14:e1007471. [PMID: 30532200 PMCID: PMC6307819 DOI: 10.1371/journal.ppat.1007471] [Citation(s) in RCA: 70] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2018] [Revised: 12/27/2018] [Accepted: 11/16/2018] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle. Hepatitis E virus (HEV) infection is believed to be the most common cause of acute hepatitis and jaundice in the world. HEV is a positive-strand RNA virus found as a non-enveloped virion in bile and feces or as a quasi-enveloped virion in blood and in cell culture. The HEV ORF3 protein is involved in viral particle secretion likely through the exosomal pathway. Here, we provide evidence for palmitoylation of ORF3 protein at its N-terminal cysteine-rich domain. Palmitoylation of ORF3 protein determines its subcellular localization and function in particle secretion. In addition, our data indicate a membrane topology where HEV ORF3 protein is entirely exposed to the cytosol, providing important insight into its interactions in the viral life cycle.
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19
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Sander AL, Corman VM, Lukashev AN, Drexler JF. Evolutionary Origins of Enteric Hepatitis Viruses. Cold Spring Harb Perspect Med 2018; 8:cshperspect.a031690. [PMID: 29610146 DOI: 10.1101/cshperspect.a031690] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The enterically transmitted hepatitis A (HAV) and hepatitis E viruses (HEV) are the leading causes of acute viral hepatitis in humans. Despite the discovery of HAV and HEV 40-50 years ago, their evolutionary origins remain unclear. Recent discoveries of numerous nonprimate hepatoviruses and hepeviruses allow revisiting the evolutionary history of these viruses. In this review, we provide detailed phylogenomic analyses of primate and nonprimate hepatoviruses and hepeviruses. We identify conserved and divergent genomic properties and corroborate historical interspecies transmissions by phylogenetic comparisons and recombination analyses. We discuss the likely non-recent origins of human HAV and HEV precursors carried by mammals other than primates, and detail current zoonotic HEV infections. The novel nonprimate hepatoviruses and hepeviruses offer exciting new possibilities for future research focusing on host range and the unique biological properties of HAV and HEV.
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Affiliation(s)
- Anna-Lena Sander
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany.,German Center for Infection Research (DZIF), Germany
| | - Victor Max Corman
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany.,German Center for Infection Research (DZIF), Germany
| | - Alexander N Lukashev
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, 119991 Moscow, Russia.,Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Preparations, 142782 Moscow, Russia
| | - Jan Felix Drexler
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany.,German Center for Infection Research (DZIF), Germany
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20
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Glitscher M, Himmelsbach K, Woytinek K, Johne R, Reuter A, Spiric J, Schwaben L, Grünweller A, Hildt E. Inhibition of Hepatitis E Virus Spread by the Natural Compound Silvestrol. Viruses 2018; 10:E301. [PMID: 29865243 PMCID: PMC6024817 DOI: 10.3390/v10060301] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Revised: 05/18/2018] [Accepted: 05/31/2018] [Indexed: 02/07/2023] Open
Abstract
Every year, there are about 20 Mio hepatitis E virus (HEV) infections and 60,000 deaths that are associated with HEV worldwide. At the present, there exists no specific therapy for HEV. The natural compound silvestrol has a potent antiviral effect against the (-)-strand RNA-virus Ebola virus, and also against the (+)-strand RNA viruses Corona-, Picorna-, and Zika virus. The inhibitory effect on virus spread is due to an inhibition of the DEAD-box RNA helicase eIF4A, which is required to unwind structured 5'-untranslated regions (UTRs). This leads to an impaired translation of viral RNA. The HEV (+)-strand RNA genome contains a 5'-capped, short 5'-UTR. This study aims to analyze the impact of silvestrol on the HEV life cycle. Persistently infected A549 cells were instrumental. This study identifies silvestrol as a potent inhibitor of the release of HEV infectious viral particles. This goes along with a strongly reduced HEV capsid protein translation, retention of viral RNA inside the cytoplasm, and without major cytotoxic effects. Interestingly, in parallel silvestrol affects the activity of the antiviral major vault protein (MVP) by translocation from the cytoplasm to the perinuclear membrane. These data further characterize the complex antiviral activity of silvestrol and show silvestrol's broad spectrum of function, since HEV is a virus without complex secondary structures in its genome, but it is still affected.
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Affiliation(s)
- Mirco Glitscher
- Department of Virology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
| | | | - Kathrin Woytinek
- Department of Virology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
| | - Reimar Johne
- Federal Institute for Risk Assessment, 10589 Berlin, Germany.
| | - Andreas Reuter
- Department of Allergology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
| | - Jelena Spiric
- Department of Allergology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
| | - Luisa Schwaben
- Department of Allergology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
| | - Arnold Grünweller
- Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg, 35037 Marburg, Germany.
| | - Eberhard Hildt
- Department of Virology, Paul-Ehrlich-Institut, 63225 Langen, Germany.
- German Center for Infection Research (DZIF), 38124 Braunschweig, Germany.
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21
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Syed SF, Zhao Q, Umer M, Alagawany M, Ujjan IA, Soomro F, Bangulzai N, Baloch AH, Abd El-Hack M, Zhou EM, Arain MA. Past, present and future of hepatitis E virus infection: Zoonotic perspectives. Microb Pathog 2018; 119:103-108. [DOI: 10.1016/j.micpath.2018.03.051] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Revised: 03/26/2018] [Accepted: 03/27/2018] [Indexed: 02/08/2023]
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22
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Fenaux H, Chassaing M, Berger S, Jeulin H, Gentilhomme A, Bensenane M, Bronowicki J, Gantzer C, Bertrand I, Schvoerer E. Molecular features of Hepatitis E Virus circulation in environmental and human samples. J Clin Virol 2018; 103:63-70. [PMID: 29656087 DOI: 10.1016/j.jcv.2018.04.003] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2018] [Revised: 03/20/2018] [Accepted: 04/03/2018] [Indexed: 12/20/2022]
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23
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Tanggis, Kobayashi T, Takahashi M, Jirintai S, Nishizawa T, Nagashima S, Nishiyama T, Kunita S, Hayama E, Tanaka T, Mulyanto, Okamoto H. An analysis of two open reading frames (ORF3 and ORF4) of rat hepatitis E virus genome using its infectious cDNA clones with mutations in ORF3 or ORF4. Virus Res 2018; 249:16-30. [DOI: 10.1016/j.virusres.2018.02.014] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 02/14/2018] [Accepted: 02/14/2018] [Indexed: 01/13/2023]
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24
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Abstract
At least 20 million hepatitis E virus (HEV) infections occur annually, with >3 million symptomatic cases and ∼60,000 fatalities. Hepatitis E is generally self-limiting, with a case fatality rate of 0.5-3% in young adults. However, it can cause up to 30% mortality in pregnant women in the third trimester and can become chronic in immunocompromised individuals, such as those receiving organ transplants or chemotherapy and individuals with HIV infection. HEV is transmitted primarily via the faecal-oral route and was previously thought to be a public health concern only in developing countries. It is now also being frequently reported in industrialized countries, where it is transmitted zoonotically or through organ transplantation or blood transfusions. Although a vaccine for HEV has been developed, it is only licensed in China. Additionally, no effective, non-teratogenic and specific treatments against HEV infections are currently available. Although progress has been made in characterizing HEV biology, the scarcity of adequate experimental platforms has hampered further research. In this Review, we focus on providing an update on the HEV life cycle. We will further discuss existing cell culture and animal models and highlight platforms that have proven to be useful and/or are emerging for studying other hepatotropic (viral) pathogens.
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Affiliation(s)
- Ila Nimgaonkar
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
| | - Qiang Ding
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
| | - Robert E Schwartz
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA
| | - Alexander Ploss
- Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA
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Paliwal D, Joshi P, Panda SK. Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR. PLoS One 2017; 12:e0187334. [PMID: 29091957 PMCID: PMC5665557 DOI: 10.1371/journal.pone.0187334] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Accepted: 10/18/2017] [Indexed: 12/18/2022] Open
Abstract
Background The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role. Methods Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein. Results ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature. Conclusions These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.
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Affiliation(s)
- Daizy Paliwal
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
| | - Prashant Joshi
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
| | - Subrat Kumar Panda
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
- * E-mail:
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ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication. J Virol 2017; 91:JVI.00621-17. [PMID: 28724761 DOI: 10.1128/jvi.00621-17] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2017] [Accepted: 07/05/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals, especially in solid-organ transplant recipients. Innate immunity, via the type I interferon (IFN) response, plays an important role during the initial stages of a viral infection. IFN-stimulated gene 15 (ISG15), an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role and can have a direct antiviral effect on a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates enhanced type I IFN-mediated signaling, resulting in an increase in the type I IFN-mediated antiviral effect during HEV replication. This observed enhanced type I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in the ISG15-mediated type I IFN sensitivity of HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role and regulates HEV sensitivity to exogenous type I IFN.IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, chronic HEV infection has recently become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid-organ transplant patients with some success. However, the mechanism behind the type I IFN-mediated antiviral effect against HEV remains unclear. This report demonstrates that ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type I IFN signaling and, thus, HEV sensitivity to type I IFN. Our results also show that PKR and OAS1 play important roles in the ISG15-mediated type I IFN sensitivity of HEV.
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Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles. Proc Natl Acad Sci U S A 2017; 114:1147-1152. [PMID: 28096411 DOI: 10.1073/pnas.1614955114] [Citation(s) in RCA: 174] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.
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Takahashi M, Kobayashi T, Tanggis, Jirintai S, Mulyanto, Nagashima S, Nishizawa T, Kunita S, Okamoto H. Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles. Arch Virol 2016; 161:3391-3404. [DOI: 10.1007/s00705-016-3047-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2016] [Accepted: 09/01/2016] [Indexed: 02/07/2023]
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Nan Y, Zhang YJ. Molecular Biology and Infection of Hepatitis E Virus. Front Microbiol 2016; 7:1419. [PMID: 27656178 PMCID: PMC5013053 DOI: 10.3389/fmicb.2016.01419] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2016] [Accepted: 08/26/2016] [Indexed: 12/13/2022] Open
Abstract
Hepatitis E virus (HEV) is a viral pathogen transmitted primarily via fecal-oral route. In humans, HEV mainly causes acute hepatitis and is responsible for large outbreaks of hepatitis across the world. The case fatality rate of HEV-induced hepatitis ranges from 0.5 to 3% in young adults and up to 30% in infected pregnant women. HEV strains infecting humans are classified into four genotypes. HEV strains from genotypes 3 and 4 are zoonotic, whereas those from genotypes 1 and 2 have no known animal reservoirs. Recently, notable progress has been accomplished for better understanding of HEV biology and infection, such as chronic HEV infection, in vitro cell culture system, quasi-enveloped HEV virions, functions of the HEV proteins, mechanism of HEV antagonizing host innate immunity, HEV pathogenesis and vaccine development. However, further investigation on the cross-species HEV infection, host tropism, vaccine efficacy, and HEV-specific antiviral strategy is still needed. This review mainly focuses on molecular biology and infection of HEV and offers perspective new insight of this enigmatic virus.
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Affiliation(s)
- Yuchen Nan
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F UniversityYangling, China; Molecular Virology Laboratory, VA-MD College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, College Park, College ParkMD, USA
| | - Yan-Jin Zhang
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, College Park, College Park MD, USA
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Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses. J Virol 2016; 90:4232-4242. [PMID: 26865708 DOI: 10.1128/jvi.02804-15] [Citation(s) in RCA: 193] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2015] [Accepted: 02/04/2016] [Indexed: 12/16/2022] Open
Abstract
UNLABELLED The hepatitis E virus (HEV) sheds into feces as nonenveloped virions but circulates in the blood in a membrane-associated, quasi-enveloped form (eHEV). Since the eHEV virions lack viral proteins on the surface, we investigated the entry mechanism for eHEV. We found that compared to nonenveloped HEV virions, eHEV attachment to the cell was much less efficient, requiring a longer inoculation time to reach its maximal infectivity. A survey of cellular internalization pathways identified clathrin-mediated endocytosis as the main route for eHEV entry. Unlike nonenveloped HEV virions, eHEV entry requires Rab5 and Rab7, small GTPases involved in endosomal trafficking, and blocking endosomal acidification abrogated eHEV infectivity. However, low pH alone was not sufficient for eHEV uncoating, suggesting that additional steps are required for entry. Supporting this concept, eHEV infectivity was substantially reduced in cells depleted of Niemann-Pick disease type C1, a lysosomal protein required for cholesterol extraction from lipid, or in cells treated with an inhibitor of lysosomal acid lipase. These data support a model in which the quasi-envelope is degraded within the lysosome prior to virus uncoating, a potentially novel mechanism for virus entry. IMPORTANCE The recent discovery of quasi-enveloped viruses has shifted the paradigm of virus-host interactions. The impact of quasi-envelopment in the virus life cycle and pathogenesis is largely unknown. HEV is a highly relevant model to study these questions. HEV circulates as quasi-enveloped virions in the blood that are hidden from neutralizing antibodies. eHEV particles most likely are responsible for the cell-to-cell spread of the virus. Given the increasing concerns about persistent HEV infection and its potential for transmission via the blood supply, understanding how eHEV infects cells is important for understanding its pathogenesis and developing therapies. Our data provide evidence that eHEV uses a potentially novel mechanism for cellular entry. Several steps critical to eHEV entry were identified and may provide a basis for developing treatments for hepatitis E. Because quasi-enveloped viruses resemble exosomes, these data also may provide insights into the exosome-mediated intercellular communications.
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Sobhy H. A Review of Functional Motifs Utilized by Viruses. Proteomes 2016; 4:proteomes4010003. [PMID: 28248213 PMCID: PMC5217368 DOI: 10.3390/proteomes4010003] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2015] [Revised: 01/07/2016] [Accepted: 01/13/2016] [Indexed: 01/05/2023] Open
Abstract
Short linear motifs (SLiM) are short peptides that facilitate protein function and protein-protein interactions. Viruses utilize these motifs to enter into the host, interact with cellular proteins, or egress from host cells. Studying functional motifs may help to predict protein characteristics, interactions, or the putative cellular role of a protein. In virology, it may reveal aspects of the virus tropism and help find antiviral therapeutics. This review highlights the recent understanding of functional motifs utilized by viruses. Special attention was paid to the function of proteins harboring these motifs, and viruses encoding these proteins. The review highlights motifs involved in (i) immune response and post-translational modifications (e.g., ubiquitylation, SUMOylation or ISGylation); (ii) virus-host cell interactions, including virus attachment, entry, fusion, egress and nuclear trafficking; (iii) virulence and antiviral activities; (iv) virion structure; and (v) low-complexity regions (LCRs) or motifs enriched with residues (Xaa-rich motifs).
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Affiliation(s)
- Haitham Sobhy
- Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
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