Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

ABSTRACT Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

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Evidence of the Extrahepatic Replication of Hepatitis E Virus in Human Endometrial Stromal Cells

Pathogens ◽

10.3390/pathogens9040295 ◽

2020 ◽

Vol 9 (4) ◽

pp. 295 ◽

Cited By ~ 5

Author(s):

Mohamed A. El-Mokhtar ◽

Essam R. Othman ◽

Maha Y. Khashbah ◽

Ali Ismael ◽

Mohamed AA Ghaliony ◽

...

Keyword(s):

Capsid Protein ◽

Stromal Cells ◽

Hepatitis E Virus ◽

Inflammatory Responses ◽

Hepatitis E ◽

Endometrial Stromal Cells ◽

Extrahepatic Replication ◽

Efficient Replication ◽

Over Time

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The tropism of HEV is not restricted to the liver, and the virus replicates in other organs. Not all the extrahepatic targets for HEV are identified. Herein, we found that non-decidualized primary human endometrial stromal cells (PHESCs), which are precursors for the decidua and placenta, are susceptible to HEV infection. PHESCs, isolated from healthy non-pregnant women (n = 5), were challenged with stool-derived HEV-1 and HEV-3. HEV RNA was measured by qPCR, and HEV capsid protein was assessed by flow cytometry, immunofluorescence (IF), and ELISA. HEV infection was successfully established in PHESCs. Intracellular and extracellular HEV RNA loads were increased over time, indicating efficient replication in vitro. In addition, HEV capsid protein was detected intracellularly in the HEV-infected PHESCs and accumulated extracellularly over time, confirming the viral assembly and release from the infected cells. HEV-1 replicated more efficiently in PHESCs than HEV-3 and induced more inflammatory responses. Ribavirin (RBV) treatment abolished the replication of HEV in PHESCs. In conclusion, PHESCs are permissive to HEV infection and these cells could be an endogenous source of HEV infection during pregnancy and mediate HEV vertical transmission.

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Production of monoclonal antibodies against hepatitis E virus capsid protein and evaluation of their neutralizing activity in a cell culture system

Archives of Virology ◽

10.1007/s00705-008-0045-6 ◽

2008 ◽

Vol 153 (4) ◽

pp. 657-666 ◽

Cited By ~ 48

Author(s):

Masaharu Takahashi ◽

Yu Hoshino ◽

Toshinori Tanaka ◽

Hideyuki Takahashi ◽

Tsutomu Nishizawa ◽

...

Keyword(s):

Cell Culture ◽

Monoclonal Antibodies ◽

Capsid Protein ◽

Culture System ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Cell Culture System ◽

Virus Capsid ◽

A Cell ◽

Virus Capsid Protein

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Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture

Viruses ◽

10.3390/v13010075 ◽

2021 ◽

Vol 13 (1) ◽

pp. 75

Author(s):

Kush K. Yadav ◽

Patricia A. Boley ◽

Zachary Fritts ◽

Scott P. Kenney

Keyword(s):

Cell Culture ◽

Viral Replication ◽

Pregnant Women ◽

Cell Lines ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Target Cells ◽

Genotype 1 ◽

Orf4 Protein

Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

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Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721345115 ◽

2018 ◽

Vol 115 (18) ◽

pp. 4773-4778 ◽

Cited By ~ 39

Author(s):

Xin Yin ◽

Dong Ying ◽

Sébastien Lhomme ◽

Zimin Tang ◽

Christopher M. Walker ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Secretory Pathway ◽

Signal Sequence ◽

Cell Receptor ◽

Hepatitis E ◽

Start Codon ◽

Free Form ◽

Virus Particles ◽

And Function

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as “quasi-enveloped” particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2Sresults from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2Ssecretion via the secretory pathway. Unlike ORF2C, ORF2Sis glycosylated and exists as a dimer. Nonetheless, ORF2Sexhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2Sdoes not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

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Characterization of capsid protein (p495) of hepatitis E virus expressed in Escherichia coli and assembling into particles in vitro

Vaccine ◽

10.1016/j.vaccine.2018.03.005 ◽

2018 ◽

Vol 36 (16) ◽

pp. 2104-2111 ◽

Cited By ~ 2

Author(s):

Minghua Zheng ◽

Jie Jiang ◽

Xiao Zhang ◽

Nan Wang ◽

Kaihang Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E

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Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

Viruses ◽

10.3390/v13030406 ◽

2021 ◽

Vol 13 (3) ◽

pp. 406

Author(s):

Marie Pellerin ◽

Edouard Hirchaud ◽

Yannick Blanchard ◽

Nicole Pavio ◽

Virginie Doceul

Keyword(s):

Cell Culture ◽

Culture System ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Inhibitory Effect ◽

Cell Culture System ◽

Efficient Replication ◽

A Cell ◽

Vitro Model

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.

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New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

10.1101/435933 ◽

2018 ◽

Author(s):

Maliki Ankavay ◽

Claire Montpellier ◽

Ibrahim M. Sayed ◽

Jean-Michel Saliou ◽

Czeslaw Wychowski ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Molecular Mechanisms ◽

Protein A ◽

Hepatitis E ◽

Post Translational Modification ◽

C Protein ◽

Glycosylation Sites ◽

Orf2 Protein ◽

Infected Cells

AbstractHepatitis E Virus (HEV) genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Recently, we developed an efficient HEV cell culture system and demonstrated that this virus produces three different forms of its capsid protein: (i) the ORF2i form (infectious/intracellular) which is the form associated with the infectious particles, (ii) the ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are massively secreted glycoproteins not associated with infectious particles, but are the major antigens present in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). Although ORF2 protein is the most characterized viral protein, its glycosylation status and the biological relevance of this post-translational modification is still unclear. In the present study, we constructed and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. During our study, we also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. In conclusion, our study led to new insights into the molecular mechanisms of ORF2 expression.

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Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces

Acta Biológica Colombiana ◽

10.15446/abc.v24n3.79351 ◽

2019 ◽

Vol 24 (3) ◽

pp. 503-508

Author(s):

Carolina Quintero-Gil ◽

Santiago Mirazo ◽

Jaime Parra-Suescún ◽

Albeiro López-Herrera ◽

Victoria Mainardi ◽

...

Keyword(s):

Cell Culture ◽

Cell Line ◽

Post Infection ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Viral Rna ◽

Human Feces ◽

Genotype 3 ◽

Virus Genotype

Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/μL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/μL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

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Hepatitis E Virus (HEV) Genotype 3 Infection of Human Liver Chimeric Mice as a Model for Chronic HEV Infection

Journal of Virology ◽

10.1128/jvi.00114-16 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4394-4401 ◽

Cited By ~ 44

Author(s):

Martijn D. B. van de Garde ◽

Suzan D. Pas ◽

Guido van der Net ◽

Robert A. de Man ◽

Albert D. M. E. Osterhaus ◽

...

Keyword(s):

Cell Culture ◽

Human Liver ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Chimeric Mouse ◽

Chimeric Mice ◽

Genotype 3 ◽

Edta Plasma

ABSTRACTGenotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities ofin vivoHEV studies in a human liver chimeric mouse model (uPA+/+Nod-SCID-IL2Rγ−/−) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. Thesein vitroHEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 daysin vitro. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV bothin vitroandin vivo. Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans.IMPORTANCEHepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared thein vivoinfectivity of clinical samples from chronic HEV patients in human liver chimeric mice to anin vitrovirus culture system. Efficientin vivoHEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observedin vivoinfectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEV's pathobiology.

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