The intergenic-junction variant (genotype 2 isolate) of hepatitis E virus restores the CREX ‘stem-loop’ structural integrity, essential for viral life cycle

ABSTRACT Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex. IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

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P196: The HEV (genotype 2) intergenic-junction variant restores the CREX RNA ‘stem-loop’ integrity, essential for viral life cycle

Journal of Viral Hepatitis ◽

10.1111/jvh.179_12425 ◽

2015 ◽

Vol 22 ◽

pp. 118-118

Keyword(s):

Life Cycle ◽

Viral Life Cycle ◽

Stem Loop ◽

Genotype 2

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Identification and characterization of avian hepatitis E virus genotype 2 from chickens with hepatitis-splenomegaly syndrome in Korea

Virus Genes ◽

10.1007/s11262-016-1351-9 ◽

2016 ◽

Vol 52 (5) ◽

pp. 738-742 ◽

Cited By ~ 9

Author(s):

Hyun-Woo Moon ◽

Byung-Woo Lee ◽

Haan Woo Sung ◽

Byung-Il Yoon ◽

Hyuk Moo Kwon

Keyword(s):

Hepatitis E Virus ◽

Hepatitis E ◽

Virus Genotype ◽

Genotype 2 ◽

Avian Hepatitis E Virus ◽

Identification And Characterization

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Life cycle and morphogenesis of the hepatitis E virus

Emerging Microbes & Infections ◽

10.1038/s41426-018-0198-7 ◽

2018 ◽

Vol 7 (1) ◽

pp. 1-12 ◽

Cited By ~ 12

Author(s):

Kiyoshi Himmelsbach ◽

Daniela Bender ◽

Eberhard Hildt

Keyword(s):

Life Cycle ◽

Hepatitis E Virus ◽

Hepatitis E

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Novel hepatitis E like virus found in Swedish moose

Journal of General Virology ◽

10.1099/vir.0.059238-0 ◽

2014 ◽

Vol 95 (3) ◽

pp. 557-570 ◽

Cited By ~ 42

Author(s):

Jay Lin ◽

Heléne Norder ◽

Henrik Uhlhorn ◽

Sándor Belák ◽

Frederik Widén

Keyword(s):

Hepatitis E Virus ◽

Sequence Similarity ◽

Mammalian Species ◽

Hepatitis E ◽

Human Infection ◽

Stem Loop ◽

Novel Virus ◽

Human Infections ◽

Transmission Pathways

A novel virus was detected in a sample collected from a Swedish moose (Alces alces). The virus was suggested as a member of the Hepeviridae family, although it was found to be highly divergent from the known four genotypes (gt1–4) of hepatitis E virus (HEV). Moose are regularly hunted for consumption in the whole of Scandinavia. Thus, the finding of this virus may be important from several aspects: (a) as a new diverged HEV in a new animal species, and (b) potential unexplored HEV transmission pathways for human infections. Considering these aspects, we have started the molecular characterization of this virus. A 5.1 kb amplicon was sequenced, and corresponded to the partial ORF1, followed by complete ORF2, ORF3 and poly(A) sequence. In comparison with existing HEVs, the moose HEV genome showed a general nucleotide sequence similarity of 37–63 % and an extensively divergent putative ORF3 sequence. The junction region between the ORFs was also highly divergent; however, two putative secondary stem–loop structures were retained when compared to gt1–4, but with altered structural appearance. In the phylogenetic analysis, the moose HEV deviated and formed its own branch between the gt1–4 and other divergent animal HEVs. The characterization of this highly divergent genome provides important information regarding the diversity of HEV infecting various mammalian species. However, further studies are needed to investigate its prevalence in the moose populations and possibly in other host species, including the risk for human infection.

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The viral ORF3 protein is required for hepatitis E virus apical release and efficient growth in polarized hepatocytes and humanized mice

Journal of Virology ◽

10.1128/jvi.00585-21 ◽

2021 ◽

Author(s):

Gulce Sari ◽

Jingting Zhu ◽

Charuta Ambardekar ◽

Xin Yin ◽

Andre Boonstra ◽

...

Keyword(s):

Life Cycle ◽

Persistent Infection ◽

Human Liver ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Productive Infection ◽

Humanized Mice ◽

Hepatocyte Culture ◽

Fecal Shedding ◽

Orf3 Protein

Hepatitis E virus (HEV), an enterically transmitted RNA virus, is a major cause of acute hepatitis worldwide. Additionally, HEV genotype (gt) 3 can frequently persist in immunocompromised individuals with an increased risk for developing severe liver disease. Currently, no HEV-specific treatment is available. The viral open reading frame 3 (ORF3) protein facilitates HEV egress in vitro and is essential for establishing productive infection in macaques. Thus, ORF3, which is unique to HEV, has the potential to be explored as a target for antiviral therapy. However, significant gaps exist in our understanding of the critical functions of ORF3 in HEV infection in vivo . Here, we utilized a polarized hepatocyte culture model and a human liver chimeric mouse model to dissect the roles of ORF3 in gt3 HEV release and persistent infection. We show that ORF3’s absence substantially decreased HEV replication and virion release from the apical surface but not the basolateral surface of polarized hepatocytes. While the wild-type HEV established a persistent infection in humanized mice, mutant HEV lacking ORF3 (ORF3null) failed to sustain the infection despite transient replication in the liver and was ultimately cleared. Strikingly, mice inoculated with the ORF3null virus displayed no fecal shedding throughout the six-week experiment. Overall, our results demonstrate that ORF3 is required for HEV fecal shedding and persistent infection, providing a rationale for targeting ORF3 as a treatment strategy for HEV infection. Importance HEV infections are associated with significant morbidity and mortality. HEV gt3 additionally can cause persistent infection which can rapidly progress to liver cirrhosis. Currently, no HEV-specific treatments are available. The poorly understood HEV life cycle hampers the development of antivirals for HEV. Here we investigated the role of the viral ORF3 protein in HEV infection in polarized hepatocyte culture and human liver chimeric mice. We found that two major aspects of the HEV life cycle require ORF3: fecal virus shedding and persistent infection. These results provide a rationale for targeting ORF3 to treat HEV infection.

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Hepatitis E Virus Assembly and Release

Viruses ◽

10.3390/v11060539 ◽

2019 ◽

Vol 11 (6) ◽

pp. 539 ◽

Cited By ~ 3

Author(s):

Xiaohui Ju ◽

Qiang Ding

Keyword(s):

Life Cycle ◽

Hepatitis E Virus ◽

Current Knowledge ◽

Virus Assembly ◽

Antiviral Treatment ◽

Hepatitis E ◽

Host Cells ◽

Direct Acting Antiviral ◽

Direct Acting

Hepatitis E is an underestimated threat to public health, caused by the hepatitis E virus (HEV). HEV is the most common cause of acute viral hepatitis in the world, with no available direct-acting antiviral treatment. According to a recent WHO report, 20 million people become infected with HEV annually, resulting in 44,000 deaths. However, due to the scarcity of efficient in vitro cell culture systems for HEV, our knowledge of the life cycle of HEV is incomplete. Recently, significant progress has been made towards gaining a more comprehensive view of the HEV life cycle, as several in vitro culturing systems have been developed in recent years. Here, we review current knowledge and recent advances with regard to the HEV life cycle, with a particular focus on the assembly and release of viral particles. We also discuss the knowledge gaps in HEV assembly and release. Meanwhile, we highlight experimental platforms that could potentially be utilized to fill these gaps. Lastly, we offer perspectives on the future of research into HEV virology and its interaction with host cells.

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The Nucleotides on the Stem-Loop RNA Structure in the Junction Region of the Hepatitis E Virus Genome Are Critical for Virus Replication

Journal of Virology ◽

10.1128/jvi.01475-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 13040-13044 ◽

Cited By ~ 48

Author(s):

Dianjun Cao ◽

Yao-Wei Huang ◽

Xiang-Jin Meng

Keyword(s):

Virus Replication ◽

Intergenic Region ◽

Rna Structure ◽

Hepatitis E Virus ◽

Reporter Genes ◽

Hepatitis E ◽

Virus Genome ◽

Junction Region ◽

Stem Loop ◽

Huh7 Cells

ABSTRACT The roles of conserved nucleotides on the stem-loop (SL) structure in the intergenic region of the hepatitis E virus (HEV) genome in virus replication were determined by using Huh7 cells transfected with HEV SL mutant replicons containing reporter genes. One or two nucleotide mutations of the AGA motif on the loop significantly reduced HEV replication, and three or more nucleotide mutations on the loop abolished HEV replication. Mutations on the stem and of the subgenome start sequence also significantly inhibited HEV replication. The results indicated that both the sequence and the SL structure in the junction region play important roles in HEV replication.

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