Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein

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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P0631 : The hepatitis E virus genotype 2 junction variant restores the crex ’stem-loop’ structural integrity, essential for viral life cycle

Journal of Hepatology ◽

10.1016/s0168-8278(15)30836-9 ◽

2015 ◽

Vol 62 ◽

pp. S555

Author(s):

M.K. Parvez

Keyword(s):

Life Cycle ◽

Hepatitis E Virus ◽

Structural Integrity ◽

Hepatitis E ◽

Viral Life Cycle ◽

Virus Genotype ◽

Stem Loop ◽

Genotype 2

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The Hepatitis E Virus Capsid C-Terminal Region Is Essential for the Viral Life Cycle: Implication for Viral Genome Encapsidation and Particle Stabilization

Journal of Virology ◽

10.1128/jvi.00444-13 ◽

2013 ◽

Vol 87 (10) ◽

pp. 6031-6036 ◽

Cited By ~ 24

Author(s):

T. Shiota ◽

T.-C. Li ◽

S. Yoshizaki ◽

T. Kato ◽

T. Wakita ◽

...

Keyword(s):

Life Cycle ◽

Viral Genome ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Viral Life Cycle ◽

Terminal Region ◽

Virus Capsid ◽

Particle Stabilization ◽

Genome Encapsidation

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Full-length genomic sequence analysis of new subtype 3k hepatitis E virus isolates with 99.97% nucleotide identity obtained from two consecutive acute hepatitis patients in a city in northeast Japan

Journal of Medical Virology ◽

10.1002/jmv.24743 ◽

2016 ◽

Vol 89 (6) ◽

pp. 1116-1120 ◽

Cited By ~ 12

Author(s):

Masahito Miura ◽

Jun Inoue ◽

Mio Tsuruoka ◽

Tsutomu Nishizawa ◽

Shigeo Nagashima ◽

...

Keyword(s):

Sequence Analysis ◽

Acute Hepatitis ◽

Hepatitis E Virus ◽

Genomic Sequence ◽

Hepatitis E ◽

Full Length ◽

Nucleotide Identity ◽

Genomic Sequence Analysis ◽

Virus Isolates

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

Gene ◽

10.1016/j.gene.2015.01.033 ◽

2015 ◽

Vol 559 (2) ◽

pp. 149-154 ◽

Cited By ~ 11

Author(s):

Mohammad Khalid Parvez

Keyword(s):

Life Cycle ◽

Hepatitis E Virus ◽

Structural Integrity ◽

Hepatitis E ◽

Viral Life Cycle ◽

Stem Loop ◽

Genotype 2 ◽

Variant Genotype

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Full-length genomic sequences of new subtype 1g hepatitis E virus strains obtained from four patients with imported or autochthonous acute hepatitis E in Japan

Infection Genetics and Evolution ◽

10.1016/j.meegid.2017.10.007 ◽

2017 ◽

Vol 55 ◽

pp. 343-349 ◽

Cited By ~ 4

Author(s):

Tsutomu Nishizawa ◽

Putu Prathiwi Primadharsini ◽

Masashi Namikawa ◽

Yuichi Yamazaki ◽

Satoko Uraki ◽

...

Keyword(s):

Acute Hepatitis ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Full Length ◽

Genomic Sequences ◽

Acute Hepatitis E ◽

Virus Strains

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Hepatitis E Virus Quasispecies and the Outcome of Acute Hepatitis E in Solid-Organ Transplant Patients

Journal of Virology ◽

10.1128/jvi.01003-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 10006-10014 ◽

Cited By ~ 71

Author(s):

Sebastien Lhomme ◽

Florence Abravanel ◽

Martine Dubois ◽

Karine Sandres-Saune ◽

Lionel Rostaing ◽

...

Keyword(s):

Liver Fibrosis ◽

Acute Hepatitis ◽

Hepatitis E Virus ◽

Organ Transplant ◽

Hepatitis E ◽

Solid Organ Transplant ◽

First Year ◽

Solid Organ ◽

Immunocompromised Patients ◽

Serum Concentrations

Hepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lowerKa/Ksratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.

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