Characterization of the hepatitis E virus replicase

AbstractHepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing 3 open reading frames (ORFs). ORF1 encodes the ORF1 non– structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all 3 systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

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Hepatitis E Virus Replication

Viruses ◽

10.3390/v11080719 ◽

2019 ◽

Vol 11 (8) ◽

pp. 719 ◽

Cited By ~ 9

Author(s):

Robert LeDesma ◽

Ila Nimgaonkar ◽

Alexander Ploss

Keyword(s):

Hepatitis E Virus ◽

Rna Virus ◽

Hypervariable Region ◽

Hepatitis E ◽

Open Reading Frames ◽

Health Concern ◽

Viral Gene ◽

Genome Replication ◽

Replication Strategy ◽

Viral Gene Product

Hepatitis E virus (HEV) is a small quasi-enveloped, (+)-sense, single-stranded RNA virus belonging to the Hepeviridae family. There are at least 20 million HEV infections annually and 60,000 HEV-related deaths worldwide. HEV can cause up to 30% mortality in pregnant women and progress to liver cirrhosis in immunocompromised individuals and is, therefore, a greatly underestimated public health concern. Although a prophylactic vaccine for HEV has been developed, it is only licensed in China, and there is currently no effective, non-teratogenic treatment. HEV encodes three open reading frames (ORFs). ORF1 is the largest viral gene product, encoding the replicative machinery of the virus including a methyltransferase, RNA helicase, and an RNA-dependent RNA polymerase. ORF1 additionally contains a number of poorly understood domains including a hypervariable region, a putative protease, and the so-called ‘X’ and ‘Y’ domains. ORF2 is the viral capsid essential for formation of infectious particles and ORF3 is a small protein essential for viral release. In this review, we focus on the domains encoded by ORF1, which collectively mediate the virus’ asymmetric genome replication strategy. We summarize what is known, unknown, and hotly debated regarding the coding and non-coding regions of HEV ORF1, and present a model of how HEV replicates its genome.

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Hepatitis E virus

10.1093/med/9780198570028.003.0048 ◽

2011 ◽

Author(s):

X. J. Meng

Keyword(s):

Hepatitis E Virus ◽

Animal Species ◽

Rna Virus ◽

Hepatitis E ◽

Open Reading Frames ◽

Industrialized Countries ◽

Efficient Cell Culture System ◽

The Family ◽

Avian Hev ◽

Animal Strains

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.

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Classification, structure and molecular diagnostics of avian hepatitis E virus

Medycyna Weterynaryjna ◽

10.21521/mw.6228 ◽

2019 ◽

Vol 75 (02) ◽

pp. 6228-2019

Author(s):

JOANNA NERC ◽

PIOTR SZELESZCZUK

Keyword(s):

Hepatitis E Virus ◽

Rna Virus ◽

Genetic Material ◽

Hepatitis E ◽

Open Reading Frames ◽

Aetiological Factor ◽

Base Pairs ◽

Avian Hepatitis E Virus ◽

And Function ◽

Overlapping Open Reading Frames

Pathological syndromes caused by avian hepatitis E virus have been described as big liver and spleen disease; necrotic haemorrhagic hepatitis-splenomegaly syndrome; necrotic, haemorrhagic, hepatomegalic hepatitis; or hepatitis-liver haemorrhage syndrome. The aetiological factor of this syndrome belongs to the Hepeviridae family. Avian hepatitis E virus is a single-strand RNA virus whose genome consists of approx. 7,200 base pairs and contains a short non-coding 5’ terminus (27-35 nucleotides) followed by three partially overlapping open reading frames: ORF1, ORF3 and ORF2. Since the avian strains of hepatitis viruses do not replicate in cell cultures, molecular biology techniques are used in the diagnosis of infections. This article discusses in detail the structure and function of each ORF of avian hepatitis E virus, as well as methods for the identification of the genetic material of this pathogen.

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A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E Virus

Journal of Virology ◽

10.1128/jvi.00046-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5919-5926 ◽

Cited By ~ 170

Author(s):

Judith Graff ◽

Udana Torian ◽

Hanh Nguyen ◽

Suzanne U. Emerson

Keyword(s):

Resistance Gene ◽

Cell Lines ◽

Northern Blot ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Open Reading Frames ◽

Subgenomic Rna ◽

Neomycin Resistance Gene ◽

Neomycin Resistance ◽

Reading Frames

ABSTRACT Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

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Full-length genome of a novel genotype 3 hepatitis E virus strain obtained from domestic pigs in Japan

Virus Research ◽

10.1016/j.virusres.2017.08.003 ◽

2017 ◽

Vol 240 ◽

pp. 147-153 ◽

Cited By ~ 2

Author(s):

Putu Prathiwi Primadharsini ◽

Masao Miyake ◽

Satoshi Kunita ◽

Tsutomu Nishizawa ◽

Masaharu Takahashi ◽

...

Keyword(s):

Virus Strain ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Full Length ◽

Genotype 3 ◽

Domestic Pigs ◽

Novel Genotype ◽

Full Length Genome

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Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants

Journal of Clinical Microbiology ◽

10.1128/jcm.01686-17 ◽

2018 ◽

Vol 56 (5) ◽

Cited By ~ 7

Author(s):

Bo Wang ◽

Dominik Harms ◽

C. Patrick Papp ◽

Sandra Niendorf ◽

Sonja Jacobsen ◽

...

Keyword(s):

Chronic Hepatitis ◽

Hepatitis E Virus ◽

Phylogenetic Analyses ◽

Hepatitis E ◽

Full Length ◽

Molecular Approach ◽

Genotype 3 ◽

Genome Sequences ◽

Virus Genotype ◽

Pcr Targeting

ABSTRACT Autochthonous hepatitis E virus genotype 3 (HEV-3) infections in industrialized countries are more frequent than previously assumed. HEV-3 is zoonotic and the causal pathogen of chronic hepatitis E. According to the latest classification of the family Hepeviridae , 10 designated HEV-3 subtypes (HEV-3a to HEV-3j) and 7 unassigned HEV-3 subtypes are proposed. In order to identify and characterize the HEV-3 variants in circulation, we developed a molecular approach combining a sensitive HEV-specific real-time reverse transcription-PCR (RT-PCR) targeting the overlapping region of HEV ORF2 and ORF3 (the ORF2/3 region) and two newly designed consensus nested RT-PCRs targeting the HEV ORF1 and ORF2 genes, respectively. Since complete genome sequences are required for new HEV-3 subtype assignment, we implemented a straightforward approach for full-length HEV-3 genome amplification. Twenty-nine human serum samples and six human feces samples from chronic hepatitis E patients were selected for evaluation of the system. Viral loads ranged from 1 × 10 4 to 1.9 × 10 10 copies/ml of serum and from 1.8 × 10 4 to 1 × 10 12 copies/g of feces. Sequence and phylogenetic analyses of partial ORF1 and ORF2 sequences showed that HEV strains had considerable genetic diversity and clustered into the HEV-3c (29/35), HEV-3e (2/35), HEV-3f (2/35), and unassigned HEV-3 (2/35) subtypes. Moreover, from these strains, three full-length HEV-3 genome sequences were generated and characterized. DE/15-0030 represents a typical HEV-3c strain (95.7% nucleotide identity to wbGER27), while DE/15-0031 and SW/16-0282 have <89.2% homology to known HEV-3 strains and are phylogenetically divergent, indicating novel HEV-3 subtypes. In summary, our approach will significantly facilitate the detection, quantification, and determination of HEV-3 strains and will thus help to improve molecular diagnostics and our knowledge of HEV diversity and evolution.

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Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

mBio ◽

10.1128/mbio.00769-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 13

Author(s):

Qiang Ding ◽

Ila Nimgaonkar ◽

Nicholas F. Archer ◽

Yaron Bram ◽

Brigitte Heller ◽

...

Keyword(s):

Hepatitis E Virus ◽

Regulatory Element ◽

Hepatitis E ◽

Regulatory Elements ◽

Genome Replication ◽

Genomic Rna ◽

Chronic Infections ◽

Particle Assembly ◽

Subgenomic Rna ◽

General Importance

ABSTRACT Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis -acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.

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