scholarly journals Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1

2013 ◽  
Vol 94 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Julia Perttilä ◽  
Pirjo Spuul ◽  
Tero Ahola
Keyword(s):  
Hepatitis E Virus ◽  
Secretory Pathway ◽  
Hepatitis E ◽  
Proteolytic Processing ◽  
Full Length ◽  
Replication Protein ◽  
Positive Strand ◽  
Early Secretory Pathway ◽  
Positive Strand Rna

Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse–chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER–Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

scholarly journals A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

2008 ◽  
Vol 83 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Huiquan Liu ◽  
Yanping Fu ◽  
Daohong Jiang ◽  
Guoqing Li ◽  
Jun Xie ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Hepatitis E Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Hepatitis E ◽  
Genomic Rna ◽  
Human Virus ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.

scholarly journals In Vitro and In Vivo Mutational Analysis of the 3′-Terminal Regions of Hepatitis E Virus Genomes and Replicons

2005 ◽  
Vol 79 (2) ◽  
pp. 1017-1026 ◽  
Author(s):  
Judith Graff ◽  
Hanh Nguyen ◽  
Chaiyan Kasorndorkbua ◽  
Patrick G. Halbur ◽  
Marisa St. Claire ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Rna Replication ◽  
Hepatitis E ◽  
Human Strain ◽  
Full Length ◽  
Genotype 1

ABSTRACT Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3′-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3′-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3′-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3′ termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3′ terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3′-terminal mutations in vivo. Incorporation of the 3′-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

scholarly journals Mutational Analysis of Glycosylation, Membrane Translocation, and Cell Surface Expression of the Hepatitis E Virus ORF2 Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4074-4082 ◽  
Author(s):  
Mohammad Zafrullah ◽  
Mehmet Hakan Ozdener ◽  
Ravinder Kumar ◽  
Subrat Kumar Panda ◽  
Shahid Jameel
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Hepatitis E Virus ◽  
Mutational Analysis ◽  
Hepatitis E ◽  
Surface Expression ◽  
In Vitro Translation ◽  
Cell Surface Expression ◽  
Positive Strand Rna

ABSTRACT Hepatitis E virus (HEV) is the etiological agent for viral hepatitis type E, which is a major problem in the developing world. Because HEV cannot be cultured in vitro, very little information exists on the mechanisms of HEV gene expression and genome replication. HEV is a positive-strand RNA virus with three potential open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2). We earlier showed (S. Jameel, M. Zafrullah, M. H. Ozdener, and S. K. Panda, J. Virol. 70:207–216, 1996) pORF2 to be a ∼88-kDa glycoprotein, carrying N-linked glycans and a potential endoplasmic reticulum (ER)-directing signal at its N terminus. Treatment with the drugs brefeldin A and monensin suggest that the protein may accumulate within the ER. Based on mutational analysis, we demonstrate Asn-310 to be the major site of N-glycan addition. In COS-1 cell expression and in vitro translation experiments, we confirm the ER-translocating nature of the pORF2 N-terminal hydrophobic sequence and show that the protein is cotranslationally, but not posttranslationally, translocated across the ER membrane. Earlier, we had also demonstrated cell surface localization of a fraction of the COS-1 cell-expressed pORF2. Using glycosylation- and translocation-defective mutants of pORF2, we now show that while transit of pORF2 into the ER is necessary for its cell surface expression, glycosylation of the protein is not required for such localization. These results may offer clues to the mechanisms of gene expression and capsid assembly in HEV.

1354 IN VITRO INFECTION AND REPLICATION OF HEPATITIS E VIRUS IN HUMAN HEPATOCYTES

2011 ◽  
Vol 54 ◽  
pp. S535
Author(s):  
Y. Oshiro ◽  
H. Yasue ◽  
S. Hattori ◽  
M. Chiba ◽  
T. Naito ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Human Hepatocytes

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

2017 ◽  
Vol 240 ◽  
pp. 147-153 ◽  
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

scholarly journals Hepatitis E Virus Immunopathogenesis

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1180
Author(s):  
Kush Kumar Yadav ◽  
Scott P. Kenney
Keyword(s):  
Hepatitis E Virus ◽  
Organ Transplant ◽  
Hepatitis E ◽  
Oral Route ◽  
Solid Organ ◽  
Immunocompromised Patients ◽  
In Vivo Models ◽  
Transplant Patients

Hepatitis E virus is an important emerging pathogen producing a lethal impact on the pregnant population and immunocompromised patients. Starting in 1983, it has been described as the cause for acute hepatitis transmitted via the fecal–oral route. However, zoonotic and blood transfusion transmission of HEV have been reported in the past few decades, leading to the detailed research of HEV pathogenesis. The reason behind HEV being highly virulent to the pregnant population particularly during the third trimester, leading to maternal and fetal death, remains unknown. Various host factors (immunological, nutritional, hormonal) and viral factors have been studied to define the key determinants assisting HEV to be virulent in pregnant and immunocompromised patients. Similarly, chronic hepatitis is seen particularly in solid organ transplant patients, resulting in fatal conditions. This review describes recent advances in the immunopathophysiology of HEV infections in general, pregnant, and immunocompromised populations, and further elucidates the in vitro and in vivo models utilized to understand HEV pathogenesis.

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

2018 ◽  
Vol 56 (5) ◽  
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.

scholarly journals Optimized Hepatitis E Virus (HEV) Culture and Its Application to Measurements of HEV Infectivity

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 139 ◽  
Author(s):  
Nicolas Capelli ◽  
Martine Dubois ◽  
Mélanie Pucelle ◽  
Isabelle Da Silva ◽  
Sébastien Lhomme ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Fetal Bovine Serum ◽  
Hepatitis E ◽  
Chronic Liver Diseases ◽  
Infectious Dose ◽  
Clinical Strains ◽  
Fetal Bovine ◽  
Culture Supernatants ◽  
Tissue Culture Infectious Dose

Hepatitis E virus (HEV) is a major concern in public health worldwide. Infections with HEV genotypes 3, 4, or 7 can lead to chronic hepatitis while genotype 1 infections can trigger severe hepatitis in pregnant women. Infections with all genotypes can worsen chronic liver diseases. As virions are lipid-associated in blood and naked in feces, efficient methods of propagating HEV clinical strains in vitro and evaluating the infectivity of both HEV forms are needed. We evaluated the spread of clinical strains of HEV genotypes 1 (HEV1) and 3 (HEV3) by quantifying viral RNA in culture supernatants and cell lysates. Infectivity was determined by endpoint dilution and calculation of the tissue culture infectious dose 50 (TCID50). An enhanced HEV production could be obtained varying the composition of the medium, including fetal bovine serum (FBS) and dimethylsulfoxide (DMSO) content. This increased TCID50 from 10 to 100-fold and allowed us to quantify HEV1 infectivity. These optimized methods for propagating and measuring HEV infectivity could be applied to health safety processes and will be useful for testing new antiviral drugs.

scholarly journals Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans

2001 ◽  
Vol 75 (17) ◽  
pp. 7791-7802 ◽  
Author(s):  
Claus W. Grassmann ◽  
Olaf Isken ◽  
Norbert Tautz ◽  
Sven-Erik Behrens
Keyword(s):  
Viral Replication ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Molar Ratio ◽  
Replication Capacity ◽  
Coding Region ◽  
Positive Strand ◽  
Reading Frame ◽  
Diarrhea Virus ◽  
Positive Strand Rna

ABSTRACT The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cisor in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to becis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.

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