scholarly journals The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 24
Author(s):  
Takashi Nishiyama ◽  
Koji Umezawa ◽  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Satoshi Kunita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Structure Prediction ◽  
Hepatitis E Virus ◽  
Culture Media ◽  
Molecular Size ◽  
Hepatitis E ◽  
Terminal Region ◽  
Translation Product ◽  
Orf2 Protein

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

scholarly journals Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

2021 ◽  
Vol 9 (11) ◽  
pp. 2302
Author(s):  
Katja Schilling-Loeffler ◽  
Oliver Viera-Segura ◽  
Victor Max Corman ◽  
Julia Schneider ◽  
Ashish K. Gadicherla ◽  
...  
Keyword(s):  
Cell Culture ◽  
Wild Boar ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Enriched Library ◽  
Genome Copy ◽  
Genotype 3 ◽  
Wild Boars ◽  
Copy Numbers ◽  
Cell Culture Isolation

Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

scholarly journals Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Gayatri D. Kanade ◽  
Kunal D. Pingale ◽  
Yogesh A. Karpe
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Liver Cell ◽  
Serine Proteases ◽  
Hepatitis E Virus ◽  
Factor Xa ◽  
Hepatitis E ◽  
Cleavage Sites ◽  
Polyprotein Processing ◽  
Liver Cell Line

ABSTRACTHepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstratedin vitroprocessing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCEHepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

scholarly journals Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 178
Author(s):  
Yiyang Chen ◽  
Tianxiang Chen ◽  
Yuhang Luo ◽  
Jie Fan ◽  
Meimei Zhang ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Keyhole Limpet Hemocyanin ◽  
Hepatitis E ◽  
Genotype 4 ◽  
Virus Shedding ◽  
Fecal Shedding ◽  
Orf2 Protein ◽  
Swine Hepatitis ◽  
Phosphate Buffered Saline ◽  
Neutralizing Epitopes

Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2–4 weeks post challenge (wpc), seroconversion by 4–9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1–2 rabbits/group in groups 1–4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5–6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.

scholarly journals Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli

2014 ◽  
Vol 7 (7) ◽  
Author(s):  
Fatemeh Farshadpour ◽  
Reza Taherkhani ◽  
Manoochehr Makvandi ◽  
Hamid Rajabi Memari ◽  
Ali Reza Samarbafzadeh
Keyword(s):  
Escherichia Coli ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Expression And Purification ◽  
Orf2 Protein

scholarly journals Hepatitis E Virus ORF2 Protein Activates the Pro-Apoptotic Gene CHOP and Anti-Apoptotic Heat Shock Proteins

PLoS ONE ◽  
2011 ◽  
Vol 6 (9) ◽  
pp. e25378 ◽  
Author(s):  
Lijo John ◽  
Saijo Thomas ◽  
Ottmar Herchenröder ◽  
Brigitte M. Pützer ◽  
Stephan Schaefer
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Apoptotic Gene ◽  
Orf2 Protein ◽  
Shock Proteins

scholarly journals Robust hepatitis E virus infection and transcriptional response in human hepatocytes

2020 ◽  
Vol 117 (3) ◽  
pp. 1731-1741 ◽  
Author(s):  
Daniel Todt ◽  
Martina Friesland ◽  
Nora Moeller ◽  
Dimas Praditya ◽  
Volker Kinast ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
Hepatoma Cell ◽  
Hepatitis E ◽  
Human Hepatocytes ◽  
Cell Culture Model ◽  
Genotype 3 ◽  
Human Hepatoma Cell ◽  
Culture Model ◽  
Viral Titers

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral–host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

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