A Yeast Two-Hybrid Study on Self-Association of the ORF2 Protein of Hepatitis E Virus

2001 ◽  
Vol 284 (3) ◽  
pp. 614-621 ◽  
Author(s):  
Shweta Tyagi ◽  
Shahid Jameel ◽  
Sunil K. Lal
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Yeast Two Hybrid ◽  
Orf2 Protein ◽  
Self Association ◽  
Two Hybrid

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

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 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 Cytoplasmic Localization of the ORF2 Protein of Hepatitis E Virus Is Dependent on Its Ability To Undergo Retrotranslocation from the Endoplasmic Reticulum

2007 ◽  
Vol 81 (7) ◽  
pp. 3339-3345 ◽  
Author(s):  
Milan Surjit ◽  
Shahid Jameel ◽  
Sunil K. Lal
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis E Virus ◽  
Major Capsid Protein ◽  
Rna Virus ◽  
Hepatitis E ◽  
26S Proteasome ◽  
Orf2 Protein ◽  
Surface Localization ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Hepatitis E virus (HEV) is a positive-strand RNA virus that is prevalent in much of the developing world. ORF2 is the major capsid protein of HEV. Although ORF2 is an N-linked glycoprotein, it is abundantly located in the cytoplasm in addition to having membrane and surface localization. The mechanism by which ORF2 protein obtains access to the cytoplasm is unknown. In this report, we prove that initially all ORF2 protein is present in the endoplasmic reticulum and a fraction of it becomes retrotranslocated to the cytoplasm. The ability of ORF2 to be retrotranslocated is dependent on its glycosylation status and follows the canonical dislocation pathway. However, in contrast to general substrates of the dislocation pathway, retrotranslocated ORF2 protein is not a substrate of the 26S proteasome complex and is readily detectable in the cytoplasm in the absence of any protease inhibitor, suggesting that the retrotranslocated protein is stable in the cytoplasm. This study thus defines the pathway by which ORF2 obtains access to the cytoplasm.

scholarly journals In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil

PLoS ONE ◽  
2017 ◽  
Vol 12 (5) ◽  
pp. e0176409 ◽  
Author(s):  
Rafael Pandolfi ◽  
Denise Ramos de Almeida ◽  
Marcelo Alves Pinto ◽  
Luiz Carlos Kreutz ◽  
Rafael Frandoloso
Keyword(s):  
Hepatitis E Virus ◽  
Blood Donors ◽  
Hepatitis E ◽  
Recombinant Orf2 Protein ◽  
Orf2 Protein ◽  
High Prevalence ◽  
South Brazil

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 The fate of Hepatitis E virus capsid protein is regulated by an Arginine-Rich Motif

2021 ◽  
Author(s):  
Kévin Hervouet ◽  
Martin Ferrié ◽  
Maliki Ankavay ◽  
Claire Montpellier ◽  
Charline Camuzet ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Hepatitis E Virus ◽  
Nuclear Translocation ◽  
Hepatitis E ◽  
Particle Assembly ◽  
Humoral Immune ◽  
Antiviral Responses ◽  
Orf2 Protein ◽  
Molecular Determinants ◽  
Arginine Rich Motif

Producing multifunctional proteins is one of the major strategies developed by viruses to condense their genetic information. Here, we investigated the molecular determinants of the multifunctionality of hepatitis E virus (HEV) ORF2 capsid protein. We previously identified 3 isoforms of ORF2 which are partitioned in different subcellular compartments to perform distinct functions. Notably, the infectious ORF2 (ORF2i) protein is the structural component of the virion, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. We identified a 5 amino acid Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region as a central regulator of the subcellular localizations and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. Furthermore, the ARM likely serves as a cleavage site of the glycosylated ORF2 protein. Finally, it promotes ORF2 membrane association that is likely essential for particle assembly. In conclusion, our observations highlight ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

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