The fate of Hepatitis E virus capsid protein is regulated by an 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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Hepatitis E virus truncated capsid protein as a potential carrier of exogenous antigens for the development of chimeric virus-like particle vaccines

10.21203/rs.2.17916/v1 ◽

2019 ◽

Author(s):

Tianyu LU ◽

Nouredine BEHLOUL ◽

Yi ZHOU ◽

Sarra BAHA ◽

Zhenzhen LIU ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Foot And Mouth Disease ◽

Vaccine Design ◽

Hepatitis E ◽

Mouth Disease ◽

Virus Like Particle ◽

Orf2 Protein ◽

Mouth Disease Virus ◽

Foot And Mouth

Abstract Background: Virus like particle (VLP), a multiprotein structure which is assembled automatically, can stimulate robust immune responses due to an appropriate size, repetitive epitopes, and a structure similar to native virions. Utilizing VLPs as vaccine carriers to present exogenous antigens is a promising and challenging field in vaccine design. Hence, this study aims to investigate the potential of Hepatitis E virus (HEV) truncated capsid protein as a VLP carrier presenting foreign antigens in vaccine design.Results: S and M domains of HEV ORF2 protein (aa112-455) were selected as an optimal carrier (CaSM). The exogenous antigen Seq8 containing three immunogenic domains from three different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP vaccine candidate (CaSM-Seq8). Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs with a diameter of approximately 26 nm, similar to the VLPs of CaSM alone but smaller than native HEV virions. Further, the thermal stability and the proteolysis resistance of Seq8 were enhanced when carried by CaSM. The antigenicity analysis revealed a more robust reactivity against anti-FMDV specific antibodies when Seq8 was presented on the CaSM particles. Upon injection into mice, anti-FMDV IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 antigen alone or a commercial inactivated FMDV vaccine.Conclusions: This study demonstrates the potential of the HEV truncated capsid protein VLPs as a presenting-platform of exogenous antigens in vaccine design and promising preliminary results on chimeric VLP vaccine against foot-and-mouth disease.

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New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

10.1101/435933 ◽

2018 ◽

Author(s):

Maliki Ankavay ◽

Claire Montpellier ◽

Ibrahim M. Sayed ◽

Jean-Michel Saliou ◽

Czeslaw Wychowski ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Molecular Mechanisms ◽

Protein A ◽

Hepatitis E ◽

Post Translational Modification ◽

C Protein ◽

Glycosylation Sites ◽

Orf2 Protein ◽

Infected Cells

AbstractHepatitis E Virus (HEV) genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Recently, we developed an efficient HEV cell culture system and demonstrated that this virus produces three different forms of its capsid protein: (i) the ORF2i form (infectious/intracellular) which is the form associated with the infectious particles, (ii) the ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are massively secreted glycoproteins not associated with infectious particles, but are the major antigens present in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). Although ORF2 protein is the most characterized viral protein, its glycosylation status and the biological relevance of this post-translational modification is still unclear. In the present study, we constructed and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. During our study, we also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. In conclusion, our study led to new insights into the molecular mechanisms of ORF2 expression.

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The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus

Journal of General Virology ◽

10.1099/0022-1317-83-9-2201 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2201-2209 ◽

Cited By ~ 73

Author(s):

G. Haqshenas ◽

F. F. Huang ◽

M. Fenaux ◽

D. K. Guenette ◽

F. W. Pierson ◽

...

Keyword(s):

Capsid Protein ◽

Disease Virus ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Cross Reactivity ◽

Putative Open Reading Frame ◽

Orf2 Protein ◽

Avian Hepatitis E Virus ◽

Avian Hev ◽

Antigenic Epitopes

We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis–splenomegaly (HS) syndrome in the USA. We showed that avian HEV is genetically related to swine and human HEVs. Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human HEVs and the Australian chicken big liver and spleen disease virus (BLSV). The region encoding the C-terminal 268 amino acid residues of avian HEV ORF2 was cloned into expression vector pRSET-C. The truncated ORF2 protein was expressed in E. coli as a fusion protein and purified by affinity chromatography. Western blot analysis revealed that the avian HEV ORF2 protein reacted with antisera against the Sar-55 strain of human HEV and with convalescent antisera against swine HEV and the US2 strain of human HEV, as well as with antiserum against BLSV. Convalescent sera from specific-pathogen-free chickens experimentally infected with avian HEV also reacted with the recombinant capsid proteins of swine HEV and Sar-55 human HEV. Antisera against the US2 human HEV also reacted with recombinant ORF2 proteins of both swine HEV and Sar-55 human HEV. The antigenic cross-reactivity of the avian HEV putative capsid protein with those of swine and human HEVs was further confirmed, for the most part, by ELISA assays. The data indicate that avian HEV shares certain antigenic epitopes in its putative capsid protein with swine and human HEVs, as well as with BLSV. The results have implications for HEV diagnosis and taxonomy.

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The Capsid Protein of Hepatitis E Virus Inhibits Interferon Induction via Its N-Terminal Arginine-Rich Motif

Viruses ◽

10.3390/v11111050 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1050 ◽

Cited By ~ 3

Author(s):

Shaoli Lin ◽

Yonglin Yang ◽

Yuchen Nan ◽

Zexu Ma ◽

Liping Yang ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Early Stage ◽

Case Fatality ◽

Hepatitis E ◽

Open Reading Frames ◽

Multifunctional Protein ◽

Terminal Domain ◽

Arginine Residues ◽

Arginine Rich Motif

Hepatitis E virus (HEV) causes predominantly acute and self-limiting hepatitis. However, in HEV-infected pregnant women, the case fatality rate because of fulminant hepatitis can be up to 30%. HEV infection is zoonotic for some genotypes. The HEV genome contains three open reading frames: ORF1 encodes the non-structural polyprotein involved in viral RNA replication; ORF2 encodes the capsid protein; ORF3 encodes a small multifunctional protein. Interferons (IFNs) play a significant role in the early stage of the host antiviral response. In this study, we discovered that the capsid protein antagonizes IFN induction. Mechanistically, the capsid protein blocked the phosphorylation of IFN regulatory factor 3 (IRF3) via interaction with the multiprotein complex consisting of mitochondrial antiviral-signaling protein (MAVS), TANK-binding kinase 1 (TBK1), and IRF3. The N-terminal domain of the capsid protein was found to be responsible for the inhibition of IRF3 activation. Further study showed that the arginine-rich-motif in the N-terminal domain is indispensable for the inhibition as mutations of any of the arginine residues abolished the blockage of IRF3 phosphorylation. These results provide further insight into HEV interference with the host innate immunity.

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Purification and diagnostic utility of a recombinant hepatitis E virus capsid protein expressed in insect larvae

Protein Expression and Purification ◽

10.1016/s1046-5928(02)00574-0 ◽

2003 ◽

Vol 27 (1) ◽

pp. 27-34 ◽

Cited By ~ 20

Author(s):

Deepak Sehgal ◽

Punjab Singh Malik ◽

Shahid Jameel

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Diagnostic Utility ◽

Recombinant Hepatitis ◽

Insect Larvae ◽

Virus Capsid ◽

Virus Capsid Protein

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Identification of two neutralization epitopes on the capsid protein of avian hepatitis E virus

Journal of General Virology ◽

10.1099/vir.0.83366-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 500-508 ◽

Cited By ~ 19

Author(s):

E.-M. Zhou ◽

H. Guo ◽

F. F. Huang ◽

Z. F. Sun ◽

X. J. Meng

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Avian Hepatitis E Virus

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Production and characterization of a fusion form of hepatitis E virus tORF2 capsid protein in Escherichia coli

Preparative Biochemistry & Biotechnology ◽

10.1080/10826068.2020.1836656 ◽

2020 ◽

pp. 1-8

Author(s):

Mohamed Boumaiza ◽

Khaled Trabelsi ◽

Zeineb Choucha ◽

Ines Akrouti ◽

Serena Leone ◽

...

Keyword(s):

Escherichia Coli ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E

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

Pathogens ◽

10.3390/pathogens11010024 ◽

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.

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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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