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.

Molecular biology and pathogenesis of hepatitis E virus

1999 ◽  
Vol 1 (18) ◽  
pp. 1-16 ◽  
Author(s):  
Shahid Jameel
Keyword(s):  
Viral Genome ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Host Interactions ◽  
Processing Enzymes ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) infection results in hepatitis E, an acute and self-limited disease. The virus is transmitted in a faecal–oral manner and is a major cause of viral hepatitis in much of the developing world, where it causes rampant sporadic infections and large epidemics. A curious feature of hepatitis E is the unusually high rates of mortality that are observed in pregnant women, in whom the disease is exacerbated by the development of fulminant liver disease. In the absence of viable in vitro propagation systems, several geographical isolates of HEV have been maintained in vivo in nonhuman primates and, subsequently, the viral genome has been cloned and sequenced. HEV has been classified provisionally into a separate family known as the HEV-like viruses, which has at least four recognised genotypes, but has only a single serotype. The viral genome is a positive-stranded (+)RNA of ~7.5 kb and encodes at least three proteins. Open reading frame 1 (ORF1) encodes the viral nonstructural polyprotein, which has domains that are homologous to some of the replication and processing enzymes found in other +RNA viruses. The HEV protein itself remains poorly characterised. The protein encoded by open reading frame 2 (ORF2) is the major HEV capsid protein, and the protein encoded by open reading frame 3 (ORF3) appears to be involved in virus–host interactions. Several questions related to the biology, epidemiology and pathogenesis of HEV remain unanswered; the progress of a few of these is reviewed here.

scholarly journals Identification by Phage Display and Characterization of Two Neutralizing Chimpanzee Monoclonal Antibodies to the Hepatitis E Virus Capsid Protein

2000 ◽  
Vol 74 (12) ◽  
pp. 5548-5555 ◽  
Author(s):  
D. J. Schofield ◽  
J. Glamann ◽  
S. U. Emerson ◽  
R. H. Purcell
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Hepatitis E Virus ◽  
Dissociation Constants ◽  
Hepatitis E ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Equilibrium Dissociation Constants

ABSTRACT Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) γ1/κ antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, Kd ) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.

scholarly journals The Hepatitis E Virus Open Reading Frame 2 Protein: Beyond Viral Capsid

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhaobin Zhou ◽  
Yinqian Xie ◽  
Chunyan Wu ◽  
Yuchen Nan
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frames ◽  
Viral Capsid ◽  
Reading Frame ◽  
Host Tropism ◽  
Orf2 Protein ◽  
Acute Hepatitis E ◽  
Open Reading Frame 2 ◽  
Reading Frames

Hepatitis E virus (HEV) is a zoonotic pathogen causing hepatitis in both human and animal hosts, which is responsible for acute hepatitis E outbreaks worldwide. The 7.2 kb genome of the HEV encodes three well-defined open reading frames (ORFs), where the ORF2 translation product acts as the major virion component to form the viral capsid. In recent years, besides forming the capsid, more functions have been revealed for the HEV-ORF2 protein, and it appears that HEV-ORF2 plays multiple functions in both viral replication and pathogenesis. In this review, we systematically summarize the recent research advances regarding the function of the HEV-ORF2 protein such as application in the development of a vaccine, regulation of the innate immune response and cellular signaling, involvement in host tropism and participation in HEV pathogenesis as a novel secretory factor. Progress in understanding more of the function of HEV-ORF2 protein beyond the capsid protein would contribute to improved control and treatment of HEV infection.

scholarly journals Antigenic Domains of the Open Reading Frame 2-Encoded Protein of Hepatitis E Virus

1999 ◽  
Vol 37 (9) ◽  
pp. 2863-2871 ◽  
Author(s):  
Yury E. Khudyakov ◽  
Elena N. Lopareva ◽  
Danny L. Jue ◽  
Tamara K. Crews ◽  
S. P. Thyagarajan ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Positive Serum ◽  
Antigenic Domains ◽  
Open Reading Frame 2 ◽  
Antigenic Epitopes

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

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 OASL Triggered by Novel Goose Astrovirus via ORF2 Restricts Its Replication

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Dan Ren ◽  
Tuofan Li ◽  
Xinyu Zhang ◽  
Xiaohui Yao ◽  
Wei Gao ◽  
...  
Keyword(s):  
Immune Response ◽  
Reading Frame ◽  
Enteric Diseases ◽  
C Terminus ◽  
Orf2 Protein ◽  
High Level ◽  
Antiviral Targets ◽  
Open Reading Frame 2

ABSTRACT Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo. The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses. IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.

scholarly journals Hepatitis E Virus (HEV) Open Reading Frame 2 Antigen Kinetics in Human-Liver Chimeric Mice and Its Impact on HEV Diagnosis

2019 ◽  
Vol 220 (5) ◽  
pp. 811-819 ◽  
Author(s):  
Ibrahim M Sayed ◽  
Lieven Verhoye ◽  
Claire Montpellier ◽  
Florence Abravanel ◽  
Jacques Izopet ◽  
...  
Keyword(s):  
Density Gradient ◽  
Hepatitis E Virus ◽  
Gradient Analysis ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Humanized Mice ◽  
Molecular Diagnostic ◽  
Reading Frame ◽  
Open Reading Frame 2

Abstract Background Hepatitis E virus infection (HEV) is an emerging problem in developed countries. Diagnosis of HEV infection is based on the detection of HEV-specific antibodies, viral RNA, and/or antigen (Ag). Humanized mice were previously reported as a model for the study of HEV infection, but published data were focused on the quantification of viral RNA. However, the kinetics of HEV Ag expression during infection remains poorly understood. Methods Plasma specimens and suspensions of fecal specimens from HEV-infected and ribavirin-treated humanized mice were analyzed using HEV antigen–specific enzyme-linked immunosorbent assay, reverse transcription–quantitative polymerase chain reaction analysis, density gradient analysis, and Western blotting. Result Open reading frame 2 (ORF2) Ag was detected in both plasma and stool from HEV-infected mice, and levels increased over time. Contrary to HEV RNA, ORF2 Ag levels were higher in mouse plasma than in stool. Interestingly, ORF2 was detected in plasma from mice that tested negative for HEV RNA in plasma but positive for HEV RNA in stool and was detected after viral clearance in mice that were treated with ribavirin. Plasma density gradient analysis revealed the presence of the noninfectious glycosylated form of ORF2. Conclusion ORF2 Ag can be used as a marker of active HEV infection and for assessment of the effect of antiviral therapy, especially when fecal samples are not available or molecular diagnostic tests are not accessible.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

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