The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated.
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ABSTRACT:
SUBMITTER: Nishiyama T
PROVIDER: S-EPMC8779013 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:AimTo cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice.MethodsPlastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA.ResultsTransplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 microg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies.ConclusionHEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.
| S-EPMC4066044 | biostudies-literature
Project description:BACKGROUND:Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. OBJECTIVES:The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. MATERIALS AND METHODS:The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-?-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS:The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37? for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. CONCLUSIONS:In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.
| S-EPMC4216576 | biostudies-literature
Project description:BACKGROUND:Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. OBJECTIVES:The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. MATERIALS AND METHODS:A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. RESULTS:Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immuno?uorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. CONCLUSIONS:The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations.
| S-EPMC4744464 | biostudies-literature
Project description:Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated 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. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.
| S-EPMC6472401 | biostudies-literature
Project description:The hepatitis E virus (HEV), a non enveloped RNA virus, causes viral hepatitis. The viral open reading frame 2 (ORF2) protein represents the capsid protein of HEV which is known to cause endoplasmic reticulum stress in ORF2 expressing cells. The initiation of endoplasmic reticulum stress induced apoptosis mainly involves the transcriptional activation of pro-apoptotic gene CHOP which will further trigger the major apoptotic pathways. However, the activation of CHOP by ORF2 protein in this study does not induce apoptotic markers such as Bax translocation to mitochondria. We have used the Affymetrix microarray platform to screen the pro-apoptotic effects induced by the expression of ORF2 protein in human hepatic cell lines (Huh7). The Huh7 cells were transduced either with recombinant adenovirus encoding the HEV ORF2 (Ad-ORF2) or an adenovirus encoding the green fluorescent protein (Ad-GFP). The array results consistently showed an ORF2 specific induction of mRNA corresponding to the chaperones Hsp72, Hsp70B’ and co-chaperone Hsp40. These studies provide further mechanisms of the ER stress mediated pro apoptotic effects caused by the ORF2 protein and its potential role for the activation of anti-apoptotic activity of the host cell. We used microarray to screen the host genes were regulated by the expression of the hepatitis E virus capsid protein. Huh7 cells transduced with Ad-GFP (control) or with Ad-HEV ORF2.
2012-12-01 | E-GEOD-29061 | biostudies-arrayexpress
Project description:Hepatitis E virus infection is responsible for acute viral hepatitis and associated with high mortality and still birth in pregnant women in developing countries. We report expression of truncated forms of HEV ORF2 as potential vaccine candidates for nanoparticle-based delivery. These two truncated ORF2 proteins (54 kDa and 26 kDa) have been reported to be highly immunogenic and can be used as nanoparticle-based vaccine candidate. The bacterial expressed protein was purified by affinity chromatography and further confirmed by western blot using anti-HEV antibody. The chitosan nanoemulsion was synthesized using ultrasonic waves. The nanoparticle size was found to be 120-160 nm and the entrapment efficiency of purified truncated ORF2 proteins within these nanoparticles was 70% (26 kDa) and 59% (54 kDa). In cell cytotoxicity analysis, 100 µg/mL nanoemulsion was found suitable for cell viability in both HeLa and THP1 cell lines. Release kinetics of encapsulated proteins at physiological pH 7.4 showed 26-59% and 9.7-40% release of 26 kDa and 54 kDa protein within 1 h that gradually increased with time (48 h). Encapsulated proteins were found to be unstable at pH 1.2.
| S-EPMC6139098 | biostudies-literature
Project description:This study presents nanoparticle-based vaccine development for Hepatitis E virus (HEV). Gold nanoparticles (GNP) of average size 12 nm were synthesized by citrate reduction method followed by functionalization with cysteamine hydrochloride for nano-conjugation. Immune response of nano-conjugates of GNP with 26 kDa protein (368-606 amino acids) and 54 kDa protein (112-606 amino acids) were evaluated. In vitro release kinetics of GNP-conjugated 54 kDa (GNP54) and 26 kDa (GNP26) proteins showed slower rate of release of 54 kDa protein as compared to 26 kDa protein. Humoral immune response of mice immunized intramuscularly with GNP54, GNP26 and GNP alone, exhibited HEV-specific IgG titer of 7.9 ± 2.9, 5.686 ± 4.098 and 0.698 ± 0.089, respectively, after 14 days of booster immunization. In addition to this, HEV-specific cell-mediated immune response was demonstrated by splenocyte proliferation assay. Analysis of results using one-way ANOVA, showed statistically significant (p value < 0.05) increase in splenocyte proliferation for GNP54- and GNP26-immunized mice in comparison to GNP alone immunized mice. Stimulation index of HEV ORF2 proteins in GNP54/GNP26-immunized mice were comparable to Concanavalin A-treated positive control. These results indicate GNP-based vaccine as a promising candidate for efficiently mediating both humoral and cell-mediated immune response against HEV.
| S-EPMC7799426 | biostudies-literature
Project description:Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C(pro)). However, PABP is cleaved by HAV 3C(pro) in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5' nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.
| S-EPMC2034478 | biostudies-literature
Project description:The hepatitis E virus (HEV), a non enveloped RNA virus, causes viral hepatitis. The viral open reading frame 2 (ORF2) protein represents the capsid protein of HEV which is known to cause endoplasmic reticulum stress in ORF2 expressing cells. The initiation of endoplasmic reticulum stress induced apoptosis mainly involves the transcriptional activation of pro-apoptotic gene CHOP which will further trigger the major apoptotic pathways. However, the activation of CHOP by ORF2 protein in this study does not induce apoptotic markers such as Bax translocation to mitochondria. We have used the Affymetrix microarray platform to screen the pro-apoptotic effects induced by the expression of ORF2 protein in human hepatic cell lines (Huh7). The Huh7 cells were transduced either with recombinant adenovirus encoding the HEV ORF2 (Ad-ORF2) or an adenovirus encoding the green fluorescent protein (Ad-GFP). The array results consistently showed an ORF2 specific induction of mRNA corresponding to the chaperones Hsp72, Hsp70B’ and co-chaperone Hsp40. These studies provide further mechanisms of the ER stress mediated pro apoptotic effects caused by the ORF2 protein and its potential role for the activation of anti-apoptotic activity of the host cell.
2012-12-01 | GSE29061 | GEO
Project description:The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an approximately 56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.
| S-EPMC2786843 | biostudies-literature