Characterization of capsid protein (p495) of hepatitis E virus expressed in Escherichia coli and assembling into particles in vitro

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The tropism of HEV is not restricted to the liver, and the virus replicates in other organs. Not all the extrahepatic targets for HEV are identified. Herein, we found that non-decidualized primary human endometrial stromal cells (PHESCs), which are precursors for the decidua and placenta, are susceptible to HEV infection. PHESCs, isolated from healthy non-pregnant women (n = 5), were challenged with stool-derived HEV-1 and HEV-3. HEV RNA was measured by qPCR, and HEV capsid protein was assessed by flow cytometry, immunofluorescence (IF), and ELISA. HEV infection was successfully established in PHESCs. Intracellular and extracellular HEV RNA loads were increased over time, indicating efficient replication in vitro. In addition, HEV capsid protein was detected intracellularly in the HEV-infected PHESCs and accumulated extracellularly over time, confirming the viral assembly and release from the infected cells. HEV-1 replicated more efficiently in PHESCs than HEV-3 and induced more inflammatory responses. Ribavirin (RBV) treatment abolished the replication of HEV in PHESCs. In conclusion, PHESCs are permissive to HEV infection and these cells could be an endogenous source of HEV infection during pregnancy and mediate HEV vertical transmission.

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Production of capsid proteins of rat hepatitis E virus in Escherichia coli and characterization of self-assembled virus-like particles

Virus Research ◽

10.1016/j.virusres.2021.198483 ◽

2021 ◽

pp. 198483

Author(s):

Tominari Kobayashi ◽

Masaharu Takahashi ◽

Satoshi Ohta ◽

Shigeo Nagashima ◽

Putu Prathiwi Primadharsini ◽

...

Keyword(s):

Escherichia Coli ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Capsid Proteins ◽

Virus Like Particles ◽

Self Assembled

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Generation in yeast and antigenic characterization of hepatitis E virus capsid protein virus-like particles

Applied Microbiology and Biotechnology ◽

10.1007/s00253-017-8622-9 ◽

2017 ◽

Vol 102 (1) ◽

pp. 185-198 ◽

Cited By ~ 5

Author(s):

Martynas Simanavicius ◽

Paulius Lukas Tamosiunas ◽

Rasa Petraityte-Burneikiene ◽

Reimar Johne ◽

Rainer G. Ulrich ◽

...

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Virus Capsid ◽

Virus Like Particles ◽

Antigenic Characterization ◽

Virus Capsid Protein

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Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Journal of Virology ◽

10.1128/jvi.01219-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1185-1194 ◽

Cited By ~ 73

Author(s):

Judith Graff ◽

Yi-Hua Zhou ◽

Udana Torian ◽

Hanh Nguyen ◽

Marisa St. Claire ◽

...

Keyword(s):

Cell Culture ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Signal Sequence ◽

Rhesus Macaques ◽

Hepatitis E ◽

Velocity Sedimentation ◽

Genome Replication ◽

Glycosylation Sites

ABSTRACT Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

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Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54871-7 ◽

1991 ◽

Vol 266 (30) ◽

pp. 19925-19929

Author(s):

K. Keshavjee ◽

C. Pyne ◽

A.L. Bognar

Keyword(s):

Escherichia Coli ◽

Folylpolyglutamate Synthetase

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

Viruses ◽

10.3390/v13071385 ◽

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.

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In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of transcription products.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)33940-1 ◽

1982 ◽

Vol 257 (18) ◽

pp. 11113-11120 ◽

Cited By ~ 2

Author(s):

N Nakajima ◽

H Ozeki ◽

Y Shimura

Keyword(s):

Escherichia Coli ◽

In Vitro Transcription

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Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

Journal of Fungi ◽

10.3390/jof7050325 ◽

2021 ◽

Vol 7 (5) ◽

pp. 325

Author(s):

Laura Isabel de de Eugenio ◽

Rosa Peces-Pérez ◽

Dolores Linde ◽

Alicia Prieto ◽

Jorge Barriuso ◽

...

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Veratryl Alcohol ◽

Dye Decolorization ◽

Irpex Lacteus ◽

Type D ◽

Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

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