Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.

Torresi, Joseph; Li, Fan; Locarnini, Stephen; Anderson, David

doi:10.1099/0022-1317-80-5-1185

Cited by 54 publications

(41 citation statements)

References 21 publications

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“…Although these results cannot rule out addition of short oligosaccharides, they do suggest that at least the majority, if not all, of the ORF2 protein accumulating within transfected S10-3 cells is not glycosylated. This result is in agreement with a report by Torresi et al (36) that the nonglycosylated ORF2 protein is the stable form in mammalian cells.…”

Section: Resultssupporting

confidence: 94%

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

Graff

Zhou

Torian

et al. 2008

J Virol

101

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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.Hepatitis E virus (HEV) is transmitted via the fecal-oral route, predominantly through contaminated water. HEV causes acute self-limiting hepatitis and in much of the developing world is responsible for sporadic infections, as well as large epidemics, of acute hepatitis E, especially in Asia and Africa.Four genotypes comprising a single serotype of mammalian HEV have been identified (26,28). HEV was recently classified as the sole member of the genus Hepevirus, family Hepeviridae (3); sequence analysis suggests it is most closely related to rubella virus, an enveloped virus in the family Togaviridae. However, HEV does not contain a lipid envelope (26). The virion is 27 to 30 nm in diameter (1, 26) and has a sedimentation coefficient of 183S (2). The HEV genome is a positivesense RNA, approximately 7.2 kb in length, with a short, capped 5Ј noncoding region and a 3Ј noncoding region preceding a poly(A) tail. Viral genomic RNA is infectious for some cultured cells and nonhuman primates: transfection with capped recombinant genomes results in production of infectious virions in vitro and acute hepatitis and/or seroconversion in vivo (6-8). The coding region of HEV contains three partially overlapping open reading frames (ORFs): ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3, which overlaps the N terminus of ORF2, encodes a small protein of 114 amino acids (aa) (13, 15) that might be a regulatory protein (7,19,33,44). The ORF2 and ORF3 proteins are encoded by a bicistronic subgenomic RNA (13,35,45).HEV does not grow sufficiently well either in cell culture or in nonhuman primates to permit direct biochemical analysis. Therefore, various in vitro systems have been utili...

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Section: Resultssupporting

confidence: 94%

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

Graff

Zhou

Torian

et al. 2008

J Virol

101

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“…Jameel et al (1996) reported that the ORF2 protein was detected in three molecular forms, including a 74 kDa non-glycosylated form and 82 and 88 kDa glycosylated forms. It was reported that the glycosylated form of ORF2 protein gradually shifted to the nonglycosylated form in the cytoplasm through the retrotranslocation pathway (Surjit et al, 2007) and that only the non-glycosylated form was stable in the cytoplasm of mammalian cells (Torresi et al, 1999). However, the 83 kDa ORF2 protein detected in the culture supernatants and lysates of pJE03-1760F/wt RNA-transfected cells (Fig.…”

mentioning

confidence: 88%

Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells

Yamada

Takahashi

Hoshino

et al. 2009

Journal of General Virology

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A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed in this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 107 copies ml−1 on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 106 copies ml−1 at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.

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“…ORF2 encodes the viral capsid protein; ORF3, which contains one potential phosphorylation site, encodes a very small protein of about 123 amino acids (59). ORF3 proteins partition with the cytoskeleton, and it has been suggested that an interaction between ORF2 and ORF3 is associated with the assembly of virions (47,50). ORF3 is also believed to interact with proteins involved in the host cell signaling pathway (24).…”

mentioning

confidence: 99%

Evidence of Recombination between Divergent Hepatitis E Viruses

Cuyck¹,

Fan

Robertson

et al. 2005

J Virol

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Phylogenetic and recombination analysis was performed on 32 complete hepatitis E virus (HEV) genomes from infected humans and pigs. For the first time, evidence for recombination between divergent HEV strains was obtained, with at least two strains being found to have discordant phylogenetic relationships consistent with the occurrence of intragenotype recombination. This finding confirms that humans can be dually infected with divergent HEV strains and has implications for the emergence and evolution of new HEV epidemics.Hepatitis E virus (HEV) is a causative agent of enterically transmitted hepatitis disease and, apart from infections of pregnant women, is generally associated with low mortality (11,17). Outbreaks of HEV have been described to occur in many regions of the world, including Asia, Africa, and Mexico (1, 33, 53), while sporadic cases have been documented in the United States, Africa (9), and Europe (27,37,38,60). HEV is presumed to be a zoonotic disease, as surveys of both wild and domestic animals have found evidence for human-related HEVs in rats (12, 22), pigs (2,7,21,29,32,56), deer (45), and wild boar (41). Direct animal-to-human transmission of HEV has been demonstrated, for example, from deer to human as a result of eating uncooked meat (45). Interestingly, HEV characterized from swine in countries where HEV is not endemic is usually closely related to cases of sporadic human HEVs from the same country, for example, in the United States and Japan (29,30,42,57), while in India, where HEV is endemic, human and swine HEV strains are not closely related (2). This suggests that in certain regions HEV is more established in the human population and that in other regions HEV is emerging as a result of direct contact with animal reservoirs for the virus. Divergent HEVs with no human counterparts have also been found in chickens (avian HEV). Avian HEV shares only 50 to 60% nucleotide sequence identity with HEV from humans and swine (15,16).HEV is a positive single-stranded RNA virus that is approximately 7,200 nucleotides (nt) in length. HEV's genome is capped and polyadenylated and has three open reading frames (ORF) (Fig. 1). As the organization of HEV's genome resembles that of Caliciviridae, at one time it was considered a mem-FIG. 1. Genomic organization of HEV. The scale at the bottom of the figure corresponds to nucleotides (in thousands). ORF1 encodes a nonstructural polyprotein which provides guanylyl-methyltransferase and RNA-dependent RNA polymerase activities and possibly other functions (23,25). The ORF2 and ORF3 proteins are believed to be encoded by individual subgenomic RNAs generated during replication (44, 58). ORF2 encodes the viral capsid protein; ORF3, which contains one potential phosphorylation site, encodes a very small protein of about 123 amino acids (59). ORF3 proteins partition with the cytoskeleton, and it has been suggested that an interaction between ORF2 and ORF3 is associated with the assembly of virions (47,50). ORF3 is also believed to interact with proteins involv...

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Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.

Cited by 54 publications

References 21 publications

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

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

Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells

Evidence of Recombination between Divergent Hepatitis E Viruses

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