The ORF3 protein of HEV is a phosphoprotein that associates with cytoskeleton

Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Journal of Virology (Impact Factor: 4.44). 01/1998; 71(12):9045-53.
Source: PubMed

ABSTRACT Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.

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Available from: Mehmet Hakan Ozdener, Sep 26, 2015
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    • "The hepatitis E virus (HEV) was discovered in 1983 and assigned to the family Hepeviridae as the sole member of the genus hepevirus (Balayan et al., 1983; Emerson et al., 2004). HEV is a non-enveloped, single-stranded RNA virus with a positive sense genome of 7.2 kb comprising 3 open reading frames (ORFs) that code for the non-structural protein (ORF1), the capsid (ORF2) and a phosphoprotein associated with the cytoskeleton (ORF3) (Zafrullah et al., 1997). Currently, HEV isolates infecting humans are classified into four major genotypes with only one serotype (Purcell and Emerson, 2008). "
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    ABSTRACT: Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.
    Zoonoses and Public Health 02/2014; 61(8). DOI:10.1111/zph.12103 · 2.37 Impact Factor
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    • "The subsequent hydrophobic domain 2 (D2) has been associated with haemopexin binding (Ratra et al., 2008), and both domains contained seven and eight unique residues. The two overlapping motifs PMSP and SPLR, as part of the P1 domain, acting as potential kinase substrates (Zafrullah et al., 1997) were only observed in gt1 and some gt3s, while moose HEV displayed many unique residue substitutions making the target serine absent for possible kinase phosphorylation. The two overlapping PXXP motifs in the P2 domain, which are associated with SH3 protein domain binding and ORF3 interaction (Korkaya et al., 2001), were detected in the gt1–4, but mutated in the moose HEV and in other animal HEVs. "
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    ABSTRACT: A novel virus was detected in a sample collected from a Swedish moose (Alces alces). The virus was classified as a member in the Hepeviridae family, however it was found to be highly divergent from the known four genotypes (gt1-4) of hepatitis E virus (HEV). Moose are regularly hunted for consumption in whole Scandinavia. Thus, the finding of this virus may be important from several aspects: a) as a new diverged HEV in a new animal species, b) potential unexplored HEV transmission pathways for human infections. Considering these aspects, we have started the molecular characterization of this virus. A sequenced 5.1 kb amplicon, corresponding to the partial ORF1, followed by complete ORF2, ORF3 and poly(A) sequence. In comparison with existing HEVs, the moose HEV genome showed a general nucleotide sequence similarity of 37-63% and an extensively divergent putative ORF3 sequence. The junction region between the ORFs was also highly divergent, however two putative secondary steam loop structure were retained when compared to gt1-4, but with altered structure appearance, which may be related to host range properties. In the phylogenetic analysis, the moose HEV deviated and formed its own branch between the gt1-4 and other divergent animal HEVs. The characterization of its highly divergent genome may contribute to a better understanding of the evolution, host range, zoonotic properties and virulence factors of HEV in general. However, further studies are needed to investigate its prevalence in the moose populations and possibly in other host species, including the risk for human infection.
    Journal of General Virology 12/2013; DOI:10.1099/vir.0.059238-0 · 3.18 Impact Factor
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    • "HEV ORF1 encodes non-structural proteins like the RNA-dependent RNA polymerase, methyltransferase, RNA helicase and cysteine protease (Karpe and Meng, 2012; Koonin et al., 1992). ORF2 encodes the capsid protein, while ORF3 encodes a multifunctional small phosphoprotein (Chandra et al., 2011; Kenney et al., 2012; Tam et al., 1991; Zafrullah et al., 1997). "
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    ABSTRACT: Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n=72), (B) samples from known HEV-negative pigs (negative controls, n=62) and (C) samples from pigs of unknown HEV infection status (n=182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa=0.92).
    Journal of virological methods 06/2013; 193(2). DOI:10.1016/j.jviromet.2013.06.010 · 1.78 Impact Factor
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