The hepatitis E virus ORF3 protein modulates epidermal growth factor receptor trafficking, STAT3 translocation, and the acute-phase response.

The hepatitis E virus (HEV) causes acute viral hepatitis, but its characterization is hampered by the lack of an efficient in vitro infection system that can be used to study the effects of HEV proteins on cellular processes. Previous studies suggest that the viral ORF3 protein (pORF3) is essential for infection in vivo and is likely to modulate the host response. Here, we report that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. The cytoplasmic phosphorylated signal transducer and activator of transcription 3 (pSTAT3) proteins require growth factor receptor endocytosis for their translocation from the cytoplasm to nucleus. Consequently, lower levels of pSTAT3 were found in the nuclei of ORF3-expressing Huh7 human hepatoma cells stimulated with EGF. This results in downregulation of the acute-phase response, a major determinant of inflammation in the host. We propose that through its effects on EGFR trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival. The effects on STAT3 translocation would result in a reduced inflammatory response. Both of these events are likely to contribute positively to viral replication.

ORF3 protein of hepatitis E virus interacts with the Bbeta chain of fibrinogen resulting in decreased fibrinogen secretion from HuH-7 cells.

The ORF3 protein of hepatitis E virus (HEV), the precise cellular functions of which remain obscure, was used in a yeast two-hybrid screen to identify its cellular binding partners. One of the identified interacting partners was fibrinogen Bbeta protein. The ORF3-fibrinogen Bbeta interaction was verified by co-immunoprecipitation and fluorescence resonance energy transfer in mammalian cells. Fibrinogen is a hepatic acute-phase protein and serves as a central molecule that maintains host homeostasis and haemostasis during an acute-phase response. Metabolic labelling of ORF3-transfected HuH-7 cells showed that secreted as well as intracellular levels of fibrinogen were decreased in these cells compared with vector-transfected controls. Northern hybridization and RT-PCR analyses revealed that the mRNA levels of all three chains of fibrinogen, Aalpha, Bbeta and gamma, were transcriptionally downregulated in ORF3-transfected cells. The constitutive expression of fibrinogen genes can be significantly upregulated by interleukin (IL)-6, an important mediator of liver-specific gene expression during an acute-phase response. Transcription of fibrinogen genes after IL-6 stimulation was less in ORF3-expressing cells compared with controls. This report adds one more biological function to, and advances our understanding of, the cellular role of the ORF3 protein of HEV. The possible implications of these findings in the virus life cycle are discussed.

The ORF3 protein of hepatitis E virus interacts with hemopexin by means of its 26 amino acid N-terminal hydrophobic domain II.

Hepatitis E virus (HEV) is a nonenveloped plus-stranded RNA virus that is a major cause of acute hepatitis in many developing countries. Recent work has shown HEV may be endemic in developed countries also. The 5' two-thirds of the 7.2 kb single-stranded RNA genome of HEV encodes ORF1, and the 3' end encodes the structural proteins ORF2 and ORF3. ORF1 is the nonstructural protein involved in viral RNA synthesis, and ORF2 is the major capsid protein, whereas ORF3 is a very small protein of only 123 amino acids. The precise cellular functions of ORF3 protein remain obscure, although it has been postulated to be a viral regulatory protein. To elucidate the role of ORF3 in viral pathogenesis, the yeast two-hybrid system was used to screen a human liver cDNA library for proteins interacting with ORF3. One of the ORF3-interacting partners thus isolated and identified was hemopexin, a 60 kDa acute-phase plasma glycoprotein with a high binding affinity to heme. The two-hybrid result was validated by in vitro pull-down and co-immunoprecipitation assays and finally by intracellular fluorescence resonance energy transfer. Using a deletion mapping approach, the hydrophobic domain II of ORF3 (spanning amino acids 37 to 62) was found to be responsible for binding to Hpx, with amino acids 63 to 77 possibly contributing to the strength of the interaction. The biological significance of this interaction in the virus life cycle has been discussed.

The VP1 protein of human enterovirus 71 self-associates via an interaction domain spanning amino acids 66-297.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease (HFMD). Several recent outbreaks of HFMD in East Asia were associated with neurological complications and numerous deaths. In 2000, an outbreak in Singapore afflicted thousands of children, resulting in four fatal cases from whom EV71 was isolated. The virus possesses four structural proteins VP1, VP2, VP3, and VP4, each of which is involved in forming the pentameric icosahedral structure of the virus. Here we report that the full-length VP1 structural protein of EV71 is capable of self-association. Dimerization of VP1 was tested using the yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis, in vitro coupled transcription-translation binding assays, and mammalian cell transcription-translation experiments. Dimerization of various truncated versions of the VP1 protein was also studied by mutational analysis. Systematic deletions of parts of VP1 revealed that the region spanning amino acids 66-132 of VP1 contains the major dimerization domain. However, the region between amino acids 132 and 297 was indispensable, and contributed largely to increasing the strength of the interaction. This ability of EV71 VP1 to self-associate and to participate significantly in forming the characteristic icosahedral capsid strongly enhances the pathogenicity and stability of the virus to withstand the environment of the gastrointestinal tract.

The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase.

The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem. However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood. We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily. Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway. Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase. The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1. Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1. This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.