The ORF3 Protein of Hepatitis E Virus Binds to Src Homology 3 Domains and Activates MAPK

Abstract

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85␣ regulatory subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3⅐SH3 complex. In pORF3-expressing cells, pp60 src was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.Hepatitis E virus (HEV), 1 the causative agent for hepatitis E, is a waterborne pathogen endemic to much of the developing world where it causes rampant sporadic infections and large scale epidemics (1-4). While the infection is self-limited with no associated chronicity, a fraction of the patients progress to fulminant hepatitis (5, 6), the most severe form of acute hepatitis. High mortality rates of 20 -30% reported for HEV infection during pregnancy (7,8) are also the result of fulminant hepatitis. The reasons for this and the mechanisms of viral pathogenesis are not known. The studies on HEV biology and pathogenesis have been severely restricted by the lack of a reliable cell culture system and small animal models of viral infection. We have used subgenomic expression strategies to study the properties and functions of individual HEV gene products toward understanding viral replication and pathogenicity (9 -12).The HEV genome is a ϳ7.5-kilobase polyadenylated, positive-sense RNA that contains three open reading frames (ORFs) designated ORF1, ORF2, and ORF3 (13). The ORF3 of HEV encodes a protein of ϳ13.5 kDa, called pORF3, for which no function has been assigned. When expressed in animal cells, pORF3 is phosphorylated at a single serine residue (Ser 80 ) in its 123-amino acid primary sequence (11). In vitro phosphorylation experiments suggested that pORF3 may be a substrate for the mitogen-activated protein (MAP) kinase, and subcellular fractionation revealed its association with the cytoskeleton (11). Recent results using inhibitors, activators, and dominant negative alleles show that pORF3 is a substrate for the extracellular signal-regulated kinase (ERK) as well as the stressactivated pr...

Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus

“…ORF3, mapped between ORF1 and ORF2, encodes a 13.5-kDa protein that is associated with the membrane as well as with the cytoskeleton fraction (27). This protein is shown to be phosphorylated by the cellular mitogen-activated protein kinase (6,8). The ORF3 protein may have a regulatory function (6,8).…”

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

“…Hence, phosphorylation does not play a role in the ORF3-bikunin interaction. Although the bikunin-ORF3 interaction is independent of phosphorylation, the interaction domain of ORF3 overlaps with the ORF3 dimerization domain (amino acids 81 to 123) (37) and SH3 binding domain (amino acids 75 to 113) (21). This region has also been shown to carry an immunodominant epitope, antibodies to which are universally present in infected humans and animals NOTES 12085 (24).…”

ORF3 protein of hepatitis E virus is essential for virion release from infected cells

“…It has been reported that the ORF3 protein is phosphorylated and interacts not only with the ORF2 protein (Tyagi et al, 2002) but also with several cellular proteins, including the cytoskeleton, a 1 -microglobulin/bikunin precursor, tumour susceptibility gene 101 (Tsg101) and the src homology 3 domains (Korkaya et al, 2001;Surjit et al, 2006;Tyagi et al, 2004;Zafrullah et al, 1997). However, it remains unclear whether the reported functions of ORF3 protein are authentic in the HEV life cycle and whether ORF3 protein is associated with virion morphogenesis.…”