The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK

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 p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, 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 pORF3.SH3 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.     

Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein

Hepatitis E virus (HEV) is the etiological agent for viral hepatitis type E, which is a major problem in the developing world. Because HEV cannot be cultured in vitro, very little information exists on the mechanisms of HEV gene expression and genome replication. HEV is a positive-strand RNA virus with three potential open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2). We earlier showed (S. Jameel, M. Zafrullah, M. H. Ozdener, and S. K. Panda, J. Virol. 70:207-216, 1996) pORF2 to be a approximately 88-kDa glycoprotein, carrying N-linked glycans and a potential endoplasmic reticulum (ER)-directing signal at its N terminus. Treatment with the drugs brefeldin A and monensin suggest that the protein may accumulate within the ER. Based on mutational analysis, we demonstrate Asn-310 to be the major site of N-glycan addition. In COS-1 cell expression and in vitro translation experiments, we confirm the ER-translocating nature of the pORF2 N-terminal hydrophobic sequence and show that the protein is cotranslationally, but not posttranslationally, translocated across the ER membrane. Earlier, we had also demonstrated cell surface localization of a fraction of the COS-1 cell-expressed pORF2. Using glycosylation- and translocation-defective mutants of pORF2, we now show that while transit of pORF2 into the ER is necessary for its cell surface expression, glycosylation of the protein is not required for such localization. These results may offer clues to the mechanisms of gene expression and capsid assembly in HEV.     

The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.

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.     

Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein

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 on the basic biology of the virus exists. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.     

Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.     

Transcriptional control of the human aldehyde dehydrogenase 2 promoter by hepatocyte nuclear factor 4: inhibition by cyclic AMP and COUP transcription factors.

An important regulatory element (designated FP330-3') of the ALDH2 promoter mediates activation by hepatocyte nuclear factor 4 (HNF4). This activation of promoter constructs containing this element by HNF4 was reduced by nearly half by 8-Br-cAMP in H4IIEC3 cells, an effect that was blocked by inhibitors of protein kinase A (PKA). Cotransfection assays showed that COUP-TF I, ARP-1, or PPARdelta suppressed the ability of HNF4 to activate the reporter. The repression was potentiated by 8-Br-cAMP. Electrophoretic mobility shift assays revealed that treatment of hepatoma cells or cultured rat hepatocytes with 1 mM 8-Br-cAMP or glucagon reduced binding of FP330-3' by HNF4 by half. In vitro phosphorylation of HNF4 by PKA decreased binding to FP330-3'. Fasting reduced the ALDH2 protein level in liver and kidney, two tissues expressing HNF4, but not heart. These data suggest that ALDH2 expression can be suppressed by cAMP, most likely through phosphorylation of HNF4 by PKA, and this may account for the reduction in enzyme protein during fasting.