Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics.

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

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.

Expression in animal cells and characterization of the hepatitis E virus structural proteins.

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression.

An Indian strain of hepatitis E virus (HEV): cloning, sequence, and expression of structural region and antibody responses in sera from individuals from an area of high-level HEV endemicity.

Hepatitis E virus (HEV) is responsible for a majority of sporadic and epidemic viral hepatitides in India and other developing countries. Even though the genomes of four geographically distinct strains of HEV have been cloned and sequenced, the Indian strain of HEV remains largely uncharacterized. We have cloned and sequenced about 2.2 kb of the HEV genome constituting the structural region from an Indian strain of HEV. The nucleotide and amino acid sequences show a high degree of conservation with sequences from other HEV strains. Open reading frames (ORF) 2 and 3 were expressed in Escherichia coli as N-terminal hexahistidine epitope fusions. The purified proteins were then used in an immunoblot assay to evaluate the antibody status in sera from individuals from an area of high-level HEV endemicity. The anti-ORF2 antibodies were found to be nonspecific and could not be correlated to clinical disease. The immunoglobulin M anti-ORF3 was found to be specific for the presence of acute disease. The implications of these findings in HEV diagnosis and vaccine development are discussed.