• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Journal of Microbiology
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• v.34 no.4
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• pp.311-315
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• 1996

The retroviral Gag polyprotein directs the assembly of virion particles and plays an important role in some events after entry into a host cell. The Gag polyprotein of a virus mixture is responsible for inducing murine acquired immunodeficiency syndrome (MAIDS) when injected into susceptible strains of mice. In order to identify the host cellular proteins which interact with the MAIDS virus Gag proteins and possibly mediate the function of the Gag proteins, mouse T-cell leukemic cDNA expression library was screened using the yeast GAL4 two hybrid system. Of 11 individual positive clones, the clone Y1 was selected for the study of protein-protein interaction. Its DNA sequence revealed that it was an exact match to the murine SH3 domain-containing protein SH3P8. It is expressed as 2.4 kbp transcripts in testis at higher levels and in various tissues tested at lower levels. Glutathione S-transferase-Y1 fusion protein binds tightly to $Pr60^{def-gag}$ as well as $Pr65^{eco-gag}$.

• Proceedings of the PSK Conference
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• 2001.04a
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• pp.209.1-209.1
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• 2001

• BMB Reports
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• v.34 no.4
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• pp.371-378
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• 2001

The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.

• Molecules and Cells
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• v.39 no.4
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• pp.286-291
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• 2016

Epstein-Barr virus (EBV) is the prototypical ${\gamma}$-herpesvirus and an obligate human pathogen that infects mainly epithelial cells and B cells, which can result in malignancies. EBV infects these target cells by fusing with the viral and cellular lipid bilayer membranes using multiple viral factors and host receptor(s) thus exhibiting a unique complexity in its entry machinery. To enter epithelial cells, EBV requires minimally the conserved core fusion machinery comprised of the glycoproteins gH/gL acting as the receptor-binding complex and gB as the fusogen. EBV can enter B cells using gp42, which binds tightly to gH/gL and interacts with host HLA class II, activating fusion. Previously, we published the individual crystal structures of EBV entry factors, such as gH/gL and gp42, the EBV/host receptor complex, gp42/HLA-DR1, and the fusion protein EBV gB in a postfusion conformation, which allowed us to identify structural determinants and regions critical for receptor-binding and membrane fusion. Recently, we reported different low resolution models of the EBV B cell entry triggering complex (gHgL/gp42/HLA class II) in "open" and "closed" states based on negative-stain single particle electron microscopy, which provide further mechanistic insights. This review summarizes the current knowledge of these key players in EBV entry and how their structures impact receptor-binding and the triggering of gB-mediated fusion.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.230-234
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• 2006

To study the mechanism of syncytium formation, novel syncytia-inducing ecotropic murine retrovirus was used. Our previous result showed that amino acid substitutions at the RBD (receptor binding domain) of envelope glycoprotein contribute to syncytium formation. In this study, we have investigated if this fusion phenomenon could occur with retroviral vectors pseudotyped with the novel syncytia-inducing ecotropic murine leukemia virus Env. We have found that these vectors were not able to mediate virus-to-cell fusion in M. dunni murine cell lines. These findings indicate that syncytia-inducing ecotropic murine leukemia virus is capable of generating syncytia during its replication. There was also no correlation between the level of ecotropic murine leukemia virus receptor (mCAT-1) and the fusogenic effect.

• Journal of fish pathology
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• v.24 no.2
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• pp.47-51
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• 2011

The present study demonstrated lymphocystis disease virus (LCDV) propagation through cytopathic effects (CPE) formation and LCDV detection in olive flounder fin (FFN) cells by polymerase chain reaction (PCR) and fluorescent antibody technique (FAT) methods. Tissue filtrates from the cluster cells produced CPE in FFN cells, which initially cells became enlarged and gradually underwent fusion en masse. Infectivity of culture grown LCDV using the FFN cells reached $10^{2.3}$ $TCID_{50}$/ml at 4 days post infection and the highest titer was measured $10^{6.5}$ $TCID_{50}$/ml at 12 days. The viral DNA was detected in the cell culture supernatants showing CPE and the CPE cells by PCR. Antigen specific strong fluorescence reacting with monoclonal antibody against the virus revealed the presence of viral antigen in the cytoplasm of infected FFN cells. These results suggest that the FFN cell line originated from the olive flounder has a susceptibility of the LCDV.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.468-473
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• 1996

The genome of tomato spotted wilt virus (TSWV) is composed of three RNA segments, S, M, and L RNA and the 5.0 kb M RNA encodes two glycoproteins Gl, G2 and NSm protein of unknown function. In an effort to investigate the function of the NSm protein, antibody was raised against NSm fusion protein overexpressed in Escherichia coli. This antibody was used to detect the NSm protein by using western blot analysis and electron microscopic observation after immunogold labelling. For the cloning of the NSm gene, total RNA extracted from a TSWV infected plant was used for cDNA synthesis and polymerase chain reaction (PCR) instead of going through time-consuming virus purification. A protein band specifically reacting to the NSm antibody was detected from TSWV inoculated plants. The NSm protein was detected in the cell wall fraction and in pellet from low speed centrifugation when the infected plant tissue was fractionated into 4 fractions. In the immuno-electron microscopic observation, gold particles were found around the plasmodesmata of infected plant tissue. These results suggest that the NSm protein of TSWV plays some role in cell-to-cell movement of this virus.

• Proceedings of the PSK Conference
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• 2000.04a
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• pp.180.3-180.3
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• 2000

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.310.1-310.1
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• 2002

The fusion (F) protein of respiratory syncytial virus(RSV) is an important antigen in including cross-protective immunity with neutralizing activity. Two peptides homologous to amino acid 205-225(F/205-225) and 255-278 (F/255-278) of the F glycoprotein of RSV containing B cell and T cell epitope were synthesized and then conjugated with KLH. (omitted)