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Title: マレック病ウイルスICP4ホモローグによるウイルス遺伝子発現の活性化
Other Titles: Enhancement of gene expression by Marek's disease virus homologue of the herpes simplex virus-1 ICP4
Authors: 遠藤, 大二1 Browse this author
Authors(alt): Endoh, Daiji1
Issue Date: 28-Jun-1996
Abstract: Marek's disease (MD) is a disease of domestic chickens caused by cellassociated herpes virus(MDV) and characterized prindpally by T cell lymphoma and demyelinating peripheral neuropathy. Three general types of virus-cell interactions are recognized: lytic, latent and transforming. Activation of virus genes is an important step for determining virus-cell interactions. The MDV homologue of the herpes simplex virus-1-ICP4 (MDVICP4) may be an important activator of virus genes. The aim of this study is to show the evidence for activation of virus genes by MDV ICP4 and the existence of the cells expressing MDV ICP4 in the tissues of MDV-infected chickens. Although MD tumor cell line, MDCC-MSB-1 (MSB-1) contains multiple copies of MDV genomes, the transcription of MDV DNA occurs along lmited portions of the viral genome. To observe the effect of MDV ICP4 on the expression of viral genes, I transfected MSB-1 tumor cells with plasmid including a coding region of MDV ICP4 using a cationic liposome. As caniers for intranuclear transport, high moboility group-1 and -2 proteins were bound to the plasmid DNA before forming liposomes. I detected transcripts from the plasmid 2 hr after transfection by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. I also detected abundant transcripts of MDV ICP4 from MDV genomes in MSB-1 cells (endogeneus ICP4) 2-96 hr after transfection. Although there was no significant diference, transcripts of phosphorylated protein pp38 were also increased. These findings suggested that expression of the introduced MDV ICP4 gene enhanced the expression of endogenous MDV ICP4 and pp38. On the other hand, transcripts of MDV DNA polymerase were not detected in transfected MSB-1 cells and quantitative PCR analysis for virus genome DNA indicated no significant alteration of copy number of the virus genome in transfected MSB-1 cells, suggesting that viral replication requires more th an turning on the MDV ICP4 gene. pp38 is one of few proteins that is detected in the MD lymphom a and lymphoblastoid cell lines and is also known as one of few proteins that could be detected on paraffin-embedded sections. Thus pp38 is an important indicator of MD-infected cells in MDV-infected tissues. In this study, the promoter/enhancer element of pp38 was identified by cDNA analysis and by open reading frame (ORF) analysis with recombinant protein. Namely, the cDNA coding full length pp38 was cloned and analyzed to ensure that the predicted ORF was the actual ORF in vivo. A full length recombinant protein coded by the entire open reading frame of the MDV pp38 cDNA was synthesized in E. coli and purified by affinity chromatography. Immunoblot analysis indicated that the expressed recombinant protein had the same mobility as the pp38 in infected cells. These findings and the result of primer extension analysis showed that the promoter of pp38 exists 15-19 bases upstream from the 5'-end of the cDNA and that the enhancer sequences include ICP4, Sp-1 and Oct-1 binding elements. The promoter/enhancer fragrnent was ligated into plasmid pGV-B on just upstream of the lueiferase gene as a reporter gene. The luciferase gene of the constructed plasmid was expressed in MSB-1 cells. Expression of the luciferase under the control of the promoter/enhancer of pp38 gene was enhanced when the cens were cotransfected with the MDV ICP4-expressing plasmid. These findings suggest that MDV ICP4 upregulates the enhancer of pp38. Transfection experiments using MDV ICP4-expressing plasmid suggest that the expression of MDV ICP4 preceded the expression of early or late genes. Thus a variety stage of MDV infection may be detected using MDV ICP4 as an indicator. To observe the MDV ICP4-expressing cells in vivo, I showed transcripts of the MDV ICP4 on paraformaldehyde-fixed and parafiin embedded cells by in situ hybridization (ISH). A cRNA-probe synthesized from O.55 kb DNA fragment clones from 1 kb upstream of ORF of MDV ICP4 was selected as the probe for MDV ICP4 transcripts, as it hybridized to full lengh transcripts on Northern blot. Using the digoxigeninlabeled O.55 kb cRNA probe, MDV ICP4 transcripts were detected in 920% of lytically-infected chicken embryo fibroblasts (CEF) and in 0.35% of MSB-1. No signal was observed on uninfected CEF. By this ISH method, 1 detected MDV ICP4 transcripts in the feather follicle epithelium (FFE), lymphoid cells in the liver, kidneys and nerve of infected chickens. The MDV ICP4 transcripts were also detected in the FFE, nerve and lymphocytic foci of liver and kidney by RT-PCR. In conclusion, MDV ICP4 is suggested to activate some MDV genes, while infection does not proceed to the lytic stage with MDV ICP4 alone. And I detected the MDV ICP4 transcripts in the lymphoid cells of MDV-infected chickens by the ISH method. As the significant proponion of lymphoid cells are reported to be non-lytic on the pre-tumorigenic stage, ISH sigrials in the lymphoid cells may indicate the partial activation of MDV genes by MDV ICP4. The application of the ISH method on the observation of the expression of viral transactivators may resulted in signjficant advances in understanding the mechanisms for tumorigenesis by MDV.
Conffering University: 北海道大学
Degree Report Number: 乙第5019号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 遠藤 大二

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