An in vitro model for Epstein-Barr virus infection of human epithelial cells
博士 === 國立臺灣大學 === 微生物學研究所 === 88 === Epstein-Barr virus (EBV) is associated closely with nasopharyngeal carcinoma (NPC) derived from epithelial cells. EBV has been also detected in some other human epithelial malignancies. However, it is difficult to study EBV infection of epithelial cells and the v...
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博士 === 國立臺灣大學 === 微生物學研究所 === 88 === Epstein-Barr virus (EBV) is associated closely with nasopharyngeal carcinoma (NPC) derived from epithelial cells. EBV has been also detected in some other human epithelial malignancies. However, it is difficult to study EBV infection of epithelial cells and the virus-cell interaction for lack of a proper in vitro system. Using NPC cell lines and keratinocytes as the target cells, the major aim of this study is to establish an in vitro model for EBV infection of human epithelial cells and to characterize the expression of viral genes and the configuration of viral DNA in the EBV-infected epithelial cells.
Firstly, epithelial cells that expressed CR2, the well-known EBV receptor, were established by transfection with a CR2-expressing plasmid. In a CR2-mediated route, EBV particles could bind to such epithelial cells and 5 to 10% of the cells were infected to express EBV nuclear antigens (EBNA) or EBV-encoded small RNA (EBER1). Another infection route was achieved by cocultivating EBV-donor B cells with recipient epithelial cells and 1 to 5% of the recipient cells were infected after the cocultivation. The cocultivation-mediated EBV infection was CR2-independent because CR2-negative epithelial cells could be infected in this way and CR2-neutralizing antibodies could not block the infection. Meanwhile, the contact between EBV-donor B cells and recipient epithelial cells was required for the infection through cocultivation, since blocking cell-to-cell contact by multi-porous membranes prevented the infection while enhancement of cell contact by centrifugation could accelerate the infection. Such cell contact-dependent infection was also observed when various target epithelial cells or virus-donor B cells carrying different EBV strains were tested. In the other aspect, there was no evidence of cell-to-cell fusion involved in such infection. Notably, although CR2 expression and cell-to-cell contact contributed to mutually independent infection routes with limited infection efficiency, they could cooperate to enhance EBV infection of epithelial cells synergistically. More than 50% of CR2-expressing epithelial cells were infected through the cell contact-mediated infection.
The viral gene expression in EBV-infected epithelial cells was examined in this study. As has been identified in NPC biopsies, transcripts of latent genes, including EBNA1 initiated from the promoter Qp, latent membrane protein (LMP)1, LMP2A, LMP2B and EBER1, as well as BamHI A RNA, were detected in the in vitro infected epithelial cells using reverse transcription-polymerase chain reaction. Of note, some EBNA transcripts initiated from other two viral promoters, Cp and Wp, were detected in the infected epithelial cells. Using the same approach, Cp- and Wp-initiated gene transcription was also detected in some NPC biopsies. Furthermore, expression of EBNA2, one of the Cp/Wp-initiated genes, was demonstrated at the protein level in EBV-infected keratinocytes using immunofluorescence staining. Although Cp and Wp were considered inactive in NPC and other epithelial cells, our observations have indicated that Cp/Wp-initiated EBNA genes can be expressed in epithelial cells. Some of the infected epithelial cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical activation.
Using Gardella gel analysis, the configuration of EBV DNA was examined. The viral DNA could exist as a circularized episome in the infected epithelial cells. Unexpectedly, two stably EBV-positive epithelial cell clones H2B4 and H2B17-7, which were isolated from cloning by limiting dilution, harbored no extra-chromosomal EBV DNA. Further analysis indicated that the linear EBV DNA in these cells was not circularized through joining the genomic termini. Using fluorescence in situ hybridization, it was demonstrated that the EBV DNA persisting in these cells was integrated into chromosomes. These integrated viruses showed a distinct pattern of viral gene expression.
The in vitro infection model established in this study provides good chances to explore various topics about the interaction between EBV and epithelial cells. Further studies will help us to understand the infection mechanism and the roles of EBV in development of NPC or other epithelial malignancies.
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author2 |
Ching-Hwa Tsai |
author_facet |
Ching-Hwa Tsai Yao Chang 張堯 |
author |
Yao Chang 張堯 |
spellingShingle |
Yao Chang 張堯 An in vitro model for Epstein-Barr virus infection of human epithelial cells |
author_sort |
Yao Chang |
title |
An in vitro model for Epstein-Barr virus infection of human epithelial cells |
title_short |
An in vitro model for Epstein-Barr virus infection of human epithelial cells |
title_full |
An in vitro model for Epstein-Barr virus infection of human epithelial cells |
title_fullStr |
An in vitro model for Epstein-Barr virus infection of human epithelial cells |
title_full_unstemmed |
An in vitro model for Epstein-Barr virus infection of human epithelial cells |
title_sort |
in vitro model for epstein-barr virus infection of human epithelial cells |
publishDate |
2000 |
url |
http://ndltd.ncl.edu.tw/handle/58583982826180825395 |
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ndltd-TW-088NTU013810092016-01-29T04:18:40Z http://ndltd.ncl.edu.tw/handle/58583982826180825395 An in vitro model for Epstein-Barr virus infection of human epithelial cells Epstein-Barr病毒感染人類上皮細胞之體外模式 Yao Chang 張堯 博士 國立臺灣大學 微生物學研究所 88 Epstein-Barr virus (EBV) is associated closely with nasopharyngeal carcinoma (NPC) derived from epithelial cells. EBV has been also detected in some other human epithelial malignancies. However, it is difficult to study EBV infection of epithelial cells and the virus-cell interaction for lack of a proper in vitro system. Using NPC cell lines and keratinocytes as the target cells, the major aim of this study is to establish an in vitro model for EBV infection of human epithelial cells and to characterize the expression of viral genes and the configuration of viral DNA in the EBV-infected epithelial cells. Firstly, epithelial cells that expressed CR2, the well-known EBV receptor, were established by transfection with a CR2-expressing plasmid. In a CR2-mediated route, EBV particles could bind to such epithelial cells and 5 to 10% of the cells were infected to express EBV nuclear antigens (EBNA) or EBV-encoded small RNA (EBER1). Another infection route was achieved by cocultivating EBV-donor B cells with recipient epithelial cells and 1 to 5% of the recipient cells were infected after the cocultivation. The cocultivation-mediated EBV infection was CR2-independent because CR2-negative epithelial cells could be infected in this way and CR2-neutralizing antibodies could not block the infection. Meanwhile, the contact between EBV-donor B cells and recipient epithelial cells was required for the infection through cocultivation, since blocking cell-to-cell contact by multi-porous membranes prevented the infection while enhancement of cell contact by centrifugation could accelerate the infection. Such cell contact-dependent infection was also observed when various target epithelial cells or virus-donor B cells carrying different EBV strains were tested. In the other aspect, there was no evidence of cell-to-cell fusion involved in such infection. Notably, although CR2 expression and cell-to-cell contact contributed to mutually independent infection routes with limited infection efficiency, they could cooperate to enhance EBV infection of epithelial cells synergistically. More than 50% of CR2-expressing epithelial cells were infected through the cell contact-mediated infection. The viral gene expression in EBV-infected epithelial cells was examined in this study. As has been identified in NPC biopsies, transcripts of latent genes, including EBNA1 initiated from the promoter Qp, latent membrane protein (LMP)1, LMP2A, LMP2B and EBER1, as well as BamHI A RNA, were detected in the in vitro infected epithelial cells using reverse transcription-polymerase chain reaction. Of note, some EBNA transcripts initiated from other two viral promoters, Cp and Wp, were detected in the infected epithelial cells. Using the same approach, Cp- and Wp-initiated gene transcription was also detected in some NPC biopsies. Furthermore, expression of EBNA2, one of the Cp/Wp-initiated genes, was demonstrated at the protein level in EBV-infected keratinocytes using immunofluorescence staining. Although Cp and Wp were considered inactive in NPC and other epithelial cells, our observations have indicated that Cp/Wp-initiated EBNA genes can be expressed in epithelial cells. Some of the infected epithelial cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical activation. Using Gardella gel analysis, the configuration of EBV DNA was examined. The viral DNA could exist as a circularized episome in the infected epithelial cells. Unexpectedly, two stably EBV-positive epithelial cell clones H2B4 and H2B17-7, which were isolated from cloning by limiting dilution, harbored no extra-chromosomal EBV DNA. Further analysis indicated that the linear EBV DNA in these cells was not circularized through joining the genomic termini. Using fluorescence in situ hybridization, it was demonstrated that the EBV DNA persisting in these cells was integrated into chromosomes. These integrated viruses showed a distinct pattern of viral gene expression. The in vitro infection model established in this study provides good chances to explore various topics about the interaction between EBV and epithelial cells. Further studies will help us to understand the infection mechanism and the roles of EBV in development of NPC or other epithelial malignancies. Ching-Hwa Tsai 蔡錦華 2000 學位論文 ; thesis 148 en_US |