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postgraduate thesis: Epstein-barr virus infection and viral gene expression in nasopharyngeal epithelial cells

TitleEpstein-barr virus infection and viral gene expression in nasopharyngeal epithelial cells
Authors
Advisors
Issue Date2013
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Pang, P. S. [彭佩欣]. (2013). Epstein-barr virus infection and viral gene expression in nasopharyngeal epithelial cells. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5060567
AbstractNasopharyngeal carcinoma (NPC) is prevalent in Southern China and closely associated with Epstein-Barr virus (EBV) infection. EBV can easily infect B cells and transform them into proliferative lymphoblastoid cell line (LCL). In contrast, direct infection of epithelial cells with EBV has been difficult to achieve. The low infection efficiency hampers investigations into early events relating to EBV infection of nasopharyngeal epithelial cells. Our laboratory has observed that pretreatment of nasopharyngeal epithelial cells with TGF-β sensitized them to EBV infection by co-culturing with EBVproducing Akata cells. In this study, high infection efficiency was achieved by directly infecting TGF-β-pretreated nasopharyngeal epithelial (NPE) cells with cell-free EBV-containing supernatant. Besides, concentrating EBV virions, resuspending EBV virions in full-supplemented RPMI medium and spinning the viral particles to nasopharyngeal epithelial cells under 37oC could improve EBV infection rate by 50-fold. Polybrene could also further enhance the EBV infection rate by around 15%. NPE cells immortalized with different genetic elements have variable rates of EBV infection and may be dependent on their sensitivity to TGF-β. The enhanced EBV infection rate by TGF-β may not be dependent on modulation of extracellular matrix synthesis as coating the culture dishes with collagen, fibronectin or laminin did not enhance significantly EBV infection rate in NPE cells. Treatment of TGF-β induces integrin α5 clustering associated with formation of stress fibers in NPE cells. Knockdown of integrin β1 and αv by shRNA have suppressed EBV infection in HONE1 cells (a NPC cell line) while shRNA knockdown of integrin α5 partially suppressed EBV infection in HONE1 and NP460hTert cells. Disruption of actin polymerization by cytochalasin D efficiently inhibited EBV infection. Therefore, integrins α5β1, αv and actin polymerization are crucial for EBV infection in NPE cells. The high EBV infection rate has enabled examination of dynamics of EBV episomes and viral gene expression profiles in freshly infected NPE cells. Lower levels of EBV gene transcriptions and a rapid loss of EBV genome in freshly infected NP460hTert cells were observed as compared to freshly infected Akata cells (B-cell lymphoma cell line) indicating that freshly infected NPE cells were unable to support EBV persistence although the cells were highly infected. These results indicated that EBV failed to persist in infected epithelial cells as compared to cells from B cell lineage. Generally, EBV copy number and mRNA transcription levels of latent and lytic genes of EBV in freshly infected NP460hTert cells within 7 days post-infection were much lower than the stably infected NP460hTert cells after propagated for more than 100 passages with G418 selection indicating that the establishment of stably infected cell line is a long process which requires multiple factors that are significant for EBV amplification and maintenance. Albeit the differences in transcription levels, expression of EBV genes representing latency type II infection was detected in both freshly and stably infected NP460hTert cells. This study has contributed to the understanding of early events of EBV infection in premalignant NPE cells and may have implication on the development of NPC.
DegreeDoctor of Philosophy
SubjectEpstein-Barr virus.
Epstein-Barr virus diseases.
Epithelial cells.
Nasopharynx - Cancer.
Dept/ProgramAnatomy
Persistent Identifierhttp://hdl.handle.net/10722/188743

 

DC FieldValueLanguage
dc.contributor.advisorTsao, GSW-
dc.contributor.advisorCheung, A-
dc.contributor.authorPang, Pei Shin.-
dc.contributor.author彭佩欣.-
dc.date.accessioned2013-09-08T15:07:51Z-
dc.date.available2013-09-08T15:07:51Z-
dc.date.issued2013-
dc.identifier.citationPang, P. S. [彭佩欣]. (2013). Epstein-barr virus infection and viral gene expression in nasopharyngeal epithelial cells. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5060567-
dc.identifier.urihttp://hdl.handle.net/10722/188743-
dc.description.abstractNasopharyngeal carcinoma (NPC) is prevalent in Southern China and closely associated with Epstein-Barr virus (EBV) infection. EBV can easily infect B cells and transform them into proliferative lymphoblastoid cell line (LCL). In contrast, direct infection of epithelial cells with EBV has been difficult to achieve. The low infection efficiency hampers investigations into early events relating to EBV infection of nasopharyngeal epithelial cells. Our laboratory has observed that pretreatment of nasopharyngeal epithelial cells with TGF-β sensitized them to EBV infection by co-culturing with EBVproducing Akata cells. In this study, high infection efficiency was achieved by directly infecting TGF-β-pretreated nasopharyngeal epithelial (NPE) cells with cell-free EBV-containing supernatant. Besides, concentrating EBV virions, resuspending EBV virions in full-supplemented RPMI medium and spinning the viral particles to nasopharyngeal epithelial cells under 37oC could improve EBV infection rate by 50-fold. Polybrene could also further enhance the EBV infection rate by around 15%. NPE cells immortalized with different genetic elements have variable rates of EBV infection and may be dependent on their sensitivity to TGF-β. The enhanced EBV infection rate by TGF-β may not be dependent on modulation of extracellular matrix synthesis as coating the culture dishes with collagen, fibronectin or laminin did not enhance significantly EBV infection rate in NPE cells. Treatment of TGF-β induces integrin α5 clustering associated with formation of stress fibers in NPE cells. Knockdown of integrin β1 and αv by shRNA have suppressed EBV infection in HONE1 cells (a NPC cell line) while shRNA knockdown of integrin α5 partially suppressed EBV infection in HONE1 and NP460hTert cells. Disruption of actin polymerization by cytochalasin D efficiently inhibited EBV infection. Therefore, integrins α5β1, αv and actin polymerization are crucial for EBV infection in NPE cells. The high EBV infection rate has enabled examination of dynamics of EBV episomes and viral gene expression profiles in freshly infected NPE cells. Lower levels of EBV gene transcriptions and a rapid loss of EBV genome in freshly infected NP460hTert cells were observed as compared to freshly infected Akata cells (B-cell lymphoma cell line) indicating that freshly infected NPE cells were unable to support EBV persistence although the cells were highly infected. These results indicated that EBV failed to persist in infected epithelial cells as compared to cells from B cell lineage. Generally, EBV copy number and mRNA transcription levels of latent and lytic genes of EBV in freshly infected NP460hTert cells within 7 days post-infection were much lower than the stably infected NP460hTert cells after propagated for more than 100 passages with G418 selection indicating that the establishment of stably infected cell line is a long process which requires multiple factors that are significant for EBV amplification and maintenance. Albeit the differences in transcription levels, expression of EBV genes representing latency type II infection was detected in both freshly and stably infected NP460hTert cells. This study has contributed to the understanding of early events of EBV infection in premalignant NPE cells and may have implication on the development of NPC.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B50605677-
dc.subject.lcshEpstein-Barr virus.-
dc.subject.lcshEpstein-Barr virus diseases.-
dc.subject.lcshEpithelial cells.-
dc.subject.lcshNasopharynx - Cancer.-
dc.titleEpstein-barr virus infection and viral gene expression in nasopharyngeal epithelial cells-
dc.typePG_Thesis-
dc.identifier.hkulb5060567-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineAnatomy-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2013-

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