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The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection and Immortalization

Wasil, Laura R. (2013) The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection and Immortalization. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Epstein-Barr virus (EBV) has been associated with the development of several human malignancies. Infection of B cells with wild-type EBV in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). The majority of latent genes are expressed during early infection, including latent membrane protein 2 (LMP2). Currently, the role of LMP2 in B cell proliferation is controversial; some studies have shown that LMP2 is dispensable, while others report it is important role for this process. However, each of these experimental systems were limited by either wild-type virus contamination or use of incomplete viral genomes (mini-EBV), which precluded clear assessments of the effects of LMP2 during early infection.
In this study I investigated the effect of LMP2 on early B cell infection and subsequent immortalization via complete recombinant EBV with knockouts of either or both isoforms of the LMP2 gene, LMP2A and LMP2B (Δ2A, Δ2B and Δ2A/Δ2B). Infection of B cells with LMP2A knockout viruses resulted in marked decreases in activation and proliferation relative to wild-type, and higher percentages of apoptotic B cells. Δ2B virus infection exhibited activation levels comparable to wild-type, but with fewer numbers of proliferating B cells. The stability of viral latency was determined for early B cell infection by evaluating latent and lytic gene expression with or without lytic stimulation via the B cell receptor (BCR). Infection with wild-type, Δ2A and Δ2B viruses with or without BCR stimulation did not result in changes in viral latency, whereas stimulation of BCR signaling in Δ2A/Δ2B-infected cells resulted in decreased LMP1 expression, suggesting loss of stability in viral latency. The long-term effects of LMP2 deletion on B cell outgrowth were investigated using LCL establishment assays, which revealed that LMP2A, but not LMP2B, is critical for efficient immortalization of B cells in vitro. Loss of both isoforms promoted the least activation, proliferation and LCL formation. This study enhances our knowledge of events required for B cell transformation by EBV, and clearly shows the public health relevance of understanding genes involved in tumorigenesis for the future pursuit of more effective treatments for EBV-associated malignancies.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Wasil, Laura R.lrw4@pitt.eduLRW4
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRowe, David T.rowe1@pitt.eduROWE1
Committee MemberAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Committee MemberCoyne, Carolyn Bcoynec2@pitt.eduCOYNEC2
Committee MemberKinchington, Paulkinchingtonp@upmc.eduKINCH
Date: 29 January 2013
Date Type: Publication
Defense Date: 6 December 2012
Approval Date: 29 January 2013
Submission Date: 21 November 2012
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 169
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: EBV,LMP2,B cell,activation,proliferation,LCL
Date Deposited: 29 Jan 2013 22:23
Last Modified: 01 Jan 2018 06:15
URI: http://d-scholarship.pitt.edu/id/eprint/16998

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  • The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection and Immortalization. (deposited 29 Jan 2013 22:23) [Currently Displayed]

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