Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Jennifer M. Martin

Second Advisor

Corrella Detweiler

Third Advisor

Bradley R. Olwin

Fourth Advisor

Charles Odorizzi

Fifth Advisor

Hubert Yim

Abstract

Epstein-Barr virus (EBV) is an oncogenic Human Herpesvirus that infects B lymphocytes. EBV's Latent Membrane Protein-1 (LMP-1) is a constitutively active Tumor Necrosis Factor Receptor analog that activates a myriad of signaling pathways. LMP-1 activates NF-κB signaling, cytostasis, and suppresses interferon alpha (IFNα) signaling. This thesis characterizes LMP-1 signaling and dissects LMP-1 complex formation in infected cells. Structure function analysis revealed that the first transmembrane domain of LMP-1 is required for suppression of IFNα signaling. The LMP-1 C-terminus, dispensable for suppression of IFNα signaling in B cells, is required for IFNα suppression in 293T cells. These findings indicate that the mechanisms of suppression IFNα signaling in these two cell types are different. LMP-1 signaling activity correlates with its ability to oligomerize and localize in membrane microdomains. A high molecular weight species of LMP-1 was identified by non-reducing SDS-PAGE analysis and shown to be a cysteine 238 disulfide linked homo-dimer. Dimeric LMP-1 co-purifies with TRAF3 and dimeric LMP-1 is restricted to lipid rafts. Ability to crosslink interacting LMP-1 monomers requires the LMP-1 transmembrane domain and correlates with signaling activity. These findings support the model in which complex formation is key for LMP-1 signaling activity. I explored the hypothesis that LMP-1 signals to its various effectors not by forming one large multimeric signaling complex but forming multiple distinct complexes. Analysis of extracts from EBV-infected cells by BN-PAGE demonstrated LMP-1 forms multiple high molecular weight native complexes. The larger subset (>669 kDa) of these complexes is enriched in lipid rafts, as is actively signaling LMP-1. The smaller subset of complexes (669 kDa). These results demonstrate that the intact transmembrane domain of LMP-1 is not only important in IFNα signaling suppression, but plays an essential role in LMP-1 homo-oligomerization and higher order complex formation. Understanding the mechanisms by which LMP-1 activates signaling is necessary for understanding the oncogenicity of LMP-1 and for development of effective therapeutics to disrupt this signaling activity.

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