Date of Award
Doctor of Philosophy (PhD)
Jennifer M. Martin
Bradley R. Olwin
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.
Wrobel, Christopher Martin, "The Transmembrane Domains of Latent Membrane Protein -1 Play Important Roles in Viral Signaling and Complex Formation" (2011). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations. 50.