The long-term goal of this research has been to determine the role of EBV in the etiology of nasopharyngeal carcinoma (NPC), an epithelial malignancy that develops with high incidence in Southern China, Northern Africa, and among Eskimos. To accomplish this, we have identified the viral genes that are expressed in NPC including the latent membrane proteins, LMP1 and 2, and the family of mRNAs, transcribed through the BamHI A fragment (BARTs). Sequence analysis of the intricately spliced cDNAs representing the BamHI A transcripts has revealed four previously unidentified open reading frames. To determine how EBV affects epithelial cell growth, we have expressed the viral proteins in epithelial cell lines and in primary keratinocytes. We have identified specific NFKappaB complexes induced by LMP1 in cultured epithelial cells and have detected homodimers of NFkappaB p50 complexed with bcl3 in NPC tumors. The activation of these specific forms of NFkappaB is mediated through the CTAR1 domain of LMP1. We have also shown that in epithelial cells LMP2 activates PI3 kinase and the Akt kinase and that this activation inhibits epithelial differentiation in raft cultures. Activation of Akt by LMP2 induces translocation of beta-catenin into the nucleus. We have also determined that LMP1 activates Akt but does not affect beta -catenin. We have used two hybrid analysis with one of the BART ORFs, RK-BARF0, and determined that RK-BARF0 interacts with Notch, HIC, epithelin, and scramblase. The interaction with Notch induces its degradation and we have shown that Notch is expressed at very low levels in EBV infected cells. In this grant, we will further analyze the biochemical basis of LMP1-mediated transformation signaling, the biologic and biochemical properties of LMP1 and LMP2 in epithelial cells, and the molecular properties of the BamHI A proteins.
Our specific aims are: 1.) Characterize the signal transduction pathways activated by LMP1 in epithelial cells that affect cellular gene expression and identify the cellular genes that are activated by the distinct forms of NFKappaB induced by LMP1. 2.) Determine the effects of expression of LMP1 and LMP2 on cellular gene expression and epithelial cell growth properties. 3.) Analyze expression in NPC samples and cell lines to identify coordinate expression of LMP1, LMP2 and activation of specific signaling targets. 4.) Identify the proteins encoded by the BamHI A open reading frames in cell lines and in NPC tumors and determine the effects of expression of the BamHI A proteins on viral and cellular expression and cellular growth properties.
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