Analysis of the requirement of Notch signaling for EBV LMP2A function in vitro and in vivo.

摘要

Epstein Barr Virus (EBV) is associated with malignancies of lymphoid and epithelial origin. While the precise role of EBV in oncogenesis remains elusive, latent membrane protein 2A (LMP2A) is detected in all EBV-associated malignancies, implicating LMP2A in their pathogenesis. Interestingly, LMP2A is expressed in EBV-associated malignancies such as Burkitt's Lymphoma, Hodgkin's Lymphoma and Nasopharyngeal Carcinoma in the absence of the viral transcriptional activator, EBNA2, suggesting an alternative mechanism is responsible for expression of LMP2A. The intracellular domain of Notch (Notch-IC) and EBNA2 are functional homologues and recent microarray analysis suggested that LMP2A may constitutively activate the Notch pathway in vivo. Western blot analysis and real time RT-PCR indicate that LMP2A constitutively activates the Notch pathway in B cells and epithelial cells. Expression of LMP2A alone is sufficient to drive expression of luciferase under the control of the LMP2A promoter and mutational analysis revealed signaling through the LMP2A amino-terminus is required. Deletion of the RBP-Jk consensus sequences results in a significant decrease in promoter activity, indicating that LMP2A utilizes the Notch pathway to control its own expression.;Given the importance of Notch signaling in cell fate decisions during lymphopoiesis and the ability of LMP2A to activate the Notch pathway, we sought to determine whether LMP2A uses Notch1 in vivo to alter B cell identity. Transgenic mice expressing LMP2A in B cells (TgE) were intercrossed with mice expressing loxP-flanked Notch1 gene and Cre recombinase. Using flow cytometry, it was shown that B cells from TgE LMP2A mice lacking Notch1 have phenotypic differences from TgE LMP2A mice that suggest Notch1 plays a role in the strength of the LMP2A survival and developmental signal. From these results, we propose that LMP2A exploits the Notch pathway to contribute to EBV associated malignancies in two ways. First, constitutive activation of the Notch pathway allows for EBNA2 independent expression of LMP2A, allowing LMP2A to provide aberrant growth signals to cells. Additionally, expression of LMP2A may contribute to the alterations in B cell identity seen in Hodgkin and Reed-Sternberg cells in part by exploiting the Notch pathway.