The binding of CD40 on B cells to CD154 on activated CD4 T cells is essential for regulating humoral responses in antigen-primed B cells. The EBV transforming protein, LMP1 mimics many aspects of CD40 function through the binding of an overlapping set of TNF receptor-associated factor (TRAF) signaling molecules. Co-expression of LMP1 and CD40 occurs at distinct stages of EBV infection and in tumor cells in which EBV is an etiological agent. Although both molecules share functional homology, the signaling cross talk between LMP1 and CD40 is not well understood. Here we show that LMP1 directly increases both TRAF3 and CD40 in B cells through elevated RNA expression. Additionally, LMP1 enhances the association of CD40 and TRAF3 with lipid rafts. To better understand LMP1-specific changes in CD40 signaling we evaluated the activity of specific downstream targets in response to CD154 stimulation using phospho-specific flow cytometry. Stimulated B cells produced a greater signal for p38 and p65 than those constitutively expressing LMP1. Also, CD40 signaling reduced the activity of LMP1-induced pLCγ2 whereas LMP1 significantly lowered that of Akt in cells receiving both stimuli. Together, our results demonstrate that LMP1 increases expression of CD40 and TRAF3 and specifically modifies CD40 activation responses when both proteins are concurrently active. These changes have the potential of underlying differences in B cell activation, survival and differentiation.

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