Transformation of primary B lymphocytes by Epstein-Barr virus requires the establishment of a strictly latent infection, the expression of several latent viral proteins, and sustained telomerase activity. Our previous findings indicated that induction of hTERT, the rate-limiting catalytic unit of the telomerase complex, was associated with the expression of the viral latent membrane protein 1 (LMP1). In the present study, we demonstrate that ectopic expression of LMP1 in BJAB and Ramos B cells resulted in an increase of hTERT transcripts, thus suggesting that LMP1 acts at the transcriptional level. This was confirmed by transient expression of a luciferase reporter plasmid containing the hTERT promoter cotransfected with an LMP1-expressing vector or transfected into B cells in which LMP1 expression was inducible. Consistently, silencing of LMP1 by small interfering RNA resulted in a reduction of hTERT transcripts. We also provide evidence indicating that LMP1-induced hTERT activation is independently mediated by NF-B and by mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways, whereas CD40, Akt, and mTOR signaling has no involvement. Moreover, our results do not support a role for c-Myc in mediating these effects on hTERT, since ectopic expression of LMP1 did not upregulate c-Myc and silencing of this oncogene or E box mutagenesis failed to inhibit LMP1-induced hTERT activation. These findings indicate that LMP1 simultaneously modulates multiple signal transduction pathways in B cells to transactivate the hTERT promoter and enhance telomerase activity, thus confirming the pleiotropic nature of this viral oncoprotein.Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a life-long asymptomatic infection in immunocompetent hosts by colonizing memory B lymphocytes. EBV has a potent transforming ability, being able to efficiently induce blast transformation and uncontrolled proliferation of infected B lymphocytes in vitro. Available evidence, particularly the presence of EBV genomes and the constant expression of viral proteins, strongly supports a relevant role for EBV in the pathogenesis of a wide spectrum of human malignancies, most of which are derived from B lymphocytes (12, 44). Latently EBV-infected B cells may express a defined set of latency genes that include those encoding six nuclear antigens (EBNAs) and three latent membrane proteins (latent membrane protein 1 [LMP1], LMP2A, and LMP2B). Among the EBV latency gene products, LMP1 is considered the strongest oncoprotein, being essential for immortalization of B cells. The N terminus and the six transmembrane domains of the protein form aggregates in the cytoplasmic membrane, allowing LMP1 to act like a constitutively activated receptor (5, 15). Indeed, LMP1 shares functional properties with members of the tumor necrosis factor (TNF) receptor superfamily, particularly CD40, and induces the expression of NF-B through activation of the TNF receptor-associated factor signaling pathway (31,40). Cons...