Latent Membrane Protein 1 of Epstein-Barr Virus Activates the hTERT Promoter and Enhances Telomerase Activity in B Lymphocytes

Abstract

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...

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost' nuclei and very short telomeres

“…31,33 LMP1 also activates the hTERT promoter in germinal center-derived BJAB Burkitt cells enhancing telomerase activity at the transcriptional level. 34 Moreover, EBNA1, also expressed in EBV-associated HL, promotes genomic instability in EBV-negative Burkitt cell lines BJAB and DG75 cells by the induction of reactive oxygen species, heralded by compensatory upregulation of gH2AX. 35 Thus, experimental LMP1 and EBNA1 upregulation in a germinal center-derived B-cell setting-Burkitt and RS cells are both germinal center derived 2 -mimics three in vivo and in vitro characteristics of EBV-negative RS cells, namely multinuclearity, high telomerase activity and genomic instability.…”

hTERT Inhibition Triggers Epstein–Barr Virus Lytic Cycle and Apoptosis in Immortalized and Transformed B Cells: A Basis for New Therapies

“…To normalize BATF levels for the amount of RNA, 10 mL of cDNA from each sample were amplified for the HPRT1 housekeeping gene, as described previously (19,24). Relative quantification was conducted using mean C t values according to the 2 ÀDC t formula, where…”

The Telomerase Inhibitor MST-312 Interferes with Multiple Steps in the Herpes Simplex Virus Life Cycle

“…qPCR was performed with a MiniOpticon PCR machine (Bio-Rad), using the following protocol: 95°C for 10 s and 44 cycles of 95°C for 30 s and 60°C for 1 min, followed by a melt curve from 65°C to 95°C. The relative starting quantity for each sample was determined by comparison to a standard curve generated by qPCR assays performed using 10-fold serial dilutions (1,10 Ϫ1 , 10 Ϫ2 , 10 Ϫ3 , 10 Ϫ4 , and 10 Ϫ5 ) of untreated infected-cell lysates. The average value for the DMSO-treated groups was set to 1, and all values are represented relative to this value.…”

“…Increased telomerase activity has been detected in cells infected with a variety of herpesviruses (3)(4)(5)(6)(7)(8)(9)(10)(11). There is evidence that HSV can mediate elevated telomerase activity during infection.…”