EBV-Encoded LMP1 Upregulates Igκ 3′Enhancer Activity and Igκ Expression in Nasopharyngeal Cancer Cells by Activating the Ets-1 through ERKs Signaling

Accumulating evidence indicates that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cells, express immunoglobulins (Igs). We previously found that the expression of the kappa light chain protein in NPC cells can be upregulated by the EBV-encoded latent membrane protein 1 (LMP1). In the present study, we used NPC cell lines as models and found that LMP1-augmented kappa production corresponds with elevations in ERKs phosphorylation. PD98059 attenuates LMP1-induced ERKs phosphorylation resulting in decreased expression of the kappa light chain. ERK-specific small interfering RNA blunts LMP1-induced kappa light chain gene expression. Luciferase reporter assays demonstrate that immunoglobulin κ 3′ enhancer (3′Eκ) is active in Igκ-expressing NPC cells and LMP1 upregulates the activity of 3′Eκ in NPC cells. Moreover, mutation analysis of the PU binding site in 3′Eκ and inhibition of the MEK/ERKs pathway by PD98059 indicate that the PU site is functional and LMP1-enhanced 3′Eκ activity is partly regulated by this site. PD98059 treatment also leads to a concentration-dependent inhibition of LMP1-induced Ets-1 expression and phosphorylation, which corresponds with a dose-dependent attenuation of LMP1-induced ERK phosphorylation and kappa light chain expression. Suppression of endogenous Ets-1 by small interfering RNA is accompanied by a decrease of Ig kappa light chain expression. Gel shift assays using nuclear extracts of NPC cells indicate that the transcription factor Ets-1 is recruited by LMP1 to the PU motif within 3′Eκ in vitro. ChIP assays further demonstrate Ets-1 binding to the PU motif of 3′Eκ in cells. These results suggest that LMP1 upregulates 3′Eκ activity and kappa gene expression by activating the Ets-1 transcription factor through the ERKs signaling pathway. Our studies provide evidence for a novel regulatory mechanism of kappa expression, by which virus-encoded proteins activate the kappa 3′ enhancer through activating transcription factors in non-B epithelial cancer cells

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Binds and Protects a Nuclear Noncoding RNA from Cellular RNA Decay Pathways

The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless β-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway