The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity

Abstract

In Epstein-Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of the BamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp-CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt's lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to maintain EBV-positive NPC cell lines in culture was related to Qp activity. Passaging of the NPC cell line HK666 led to activation of expression of BZLF1, which encodes Zta and loss of Qp function. Transient expression assays linked Zta expression to the down-regulation of Qp. Cotransfection of Zta reduced Qp activity in reporter assays. This negative regulation required Zta DNA-binding activity. We provide evidence that Zta up-regulation of p53 leads to p53-mediated interference with JAK/STAT activation of Qp. The data imply that JAK/STAT signaling has a role in EBV-associated malignancies.

Figure 1

Qp contains potential STAT-binding sites. (A) The relative locations on the EBV genome of the Qp, Cp, and BamHI-W promoter (Wp) latency promoters and the ORF (BKRF1) encoding EBNA-1 are illustrated. The DNA sequence from −100 to +36 that was included in the Qp-CAT reporter is shown. The position of the IRF-binding site (broken line), two potential STAT-binding sites (underlined), and RNA start site (arrow) are indicated. The nucleotides in lowercase show the mutations introduced into QpSTATm1–CAT and QpSTATm2–CAT. (B) Comparison of STAT DNA-binding sequences with the potential Qp STAT sequences (38). (C) Activation of Qp-CAT by JAK-1. HeLa cells were transfected with a control STAT–CAT reporter or Qp-CAT reporter. As indicated, cells were also cotransfected with JAK-1 or JAK-1 plus STAT-1 or treated with IL-6. The results shown are an average of two experiments with the standard deviation indicated.

Figure 2

Qp binds STAT-4. (A) Electrophoretic mobility-shift assay showing binding of purified STAT-4 to Qp probe (lane 2). The DNA–protein complex was disrupted by anti-STAT-4 antibody (lane 3) but not by control antibody (lane 4). (B) Electrophoretic mobility-shift assay illustrating the specificity of STAT-4 binding. STAT-4 binding was competed by unlabeled STAT-4 (lane 3), Qp (lane 4), and QpIRFmAT (lane 6) oligonucleotides but not by QpSTAT mutant (lane 5) or control oligonucleotides (lane 7). Lane 1 contains Qp probe alone.

Figure 3

Effect of mutations in the potential STAT-binding sites on JAK activation (A) and basal activity (B) of Qp-CAT in transfected HeLa cells. The results shown are an average of two experiments with the standard deviation indicated.

Figure 4

Constitutively activated STAT-4 is present in EBV⁺ Rael B cells and HK666 nasopharyngeal carcinoma cells. Indirect immunofluorescence assay showing the presence of nuclear STAT-4 protein in HK666 cells and nuclear plus cytoplasmic STAT-4 in Rael. Arrowheads indicate cytoplasmic staining in Rael.

Figure 5

Passaging of HK666 leads to induction of BZLF-1 and loss of Qp expression. (A) Expression of Qp-initiated EBNA-1 transcripts and BZLF-1 transcripts in HK666 cells was examined by RT-PCR. Southern blots of the DNA products were probed for BKRF-1 (EBNA-1) (Upper) and BZLF-1 (Lower). Cells at passage 4 were also tested after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) (20 ng/ml) and n-butyrate (3 mM) for 48 hr (+TPA). (B) Detection of Zta protein in HK666 cells by immunoperoxidase staining.

Figure 6

Zta down-regulates Qp-CAT expression. (A) Qp-CAT was cotransfected into HeLa cells with Zta or Zta variants carrying mutations in the DNA-binding domain (Zdbm1) or the dimerization domain (Zdmmt) or deletions in the activation domain (ZtaΔ2–25, ZtaΔ2–141, ZtaΔ25–86, ZtaΔ93–141). The HHV8-K8 effector and oriLyt-CAT target served as controls for the specificity of the Zta-mediated repression of Qp-CAT. The results shown are an average of two experiments, with the standard deviation indicated. (B) Induction of Zta in the HeLa-Zta cell line by removal of tetracycline from the medium led to reduced expression of transfected Qp-CAT. The p53-CAT reporter served as a positive control for Zta expression. CAT activity is expressed relative to that of Qp-CAT in the presence of tetracycline (set at 100%).

Figure 7

p53 is a downstream effector of Qp repression. (A) Qp-CAT was down-regulated by coexpression of p53. Qp-CAT was cotransfected into HeLa cells with either wild-type (Wt) or mutant (G245 m, R248 m) p53. p53-CAT formed a positive control for p53 function. The results shown are an average of two experiments, with the standard deviation indicated. (B) Zta is an ineffective repressor of Qp-CAT in p53⁻ cells. The effect of cotransfection of Zta or p53 on Qp-CAT activity was compared in transfected HeLa and p53⁻ Hep3B cells. Qp-CAT activity in the absence of cotransfected Zta and p53 is set at 100%.

Figure 8

Zta and p53 interfere with JAK/STAT activation of a STAT–CAT reporter. The effect of cotransfected Zta and p53 on STAT–CAT activity was compared in HeLa cells in the presence or absence of cotransfected JAK-1. The activity of STAT–CAT was set at 100% in each case.

Figure 9

Summary of Qp regulation in the different forms of EBV latency.