Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.

The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells.

Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.

The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B.

The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.

Development of glutathione peroxidase activity during dietary and genetic copper deficiency.

Copper deficiency was produced in developing rodents to study a possible interaction between copper and the selenoenzyme, glutathione peroxidase (GSH-Px). Dietary copper deficiency was investigated in Sprague-Dawley rats and in three mouse strains (C57BL, C3H/HeJ, C58); genetic copper deficiency was studied in two of the mouse strains, C57BL and C3H/HeJ, using brindled mice. In certain cases it appeared that copper deficiency was associated with depressed liver GSH-Px activity; values from copper-deficient livers were 40-70% of control values. However, the decrease in liver GSH-Px in both rats and mice was only observed when body weight was also depressed and did not necessarily correlate with copper deficiency signs, such as lower serum ceruloplasmin or liver cytochrome oxidase activities. In weanling rats, serum GSH-Px activity was normal despite a 60% reduction in liver activity. Mouse liver GSH-Px activity rose fourfold during the first 3 weeks of life to 75% of the adult level. Rat liver GSH-Px also increased during the suckling period. When perinatal copper deficiency, nutritional or genetic, was severe enough to retard growth, liver GSH-Px activity was depressed. Unlike rats, adult murine liver GSH-Px was equivalent in males and females.

The proto-oncogene Pim-1 is a target of miR-33a.

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3'-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

The Epstein-Barr virus BRLF1 gene product transactivates the murine and human c-myc promoters.

A common feature of the Epstein-Barr virus (EBV)-associated malignancy, Burkitt's lymphoma, is chromosomal translocation affecting the c-myc oncogene. We report here that an EBV immediate-early (IE) protein, BRLF1 (R), transactivates the murine and human c-myc promoters. The R transactivator enhances expression from transiently transfected murine and human c-myc promoters as determined both at the CAT reporter and at the mRNA level. Transactivation of the human c-myc promoter by R occurs in several different cell lines, and this effect is reporter-gene independent. Both the P1 and P2 c-myc promoters can be activated by the EBV R IE protein, although the R-induced transactivation of P1 is greater than P2. The portion of the human c-myc promoter from -228 to -63 (relative to the P1 mRNA start site) is necessary, but not sufficient, for transactivation by R in the Louckes B-cell line. Binding of the R protein directly to the c-myc promoter could not be demonstrated, suggesting that the effect of R on c-myc activity occurs by an indirect mechanism. The ability of an EBV protein to activate c-myc expression is likely to facilitate productive viral infection and is also potentially relevant in the genesis of EBV-associated lymphomas.

Detection of low level of human papilloma virus type 16 DNA sequences in cancer cell lines derived from two well-differentiated nasopharyngeal cancers.

Human papilloma virus type 16 (HPV16) related sequences were detected in two EBV-negative nasopharyngeal carcinoma (NPC) cell lines derived from two well-differentiated NPC by polymerase chain reaction. E2 and E6 related sequences of HPV16 were demonstrated using two pairs of primers derived from these two regions. DNA sequence analysis of amplified products excluded the possibility of laboratory viral DNA contamination.

EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus.

Among the few Epstein-Barr virus (EBV) genes expressed during latency are the Epstein-Barr nuclear antigens (EBNAs), at least one of which contributes to the ability of the virus to transform B lymphocytes. We have analyzed a promoter located in the BamHI-C fragment of EBV which is responsible for the expression of EBNA-1 in some cell lines. Deletion analysis of a 1.4-kb region 5' of the RNA start site has identified a 700-bp fragment that is required for optimal promoter activity in latently infected B lymphocytes, as shown by promoter constructs linked to the chloramphenicol acetyltransferase reporter gene. This fragment is also able to enhance activity, in an orientation-independent manner, of the simian virus 40 early promoter linked to the chloramphenicol acetyltransferase gene. The enhancer element has some constitutive activity in EBV-negative lymphoid cells, which is increased in the presence of the EBNA-2 gene product. Further deletions have shown that the EBNA-2-responsive region requires a 98-bp region that contains a degenerate octamer-binding motif. In epithelial cells there was no enhancer activity regardless of the presence of EBNA-2. These results demonstrate that BamHI-C promoter activity may be dependent not on an enhancer contained in the ori-P, as was previously assumed, but rather on EBNA-2 transactivation of this more proximal enhancer located in the upstream region of the BamHI C promoter itself.

The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor.

Disruption of viral latency in Epstein-Barr virus-infected cells is mediated through the activation of the BZLF1 (Z) immediate-early gene product. The Z protein can be derived from either of two promoters: the BZLF1 promoter, which directs transcription of a 1.0-kb mRNA encoding the Z gene product alone, or the upstream BRLF1 promoter, which directs transcription of a 2.8-kb bicistronic mRNA encoding the BRLF1 and BZLF1 immediate-early proteins. In this study we have examined the regulation of the BRLF1 promoter by viral and cellular factors. We found that the BRLF1 promoter is autoregulated by the BRLF1 transactivator through a nonbinding mechanism. We show that the BRLF1 (but not the BZLF1) promoter is highly responsive to the Sp1 transcription factor. Sp1 activation of the BRLF1 promoter is mediated through a consensus Sp1-binding site located from -39 to -44 (relative to the mRNA start site). We demonstrate that the BRLF1 promoter has high constitutive activity in C-33 cells (an epithelial cell line) and that the proximal Sp1-binding site is required for this activity. Despite the ubiquitous presence of Sp1 in many cell types, we found that the BRLF1 promoter has essentially no activity in lymphoid cell lines, suggesting that factors other than Sp1 may negatively regulate the BRLF1 promoter in these cells. Our findings demonstrate that the two potential promoters directing BZLF1 transcription are differentially regulated and that Sp1 can activate the BRLF1 promoter but not the BZLF1 promoter.

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early promoter, BMRF1.

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243-9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085-4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at -59 and -106), whereas two additional Z binding sites (located at -42 and -170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251-8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.