ABSTRACT
Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.
Subject(s)
DNA-Binding Proteins/metabolism , Down-Regulation , Herpesvirus 4, Human/pathogenicity , Receptors, Tumor Necrosis Factor/metabolism , Trans-Activators/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Viral Proteins , Apoptosis/drug effects , Cell Line , Cell Line, Transformed , Humans , Keratinocytes/virology , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The Epstein-Barr virus (EBV) immediate-early protein BZLF1 is a transcriptional activator that mediates the switch between the latent and the lytic forms of EBV infection. It was previously reported that BZLF1 inhibits p53 transcriptional function in reporter gene assays. Here we further examined the effects of BZLF1 on p53 function by using a BZLF1-expressing adenovirus vector (AdBZLF1). Infection of cells with the AdBZLF1 vector increased the level of cellular p53 but prevented the induction of p53-dependent cellular target genes, such as p21 and MDM2. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. Thus, the inhibitory effects of BZLF1 on p53 transcriptional function cannot be explained by its effects on p53 phosphorylation, acetylation, or DNA binding activity. BZLF1 substantially reduced the level of cellular TATA binding protein (TBP) in both normal human fibroblasts and A549 cells, and the inhibitory effects of BZLF1 on p53 transcriptional function could be partially rescued by the overexpression of TBP. Thus, BZLF1 has numerous effects on p53 posttranslational modification but may inhibit p53 transcriptional function in part through an indirect mechanism involving the suppression of TBP expression.
Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins , Trans-Activators/physiology , Tumor Suppressor Protein p53/physiology , Viral Proteins , Acetylation , Amino Acid Sequence , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , DNA/metabolism , Humans , Molecular Sequence Data , Phosphorylation , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-mdm2 , Serine/metabolism , TATA-Box Binding Protein/analysis , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/physiology , Transcriptional ActivationABSTRACT
The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G(1)/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G(1)/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.
Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins/physiology , Keratinocytes/metabolism , Trans-Activators/physiology , Transcription Factors/biosynthesis , Viral Proteins , Adenoviridae/genetics , Apoptosis , B-Lymphocytes/cytology , B-Lymphocytes/virology , Cells, Cultured , Cyclin E/biosynthesis , E2F Transcription Factors , E2F1 Transcription Factor , G1 Phase , Humans , Keratinocytes/cytology , Oligonucleotide Array Sequence Analysis , S Phase , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Telomerase/genetics , Tumor Suppressor Protein p53/analysisABSTRACT
The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.
Subject(s)
Adenoviruses, Human , DNA-Binding Proteins/genetics , Genetic Vectors , Herpesvirus 4, Human/genetics , Immediate-Early Proteins/genetics , Nasopharyngeal Neoplasms/physiopathology , Trans-Activators/genetics , Viral Proteins , Animals , Apoptosis , Cell Division , Cell Line , DNA Replication , DNA, Viral/biosynthesis , Disease Models, Animal , Female , Gene Expression , Humans , Mice , Mice, Nude , Nasopharyngeal Neoplasms/virology , Necrosis , Neoplasms, Experimental/physiopathology , Neoplasms, Experimental/virology , Stomach Neoplasms/virologyABSTRACT
The Epstein-Barr virus immediate-early protein BZLF1 is a transcriptional activator that mediates the switch from latent to lytic infection. Here we demonstrate that BZLF1 induces both a G(2) block and a mitotic block in HeLa cells and inhibits chromosome condensation. While the G(2) block is associated with decreased cyclin B1 in host cells and can be rescued by overexpression of cyclin B1, the mechanism for the mitotic defect is as yet undetermined.
