No abstract

Epstein-Barr virus (EBV), a human herpesvirus, is associated with several human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, T-cell lymphoma, gastric carcinoma, Hodgkin lymphoma, and immunoblastic lymphoma in immunocompromised individuals. The virus has the ability to immortalize primary B cells in vitro. In these immortalized lymphoblastoid cell lines, only a few viral genes are expressed, coding for six nuclear proteins (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP) and three latent membrane proteins (LMP1, LMP2A, and LMP2B) (for a review, see reference 35). Five of the nuclear antigens (EBNA1, -2, -3A, and -3C seem to be absolutely required for B-cell immortalization (10,23,32,68), whereas EBNA-LP and LMP2A seem to affect the efficiency of the process (6,23,47).EBNA2 plays a pivotal role in B-cell immortalization, since a natural occurring EBV mutant, the P3HR1 strain, carrying a deletion of the EBNA2 gene, has lost the ability to transform primary B cells. Reintroduction of the EBNA2 gene in the viral genome by homologous recombination or complementation can restore the immortalizing capacity of the virus (23). EBNA2 contributes to B-cell immortalization most likely by its ability to act as transcriptional modulator of cellular and viral gene expression. It activates the transcription of the B-cell activation markers CD21 and CD23 (9,11,70) and the tyrosine kinase c-Fgr (37) and downregulates the expression of the immunoglobulin heavy-chain locus (Ig) (30). In addition, EBNA2 transactivates the viral promoters of the three latent membrane proteins LMP1, LMP2A, and LMP2B and the Cp promoter, which regulates the transcription of the EBNA genes (17,64,75,76). EBNA2 does not bind to DNA directly (45, 77) but is recruited to EBNA2-responsive elements by interacting with the transcriptional factors 25,69,78) and PU

Varicella-zoster virus (VZV) viremia at different stages of infection was characterized. Different approaches were used, polymerase chain reaction (PCR), isothermal transcription based nucleic acid amplification (NASBA), and immunofluorescence to describe and quantitate viral infection of peripheral blood mononuclear cells (PBMC). In patients with acute varicella 200 to 5,000 copies of the viral genome in every 150,000 PBMC were found with quantitative competitive PCR (QCPCR). With NASBA, viral transcriptional activity was detected in these cells. RNA transcribed from the immediate early gene IE 63 as well as from the late gene 68 were found, indicating a productive infection. Glycoprotein gE specific immunofluorescence visualized by confocal laser scanning microscopy revealed that only 1 in 10,000 to 100,000 PBMC was infected. T and B lymphocytes as well as monocytes expressed viral protein on their surface. Similar results were obtained with PBMC from immunocompetent zoster patients. In some cases a transient viremia was found shortly after the onset of rash, although the viral load seemed to be lower than in patients with varicella. Examination of blood samples from 16 persons with postherpetic neuralgia (PHN) signs of viral replication in PBMC were not detected. In conclusion, the data suggest that VZV viremia is a frequent event in patients with varicella and zoster, but not in those with postherpetic neuralgia. Moreover, the results indicated that subclinical reactivations occur both in immunocompromised and immunocompetent individuals.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-J/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G 2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells. Epstein-Barr virus (EBV), a lymphotropic human gammaherpesvirus, is associated with several human malignancies. It infects primary resting B cells and induces unlimited proliferation of virus-infected cells in vitro (so called EBV-immortalized lymphoblastoid cell lines). In these cell lines, only a subset of viral genes that code for six EBV nuclear proteins (EBNA1, -2, -3A, -3B, -3C, and -LP) and three latent membrane protein antigens (LMP1, -2A, and -2B) are expressed. Five of these, EBNA1, -2, -3A, and -3C and LMP1, appear to be absolutely required for B-cell immortalization (33).EBNA2 and LMP1 most likely contribute to B-cell immortalization by activation of cellular pathways physiologically dependent on ligand-receptor interactions. LMP1 mimics a constitutively activated receptor of the tumor necrosis factor receptor family (11,14,47) and shares functional characteristics with CD40 (35,64,70). Both molecules induce NF-B (5, 16, 37), stress-activated protein kinases (4,11,34), and the JAK/STAT pathway (13,18,30). Through activation of these pathways, LMP1 induces B-cell activation markers and cell adhesion molecules (33) and enhances the viability of nonproliferating B cells (70).EBNA2 is essential for initiation and maintenance of B-cell immortalization (7,17,31). Acting as a transcriptional activator, it modulates the transcription of different cellular genes, including the B-cell activation markers CD21 and CD23 and the proto-oncogenes c-fgr (33) and c-myc (28). In addition, it activates transcription of the viral RNAs coding for the various EBV nuclear and membrane proteins (33). EBNA2 does not bind to DNA directly (41, 68), but is recruited to EBNA2-responsive elements by interacting with the transcription factors RBP-J (also called ...

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jκ recognition site. RBP-Jκ recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jκ, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jκ sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch–RBP-Jκ interaction. In addition to RBP-Jκ, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.