The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5-to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.Epstein-Barr virus (EBV) is a gammaherpesvirus and a preeminent tumor virus in humans. EBV is associated with a variety of cancers, including endemic Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and lymphoma in the immunosuppressed (40). Consistent with its association with human malignancy, EBV also immortalizes human B lymphocytes with high efficiency in vitro (35). Efficient immortalization of B lymphocytes requires expression of only a subset of viral genes (22). These genes include several EBV nuclear antigens (EBNAs), EBNA1, EBNA2, EBNA3A and -C, and EBNA-LP, and an integral latent membrane protein, LMP-1. EBNA-LP is the first protein along with EBNA2 made during infection of lymphocytes by EBV (1). Despite a growing body of knowledge on the molecular mechanisms of latent protein functions, the role of EBNA-LP for EBV-induced immortalization remains enigmatic.The EBNA-LP protein (also referred to as EBNA-5 or E...
Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein.Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2-and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.Epstein-Barr virus (EBV) infection is associated with several human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and lymphomas in the immunosuppressed host (53). EBV latent infection of human B lymphocytes in vitro induces expression of B-cell activation markers, proliferation, and eventual outgrowth of continuously growing lymphoblastoid cell lines (LCLs) (36). LCLs phenotypically resemble physiologically activated primary B cells (36). The ability of EBV to ...
This retrospective study examined expression of Epstein-Barr virus (EBV) latent genes in oral epithelium from human immunodeficiency virus-seropositive subjects, to identify genes associated with the pathogenesis of oral hairy leukoplakia (HLP). Transcription of EBV latent genes was detected in tis-Epstein-Barr virus (EBV) is a human herpesvirus that establishes persistent infection and is associated with many diseases, including infectious mononucleosis syndrome, malignant lymphomas, nasopharyngeal carcinoma, and oral hairy leukoplakia (HLP). HLP is an EBV-associated oral epithelial disease that is characterized by productive EBV replication. HLP resolves with treatment that inhibits EBV replication, suggesting that replication is necessary for the pathogenesis of HLP [1]. However, EBV replication also occurs in normal oral epithelial cells, suggesting that replication is insufficient for the pathogenesis of HLP and that additional cofactors are required [2]. Several EBV latent genes are expressed during EBV replication in HLP, and some have been shown to be coexpressed in the same epithelial cells in HLP [3]. Thus, expression of EBV latent genes during EBV replication in oral epithelium may be the cofactor responsible for the pathogenesis of HLP [2,3].In the present study, expression of EBV genes was examined in HLP and normal oral epithelium from HIV-seropositive subjects. The primary objective of the present study was to identify specific EBV latent genes that may be involved in the pathogenesis of HLP by use of a retrospective case-control design and both univariate and multivariate analyses of histologic and molecular data.Subjects, materials, and methods. Thirty HIV-seropositive research subjects were enrolled in a valacyclovir-treatment protocol that involved obtaining multiple oral epithelial biopsy specimens [1]. Informed consent was obtained from each research subject participating in this research. The human-experimentation guidelines of each participating institution were followed in the conduct of this research. Each biopsy specimen was 6 mm in diameter and 2 mm in depth, to include the entire surface epithelium and only as little of the epithelial submucosa as possible. One half of each biopsy specimen was formalin-fixed, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. Specimens were classified as HLP by an oral and maxillofacial pathologist only if histologic examination demonstrated hyperparakeratosis, acanthosis, and balloon cells [4], as well as molecular evidence of productive EBV replication [5]. The other half of each biopsy specimen was snap-frozen on dry ice, and nucleic acids were later extracted and purified as described elsewhere [1].Specimens were analyzed by use of reverse-transcription and nested polymerase chain reaction (PCR) amplification (80 cycles total), using primers specific for the transcripts of the human genes CD19 and CD45 and using primers specific for the transcripts of the following EBV genes: BZLF1, BHRF1 expressed from the H promoter (BHRF1-Hp), BS...
Rhesus monkeys and other nonhuman Old World primates are naturally infected with lymphocryptoviruses (LCV) that are closely related to Epstein-Barr virus (EBV). A rhesus LCV isolate (208-95) was derived from a B-cell lymphoma in a simian immunodeficiency virus-infected rhesus macaque. The EBNA-2 homologues from 208-95 and a previous rhesus LCV isolate (LCL8664) were polymorphic on immunoblotting, so the EBNA-2 genes from these two rhesus LCV were cloned, sequenced, and compared. The EBNA-2 genes have 40% nucleotide and 41% amino acid identities, and the differences are similar to those between the type 1 and type 2 EBV EBNA-2. Sequence from a portion of the LMP1 gene which is extremely divergent among different LCV was virtually identical between the 208-95 and LCL8664 strains, confirming a common rhesus LCV background. Thus, the EBNA-2 polymorphism defines the presence of two different rhesus LCV types, and both rhesus LCV types were found to be prevalent in the rhesus monkey population at the New England Regional Primate Research Center. The existence of two rhesus LCV types suggests that the selective pressure for the evolution of two LCV types is shared by human and nonhuman primate hosts.
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