The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle.

A spliced cDNA spanning the Epstein-Barr virus BZLF1 gene expresses the BZLF1 protein and is active in inducing the virus productive cycle. A deletion mutant which lacks the N-terminal half of the protein is inactive. Cotransfection experiments in EBV-negative B-lymphocyte cell lines demonstrated that the BZLF1 gene activates the promoter for the BSLF2 + BMLF1 gene in the absence of any other EBV gene product. These results confirmed that the spliced BZLF1 gene is the transactivating gene structure in BamHI-Z.     

The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R.

The Epstein-Barr virus (EBV) DR promoter is located upstream of the PstI repeats, and besides the TATA box, it contains two cis-acting regulatory elements. One of them has enhancer properties. To define more precisely the functional region(s) in the DR enhancer, we generated 5' and 3' deletion mutants. These deletion mutants, which were transfected into various recipient cells of different origins, allowed us to identify two functionally distinct domains, A and B. Domain A was constitutively active in all cell lines tested, except in lymphoid B cells. Domain B was active in lymphoid B cells, and its activity required both EB1 (the BZLF1-encoded EBV trans-acting factor) and the presence of the EBV genome. This suggested that an EBV-encoded, EB1-inducible factor was activating the enhancer B domain. In effect, the B domain was trans-activated by R, an EBV early product encoded by the open reading frame BRLF1, and the activation by R occurred in epithelial, fibroblastic, and lymphoid cells. The R-responsive element has been reduced to 28 base pairs containing the double palindromic sequence TTGTCCCGTGGACAATGTCC. Both domains A and B act by increasing the initiation of specific RNAs.     

The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner.

The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRF1) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type.     

The latent membrane protein 1 of Epstein-Barr virus (EBV) primes EBV latency cells for type I interferon production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.     

A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1.

Recent cDNA cloning and sequencing of two Epstein-Barr virus (EBV)-specific mRNAs from latently infected cultures revealed that these RNAs are encoded across the fused terminal repeats of the viral genome and that they are likely to encode two nearly identical proteins with the same transmembrane domains. The smaller predicted protein (LMP2B) lacks 119 amino-terminal amino acids found in the larger one (LMP2A). To test whether these proteins are expressed in latently infected lymphocytes, antibodies to the LMP2 proteins were derived by immunizing rabbits with TrpE-LMP2A fusion proteins. Affinity-purified LMP2-specific antibodies recognized 54- and 40-kilodalton proteins, corresponding to LMP2A and LMP2B, in immunoblots of rodent fibroblasts stably transfected with eucaryotic expression plasmids containing either the LMP2A or LMP2B cDNA. Similar-size proteins were also identified in immunoblots of latently infected lymphocytes. LMP2A localized to membranes in cellular fractionation studies. In immunofluorescent studies, LMP2 localized in the plasma membrane of EBV-infected lymphocytes, with the majority of reactivity confined to the region of the LMP1 patch. This reactivity was detected in almost all lymphoblastoid cells latently infected with EBV.     

End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis

End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa (“field effect”) of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.     

A region of the Epstein-Barr virus (EBV) mRNA export factor EB2 containing an arginine-rich motif mediates direct binding to RNA

The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.