Rescue of the Epstein–Barr Virus BZLF1 Mutant, Z(S186A), Early Gene Activation Defect by the BRLF1 Gene Product

Expression of the Epstein-Barr virus (EBV) immediate-early protein, BZLF1 (Z), is sufficient to disrupt viral latency. Z transcriptionally activates the EBV early genes by binding to upstream Z-responsive elements (ZREs). Recently, a serine-to-alanine mutation of Z residue 186 (within the basic DNA binding domain) was shown to inhibit the ability of Z to induce lytic infection in latently infected cells, although the Z(S186A) mutant could still bind several known ZREs and activated an early EBV promoter (BMRF1) in transient reporter gene assays (Francis, A. L., Gradoville, L., and Miller, G. (1997). J. Virol. 71, 3054-3061). We now show that a specific deficiency in the ability to bind to ZRE elements in the immediate-early BRLF1 promoter may account for the inability of Z(S186A) to activate BRLF1 expression. Furthermore, we demonstrate that the ability of Z(S186A) to induce early BMRF1 and BHRF1 gene expression is rescued by cotransfection with a BRLF1 expression vector. However, the Z(S186A)/BRLF1 (R) combination cannot induce full lytic replication, suggesting that Z(S186A) may also be deficient in a replication-specific function. These results suggest that in the context of the intact viral genome, both Z and R expression are required for activation of early gene transcription in latently infected cells

Expression and function of ZEBRA: the Epstein-Barr viral replication activator

Epstein-Barr virus (EBV) encodes a protein, ZEBRA, which enables the virus to switch from a latent to a lytic life cycle. ZEBRA expression was studied in lymphoid cells harboring either standard virus or a mixture of standard and defective viruses. ZEBRA protein could not be detected in cells latently infected with standard EBV but was constitutively expressed in cells containing both defective and standard EBV genomes. Experiments indicated that regulation of transcription of the ZEBRA gene (BZLF1) is a pivotal event in the control of EBV replication. ZEBRA specific BZLF1 transcripts were spontaneously expressed in cells harboring defective virus but were only synthesized by the latent virus after chemical induction. BZLF1 transcription was sensitive to inhibition of protein synthesis but not to inhibition of lytic viral DNA synthesis. This suggests that ZEBRA is an early replicative protein whose expression is dependent on the synthesis of a viral or cellular factor.The basis domain of the ZEBRA protein is homologous to the Fos/Jun oncogene family and shares their ability to bind the canonical AP-1 site (TGAGTCA). However, ZEBRA does not contain a leucine zipper domain; a motif necessary for DNA binding of Fos/Jun proteins. Additionally, ZEBRA binds to sites which deviate from the AP-1 consensus sequence. Thus, it was of interest to study the DNA binding properties of the ZEBRA protein.Deletional mutagenesis of the BZLF1 cDNA indicated that amino acids 172-227, representing the basic and putative dimerization domains, were required for specific binding to AP-1 and divergent sites. Mutagenesis of three basic amino acids, which are conserved in Fos, abrogated ZEBRA binding to all target sequences. Additionally, ZEBRA was determined to bind DNA as a homodimer.DNA binding studies of ZEBRA and a Fos-GCN4 chimera indicated that although these proteins have homology in their basic DNA binding domains, they have different cognate binding specificities. The autoregulated BZLF1 promoter contains three divergent AP-1 sequences; Fos-GCN4 uniquely recognized one of the sites while ZEBRA bound only the other two sequences. Additionally, ZEBRA, but not Fos, was found to be phosphorylated by casein kinase II in-vitro. This phosphorylation abrogated ZEBRA binding to all of its target DNA sequences. Regulation of ZEBRA\u27s DNA binding activity may be required for the progression of the EBV replicative cycle

Sequence variation of Epstein-Barr virus: viral types, geography, codon usage and diseases

138 new Epstein-Barr virus (EBV) genome sequences have been determined. 125 of these and 116 from previous reports were combined to produce a multiple sequence alignment of 241 EBV genomes, which we have used to analyze variation within the viral genome. The type 1/type2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 SNPs at the EBNA3 locus extend into the adjacent gp350 and gp42 genes, whose products mediate infection of B cells by EBV. A small insertion within the BART miRNA region of the genome was present in 21 EBV strains. EBV from saliva of USA patients with chronic active EBV infection aligned with the wild type EBV genome, with no evidence of WZhet rearrangements. The V3 polymorphism in the Zp promoter for BZLF1 was found to be frequent in nasopharyngeal carcinoma cases both from Hong Kong and Indonesia. Codon usage was found to differ between latent and lytic cycle EBV genes and the main forms of variation of the EBNA1 protein have been identified.IMPORTANCE Epstein-Barr virus causes most cases of infectious mononucleosis and post-transplant lymphoproliferative disease. It contributes to several types of cancer including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, nasopharyngeal carcinoma and gastric carcinoma. EBV genome variation is important because some of the diseases associated with EBV have very different incidences in different populations and geographic regions - differences in the EBV genome might contribute to these diseases. Some specific EBV genome alterations that appear to be significant in EBV associated cancers are already known and current efforts to make an EBV vaccine and antiviral drugs should also take account of sequence differences in the proteins used as targets

Functional Analysis and Characterization of Epstein Barr Virus Latent Membrane Protein 2b

Epstein Barr virus persists in the human host by establishing a latent infection following primary infection. The virus periodically reactivates; producing virus that can infect new cells or be shed in saliva to infect new hosts. EBV is also implicated in malignant B cell proliferation in the immunocompromised and a variety of haemopoetic cancers, indicating that it is of public health significance.The LMP2 gene of EBV encodes 2 protein isoforms: a 497aa protein (LMP2a) and a 378aa protein (LMP2b). These isoforms are identical, with the exception of an N-terminal cytoplasmic signaling domain of 119aa encoded in the LMP2a exon 1. The remaining residues (including the entirety of LMP2b) encode an integral membrane protein consisting of 12 transmembrane spanning regions with short alternating intracellular and extracellular connection loops.Most research on the LMP2 isoforms has focused on the LMP2a protein and it's role in blocking B-cell receptor mediated signaling, degradation of associated proteins, and transformation. LMP2b, lacking the obvious signaling domain, has been largely ignored. Recently studies have suggested that LMP2b is a negative regulator of LMP2a.In the following studies, we have evaluated the contribution of LMP2b to the block in BCR signaling using LMP2b expressing BJAB cell line. Our results demonstrate that LMP2b has the ability to singularly block BCR signaling.LMP2 proteins have been described at both the plasma membrane as well as in the intracellular membranes of cells. Our studies indicate that the intact 12-TM region of the LMP2 proteins is necessary for intracellular localization. Through progressive deletions of 2TM segments from both the N- and C-terminal ends of the protein, we find that an intact 12-TM domain is necessary for localization, and there are at least 2 domains required for multimerization.The role of LMP2 in immortalization is also contested, with groups reporting that LMP2a is both necessary and dispensable for immortalization. We utilized an established system of recombinant EBV construction to demonstrate that LMP2a, but not LMP2b plays a role in establishment and maintenance of viral latency.Taken together, these results indicate a function for LMP2b in signaling and immortalization separate from LMP2a

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

High prevalence of antibodies to human herpesvirus 8 in relatives of patients with classic Kaposi's sarcoma from Sardinia

A survey for antibodies to a recombinant small viral capsid antigen (sVCA) of human herpesvirus type 8 (HHV‐8) was conducted in Sardinia, one of the world's highest incidence areas for classic Kaposi's sarcoma (KS). Prevalence of antibodies to HHV‐8 sVCA was greatest in patients with KS (95%), followed by family members (39%) and a Sardinian control population age‐ and sex‐matched to the relatives (11%). Within families, prevalence of antibodies was about equal among spouses, children, and siblings of KS patients, a finding that raises the possibilities of intrafamilial person‐to‐person or vertical transmission. Antibodies were detected 2–3 times more frequently in males than in females. The data show that prevalence of antibodies to HHV‐8 sVCA correlates with the distribution of classic KS in a high‐ incidence area. Clustering of seroprevalence within some families suggests the presence of familial risk factors for active HHV‐8 infection

To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the causative agent of three human malignancies: Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), and primary effusion lymphoma (PEL). In tumors, KSHV establishes latent infection during which it produces no infectious particles. Latently infected cells can enter the lytic replication cycle, and upon provision of appropriate cellular signals, produce progeny virus. PEL, commonly described in patients with AIDS, represents a diffuse large-cell non-Hodgkin's lymphoma, with median survival time less than six months after diagnosis. As tumor suppressor gene TP53 mutations occur rarely in PEL, the aim of this thesis was to investigate whether non-genotoxic activation of the p53 pathway can eradicate malignant PEL cells. This thesis demonstrates that Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction, efficiently restored p53 function in PEL cells, leading to cell cycle arrest and massive apoptosis. Furthermore, we found that KSHV infection activated DNA damage signaling, rendering the cells more sensitive to p53-dependent cell death. We also showed in vivo the therapeutic potential of p53 restoration that led to regression of subcutaneous and intraperitoneal PEL tumor xenografts without adversely affecting normal cells. Importantly, we demonstrated that in a small subset of intraperitoneal PEL tumors, spontaneous induction of viral reactivation dramatically impaired Nutlin-3-induced p53-mediated apoptosis. Accordingly, we found that elevated KSHV lytic transcripts correlated with PEL tumor burden in animals and that inhibition of viral reactivation in vitro restored cytotoxic activity of a small-molecule inhibitor of the p53-MDM2 interaction. Latency provides a unique opportunity for KSHV to escape host immune surveillance and to establish persistent infections. However, to maintain viral reservoirs and spread to other hosts, KSHV must be reactivated from latency and enter into the lytic growth phase. We showed that phosphorylation of nucleolar phosphoprotein nucleophosmin (NPM) by viral cyclin-CDK6 is critical for establishment and maintenance of the KSHV latency. In short, this study provides evidence that the switch between latent phase and lytic replication is a critical step that determines the outcome of viral infection and the pathogenesis of KSHV-induced malignancies. Our data may thus contribute to development of novel targeted therapies for intervention and treatment of KSHV-associated cancers.Ei saatavill

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

Regulating STING in health and disease.

The presence of cytosolic double-stranded DNA molecules can trigger multiple innate immune signalling pathways which converge on the activation of an ER-resident innate immune adaptor named "STimulator of INterferon Genes (STING)". STING has been found to mediate type I interferon response downstream of cyclic dinucleotides and a number of DNA and RNA inducing signalling pathway. In addition to its physiological function, a rapidly increasing body of literature highlights the role for STING in human disease where variants of the STING proteins, as well as dysregulated STING signalling, have been implicated in a number of inflammatory diseases. This review will summarise the recent structural and functional findings of STING, and discuss how STING research has promoted the development of novel therapeutic approaches and experimental tools to improve treatment of tumour and autoimmune diseases