Sequence analysis of the Epstein-Barr virus (EBV) BRLF1 gene in nasopharyngeal and gastric carcinomas

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr virus (EBV) has a biphasic infection cycle consisting of a latent and a lytic replicative phase. The product of immediate-early gene BRLF1, Rta, is able to disrupt the latency phase in epithelial cells and certain B-cell lines. The protein Rta is a frequent target of the EBV-induced cytotoxic T cell response. In spite of our good understanding of this protein, little is known for the gene polymorphism of BRLF1.</p> <p>Results</p> <p>BRLF1 gene was successfully amplified in 34 EBV-associated gastric carcinomas (EBVaGCs), 57 nasopharyngeal carcinomas (NPCs) and 28 throat washings (TWs) samples from healthy donors followed by PCR-direct sequencing. Fourteen loci were found to be affected by amino acid changes, 17 loci by silent nucleotide changes. According to the phylogenetic tree, 5 distinct subtypes of BRLF1 were identified, and 2 subtypes BR1-A and BR1-C were detected in 42.9% (51/119), 42.0% (50/119) of samples, respectively. The distribution of these 2 subtypes among 3 types of specimens was significantly different. The subtype BR1-A preferentially existed in healthy donors, while BR1-C was seen more in biopsies of NPC. A silent mutation A/G was detected in all the isolates. Among 3 functional domains, the dimerization domain of Rta showed a stably conserved sequence, while DNA binding and transactivation domains were detected to have multiple mutations. Three of 16 CTL epitopes, NAA, QKE and ERP, were affected by amino acid changes. Epitope ERP was relatively conserved; epitopes NAA and QKE harbored more mutations.</p> <p>Conclusions</p> <p>This first detailed investigation of sequence variations in BRLF1 gene has identified 5 distinct subtypes. Two subtypes BR1-A and BR1-C are the dominant genotypes of BRLF1. The subtype BR1-C is more frequent in NPCs, while BR1-A preferentially presents in healthy donors. BR1-C may be associated with the tumorigenesis of NPC.</p

Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection

RTA Promoter Demethylation and Histone Acetylation Regulation of Murine Gammaherpesvirus 68 Reactivation

Gammaherpesviruses have a common biological characteristic, latency and lytic replication. The balance between these two phases in murine gammaherpesvirus 68 (MHV-68) is controlled by the replication and transcription activator (RTA) gene. In this report, we investigated the effect of DNA demethylation and histone acetylation on MHV-68 replication. We showed that distinctive methylation patterns were associated with MHV-68 at the RTA promoter during latency or lytic replication. Treatment of MHV-68 latently-infected S11E cells with a DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (5-AzaC), only weakly reactivated MHV-68, despite resulted in demethylation of the viral RTA promoter. In contrast, treatment with a histone deacetylase (HDAC) inhibitor trichostatin A (TSA) strongly reactivated MHV-68 from latency, and this was associated with significant change in histone H3 and H4 acetylation levels at the RTA promoter. We further showed that HDAC3 was recruited to the RTA promoter and inhibited RTA transcription during viral latency. However, TSA treatment caused rapid removal of HDAC3 and also induced passive demethylation at the RTA promoter. In vivo, we found that the RTA promoter was hypomethylated during lytic infection in the lung and that methylation level increased with virus latent infection in the spleen. Collectively, our data showed that histone acetylation, but not DNA demethylation, is sufficient for effective reactivation of MHV-68 from latency in S11E cells

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells

A study on the suitability of cable models to simulate switching transients in a 132 kV underground cable

Switching transients resulting from the energisation of high voltage cable systems may have a significant effect on both the cables being switched as well as the power system components in the vicinity. The impacts of these transients on such cables are measured based on the stress arising as a result of the voltage and current peaks and the frequency of oscillatory transients. These quantities are typically obtained from a simulation by using a suitable cable model, normally with the capability to predict the transient behaviour in the range up to several 10 kHz. To obtain a cable model that enables the accurate determination of the switching transient behaviour of a cable system in service, comparison of simulated data with actual measured data is a vital process before the cable model is selected and then used for further transient analysis. Statistical studies are then carried out to simulate the stress experienced by the underground cable under study. In this paper, the authors have carried out studies on the suitability of various cable models available in PSCAD/EMTDC investigate the ability of the two available Frequency-Dependent (FD) cable models in predicting the peak, frequency and oscillatory nature of current energisation transients resulting from the switching of an unloaded 132 kV underground cable. Using the most accurate cable model evaluated from the first stage of the study, a statistical analysis of over-voltage distribution to analyse the over-voltage stress at the sending and receiving ends of the cable is also presented. This study employed two different techniques based on probabilistic and deterministic approaches to measure the distribution of over-voltages at the sending and receiving ends of the cable

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific

An Investigation of Phase Crystallinity in Laser Modified Yttria Stabilized Zirconia (YSZ) Thermal Barrier Coating

This paper presents laser surface modification process of plasma sprayed yttria stabilized zirconia (YSZ) thermal barrier coating (TBC) for enhanced hardness properties and low surface roughness. A 300W JK300HPS Nd: YAG laser was used to process YSZ TBC sample surface. The parameters selected for examination were laser power, pulse repetition frequency (PRF) and residence time. Micrographs of the TBC system were captured using EVO 15 Scanning Electron Microscope (SEM). Surface roughness was measured using 2-dimensional stylus profilometer. X-ray diffraction analysis (XRD) was conducted to measure phase crystallinity of the laser-modified coating surface. X-ray diffraction patterns were recorded in the 2θ range of 10 to 80° using Bruker D8 Advance system with 0.7Å wavelength from a copper source (~1.5Å). The laser modified surface exhibited higher crystallinity compared to the as-sprayed samples. The presence of tetragonal phase was detected in the as-sprayed and laser processed samples. The hardness properties of laser modified TBC increased 15% of the as-sprayed sample. These finding are significant to development of thermal barrier coating design optimization for enhanced surface properties of semi-solid forming die

The Zif268 cellular transcription factor activates expression of the Epstein-Barr virus immediate-early BRLF1 promoter.

The Epstein-Barr virus immediate-early protein BZLF1 mediates the switch from latent to lytic infection. BZLF1 transcription can be derived from either the BZLF1 promoter or the BRLF1 promoter (Rp). Productive viral infection of EBV-infected B cells can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, as well as cross-linking of surface immunoglobulin with antiimmunoglobulin antibody. Both TPA and antiimmunoglobulin antibody are known to activate expression of the cellular transcription factor Zif268 in B cells. In this study, we have examined the regulation of BZLF1 transcription by Zif268. We show that Rp (but not the BZLF1 promoter) is activated by Zif268. Bacterially synthesized Zif268 binds strongly to an upstream sequence in the Rp promoter (located from -131 to -123 relative to the start site) and more weakly to a proximal sequence (-49 to -40). Zif268 activation of Rp requires these two Zif268 binding sites. TPA treatment of B cells induces the expression of Zif268 protein, which binds to Rp. Furthermore, TPA activation of Rp requires the upstream Zif268 site. These findings indicate that Zif268 can activate a critical Epstein-Barr virus immediate-early promoter and, therefore, may play a key role in the regulation of viral latency