Takeshi Sairenji

Epstein-Barr Virus Protein Kinase BGLF4 Is a Virion Tegument Protein That Dissociates from Virions in a Phosphorylation-Dependent Process and Phosphorylates the Viral Immediate-Early Protein BZLF1

ABSTRACT Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl2 to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

Inhibition of Epstein-Barr virus (EBV) reactivation by short interfering RNAs targeting p38 mitogen-activated protein kinase or c-myc in EBV-positive epithelial cells.

ABSTRACT Latent Epstein-Barr virus (EBV) is reactivated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in EBV-infected cells. In this study, we found that TPA up-regulated phosphorylation of p38, a mitogen-activated protein kinase, and activated c-myc mRNA in EBV-positive epithelial GT38 cells. The EBV immediate-early gene BZLF1 mRNA and its product ZEBRA protein were induced following TPA treatment. Protein kinase C inhibitors, 1-(5-isoquinolinesulphonyl)-2, 5-dimethylpiperazine (H7) and staurosporine, inhibited the induction of p38 phosphorylation and the activation of c-Myc by TPA. The p38 inhibitor SB203580 blocked both p38 phosphorylation and ZEBRA expression by TPA. Pretreatment of GT38 cells with the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine inhibited p38 phosphorylation and c-Myc activation by TPA, suggesting that NO may inhibit EBV reactivation via both p38 and c-Myc. By using short interfering RNA (siRNA) targeting either p38 or c-myc, we found that p38 or c-myc siRNA specifically inhibited expression of the respective gene and also suppressed the induction of ZEBRA and EBV early antigen. The interferon (IFN)-responsive gene expression tests ruled out the possibility that the antiviral effect of siRNA is dependent on IFN. Our present study demonstrates for the first time that either p38 or c-myc siRNA can efficiently inhibit TPA-induced EBV reactivation in GT38 cells, indicating that p38- and/or c-myc-associated signaling pathways may play critical roles in the disruption of EBV latency by TPA.

Phosphorylation of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product ZEBRA is a first step in the cascade of the virus-productive cycle. ZEBRA protein was detected by immunoblotting as a single band at 38 kDa in Akata cells after crosslinkage of membrane immunoglobulin G (IgG) with anti-IgG antibody. Immunoprecipitation of [32P]phosphate-labeled, anti-IgG-stimulated Akata cells with anti-ZEBRA antibody showed that ZEBRA was phosphorylated. Phosphoamino acid analysis demonstrated phosphorylation of serine, but not threonine or tyrosine, and tryptic-peptide mapping showed multiple phosphorylated peptides of ZEBRA. Treatment with 8-bromo cAMP and blockage of phosphodiesterase by theophylline in anti-IgG-stimulated cells increased the phosphorylation of three ZEBRA peptides. Incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced the phosphorylation of these three ZEBRA peptides, while treatment with staurosporine, a protein kinase C (PKC) inhibitor, enhanced their phosphorylations. These data suggest that activation of PKC with TPA induces the ZEBRA dephosphorylation and that activation of cAMP-dependent protein kinase A enhances the ZEBRA phosphorylation at the specific sites.

Formation of intranuclear replication compartments of Epstein-Barr virus with redistribution of BZLF1 and BMRF1 gene products

The localizations of the Epstein-Barr virus immediate-early transcriptional activator BZLF1 protein ZEBRA, of the BMRF1 early antigen diffuse component (EA-D), and of viral DNA replication were studied in the Burkitts lymphoma cell line Akata treated with anti-human immunoglobulin antibodies. Prompt and sequential appearance of ZEBRA, EA-D, and viral DNA was observed in about 70% of the cells. At early times after activation, ZEBRA had a diffuse intranuclear distribution, but later it was concentrated in globular regions within the nucleus. EA-D appeared first in a finely stippled pattern and then in a diffuse pattern. At late times, EA-D concentrated in globular regions similar to those with ZEBRA. Double staining for ZEBRA and EA-D revealed that ZEBRA followed the morphological changes of EA-D with a 1-2 hr delay and that both finally coalesced in the same structures, where in situ hybridization localized replicating viral DNA. The redistribution of both ZEBRA and EA-D to these compartments depended upon the replication of lytic viral DNA. These findings indicate that these globular regions are sites for viral replication and that transcription of EBV late genes may be regulated in these structures.

Identification of hairy cell leukemia subset defining p35 as the human homologue of Ii

A molecule defining a subset of patients with hairy cell leukemia (HCL) on the basis of being abundantly labeled with [35S]methionine, was demonstrated to be the human homologue of murine Ii, a glycoprotein which lacks alloantigenic variation and is associated non-covalently with Ia antigens. In one-dimensional SDS-polyacrylamide gradient gel electrophoresis, the HCL-subset-defining molecule migrated with HLA-DR molecules which were immunoprecipitated with a specific heteroantiserum. These molecules were further defined in two-dimensional, SDS and non-equilibrium pH gradient electrophoresis of either membrane preparations or immunoprecipitates formed with various antibodies. [35S]methionine-labeling of the HCL-subset-defining molecule was greater in hairy leukemic cells than in lymphoblastoid cell lines. The subset-defining species was associated non-covalently with HLA-DR alpha and beta chains and ran electrophoretically at a position described for murine and human Ii molecules (in terms of pI and weight). Metabolic labeling of HLA-A,-B and -DR was also increased in HCL cells relative to lymphoblastoid cell lines. A separate protein, of 41,000 mol. wt and pI of 7-8, resembled another Ii-associated molecule which has been described in murine and human studies.

Isolation and Characterization of an EBV Superinfection-Enhanced, Host-Cell-Coded, 53,000-Dalton Protein

Microsomal membrane fraction proteins with enhanced synthesis after P3HR-1 Epstein-Barr virus (EBV) superinfection of Raji cells were identified with [35S]-methionine labeling and SDS-PAGE. One 53,000-dalton protein, which was found in both the microsomal membrane and cytosol fractions, was purified by ion-exchange chromatography, and specific rabbit antisera were prepared to it. This protein was found to be present in Raji cells, but its expression was enhanced after P3HR-1 EBV superinfection. It was more abundant in the cytosol than in the microsomal membrane fraction of the cell, and its synthesis was not affected by treatment of the cells with phosphonoacetic acid. It was present in several EBV-genome-negative cell lines and in activated B lymphocytes and consequently represents a host-cell-coded protein which is enhanced by EBV superinfection or by lymphocyte activation.

Roles of Accessory Molecules in Processing and Presentation of Foreign Antigens

The Ii sequence Phe146-Val164 was hypothesized to coil as an amphipathic, α helix in the desetope of class II MHC antigens until release in an acidic, foreign antigen-containing endosome to catalyze charging of the desetope with a structurally similar foreign peptide (1). A serum from one of four rabbits injected with a KLH-conjugated, synthetic peptide of Ii sequence 146–169, substituting Tyr for Phe146, immunoprecipitated a 67-kD protein from Raji cells after 3 hours [35S]methionine labeling and 69- and 67 kD-proteins after 5 or 10 hours of labeling, respectively. The 67-kD protein was not sensitive to endoglycosidase F treatment or tunicamycin and had a pI about 5.5. p67 was not surface expressed as judged by immunofluorescence analyses with the antiserum and by immunoprecipitation of surfacebiotinylated proteins. These human molecules might correspond to the murine proteins p72/74, described by Lakey et al. (2) to have a potential role in antigen presentation.

EBV Activation By Anti-IgG-Triggered, Second Messenger Pathways

The Burkitt’s lymphoma cell line, Akata, demonstrates prompt and synchronous activation of latent EBV genomes following cross-linking of its membrane immunoglobulin G (mIgG) with anti-IgG (1). In B cells, activation from crosslinkage of mlg is initiated by a mlg-triggered phospholipase C (PLC), which cleaves phosphatidylinositol (PI) to inositol-1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) (Fig. 1). IP3releases intracellular Ca++which can activate Ca++/calmodulin-dependent protein kinases, and DAG activates protein kinase C (PKC) (2). Adenylate cyclase can be activated, yielding cAMP which regulates protein kinase A (PKA). We have examined EBV activation in the Akata cells, the kinetics and synergy of these second messengers and the effect of cAMP, in the context of early antigen (EA) induction after anti-IgG crosslinking of mIgG (3).

Inhibition of Epstein-Barr Virus Release by anti-MA Antibody

We have previously reported a murine monoclonal antibody (MAb 1B6) which recognized an Epstein-Barr virus (EBV) membrane antigen (MA) and blocked release of infectious EBV from virus-producing P3HR-1 cells.