Tatsuya Tsurumi

SUMMARY: The EBV is a human gamma‐herpesvirus that is associated with a variety of neoplasms. Upon primary infection, it transiently runs a short lytic program and then predominantly establishes latent infection. Only a small percentage of infected cells switch from the latent stage into the lytic cycle and produce progeny viruses. Although EBV in cancer cells is mostly in the latent state, the lytic cycle of the virus is also expected to play a pivotal role in development and maintenance of tumors because of its association with secretion of cytokines or growth factors. Moreover, if efficient artificial induction of lytic replication could somehow be achieved, development of oncolytic therapy for EBV‐positive cancers would be conceivable. Thus, understanding the switching mechanism is of essential importance. Reactivation of the virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12‐O‐tetradecanoylphorbol‐13‐acetate, calcium ionophore, or histone deacetylase inhibitors. Transcription from the Zp is regulated by the balance between active and suppressive epigenetic histone marks, including histone acetylation, histone H3 Lysine 4 trimethylation and histone H3 lysine 27 trimethylation, being mediated by multiple transcription factors, such as myocyte enhancer factor 2, specificity protein 1, and zinc finger E‐box binding homeobox. This review will focus on such molecular mechanisms by which the EBV lytic switch is controlled and discuss the physiological significance of the switching for oncogenesis. Copyright © 2013 John Wiley & Sons, Ltd.

Reactivation of Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including those encoding MEF2B, KLF4, and some cellular b-Zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we prepared recombinant EBV-BAC, in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293 cells. The virus failed to induce a subset of early genes, such as that encoding BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could be completely lifted by exogenous supply of BRLF1, together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1, but also BRLF1 in comparison to wild-type or revertant virus cases. We also observed that MEF2 mutant EBV featured relatively high repressive histone methylation, such as H3K27me3, but CpG DNA methylation levels were comparable around Zp and the BRLF1 promoter (Rp). These findings shed light on BZLF1 expression and EBV reactivation from latency.

Productive replication of the Epstein-Barr virus (EBV) occurs in discrete sites in nuclei, called replication compartments, where viral genome DNA synthesis and transcription take place. The replication compartments include subnuclear domains, designated BMRF1 cores, which are highly enriched in the BMRF1 protein. During viral lytic replication, newly synthesized viral DNA genomes are organized around and then stored inside BMRF1 cores. Here, we examined spatial distribution of viral early and late gene mRNAs within replication compartments using confocal laser scanning microscopy and three-dimensional surface reconstruction imaging. EBV early mRNAs were mainly located outside the BMRF1 cores, while viral late mRNAs were identified inside, corresponding well with the fact that late gene transcription is dependent on viral DNA replication. From these results, we speculate that sites for viral early and late gene transcription are separated with reference to BMRF1 cores.

Epstein-Barr virus (EBV) replication proteins are transported into the nucleus to synthesize viral genomes. We here report molecular mechanisms for nuclear transport of EBV DNA polymerase. The EBV DNA polymerase catalytic subunit BALF5 was found to accumulate in the cytoplasm when expressed alone, while the EBV DNA polymerase processivity factor BMRF1 moved into the nucleus by itself. Coexpression of both proteins, however, resulted in efficient nuclear transport of BALF5. Deletion of the nuclear localization signal of BMRF1 diminished the proteins' nuclear transport, although both proteins can still interact. These results suggest that BALF5 interacts with BMRF1 to effect transport into the nucleus. Interestingly, we found that Hsp90 inhibitors or knockdown of Hsp90β with short hairpin RNA prevented the BALF5 nuclear transport, even in the presence of BMRF1, both in transfection assays and in the context of lytic replication. Immunoprecipitation analyses suggested that the molecular chaperone Hsp90 interacts with BALF5. Treatment with Hsp90 inhibitors blocked viral DNA replication almost completely during lytic infection, and knockdown of Hsp90β reduced viral genome synthesis. Collectively, we speculate that Hsp90 interacts with BALF5 in the cytoplasm to assist complex formation with BMRF1, leading to nuclear transport. Hsp90 inhibitors may be useful for therapy for EBV-associated diseases in the future.

Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ^null (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S -transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.

The Epstein-Barr virus (EBV) is an oncogenic human gamma-herpesvirus that predominantly establishes latent infection in B lymphocytes. Viral genomes exist as extrachromosomal episomes with a nucleosomal structure. Maintenance of virus latency or execution of reactivation is controlled by the expression of BZLF1, a viral immediate-early gene product, tightly controlled at the transcriptional level. In this article, we review how BZLF1 transcription is controlled, in other words how virus reactivation is regulated, especially in terms of epigenetics. We recently found that histone H3 lysine 27 trimethylation (H3K27me3) and H4K20me3 markers are crucial for suppression of BZLF1 in latent Raji cells. In addition, H3K9me2/3, HP1 and H2A ubiquitination are associated with latency, whereas positive markers, such as higher histone acetylation and H3K4me3, are concomitant with reactivation. Since lytic replication eventually causes cell cycle arrest and cell death, development of oncolytic therapy for EBV-positive cancers is conceivable using epigenetic disruptors. In addition, we note the difficulties in analyzing roles of epigenetics in EBV, including issues like cell type dependence and virus copy numbers.

SUMMARY: Because virus infections elicit various cellular responses that inhibit viral replication and growth, viruses must intervene to attenuate antiviral measures in order to thrive. The genome guardian p53 plays a central part not only in DNA damage responses, inducing cell cycle arrest or apoptosis, but also in the innate host immune control of viral infections by orchestrating diverse signaling pathways originating from many different cellular receptors and sensors. Many viruses have acquired sophisticated mechanisms to regulate p53 functions by deploying subversive proteins and modulating its post‐transcriptional status. In this review, we overview the mechanisms by which DNA and RNA viruses manage p53 signaling in favor of their continued survival. Copyright © 2012 John Wiley & Sons, Ltd.