Cyclosporin A and FK506 Block Induction of the Epstein-Barr Virus Lyric Cycle by Anti-Immunoglobulin

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib.IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors.KEYWORDS B cell receptor pathway, cyclosporine, dasatinib, Epstein-Barr virus, ibrutinib, idelalisib, lytic infection, rapamycin, tacrolimus, mTOR E pstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus infection that is maintained in a pool of resting memory B cells following primary infection (1-3).The disappearance of B cells from blood as assessed by flow cytometry following treatment with a B cell-targeting monoclonal antibody commonly results in an inability to detect EBV DNA in peripheral blood mononuclear cells (PBMCs) (4). The reservoir in B cells appears to be necessary to maintain the virus as evidenced by the observation that chronic infection is not established in patients with Bruton agammaglobulinemia who lack B cells (3). B cells not only harbor the virus, but the character of the B cells that harbor virus influence viral gene regulation. In vitro, EBV infection of B cells results in immortalized lymphoblastoid cell lines expressing eight or more latency antigens, whereas in vivo, in healthy seropositive adults, the B cells that harbor viral genomes demonstrate very restricted viral gene expression. The germinal center reaction that occurs in lymphoid tissue is hypothesized to play a key role in downmodulating viral gene expression (1). In some B cell tumor lines, B cell receptor (BCR) signaling is a potent activator of EBV lytic gene expression (5). EBV gene expression and particularly

“…It has been previously reported that anti-Ig induction of the EBV lytic cycle in Akata cells can be blocked by the immunosuppressants cyclosporine and tacrolimus (11). As shown in Fig.…”

Section: Resultsmentioning

confidence: 77%

Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation

Kosowicz

Lee

Peiffer

et al. 2017

“…each of these events, though all the events have yet to be analyzed simultaneously in one experimental system. In Akata cells, EBV lytic cycle activation, triggered by crosslinking of the B-cell antigen receptor, can be blocked by cyclosporine A and FK506 which inhibit the Ca 2ϩ /calmodulindependent protein kinase pathway (27). In B95-8 cells, EBV lytic cycle activation mediated by treatment with TPA is blocked by bisindoylmaleimide, a specific inhibitor of protein kinase C (29).…”

Section: Discussionmentioning

confidence: 99%

Histone Hyperacetylation Occurs on Promoters of Lytic Cycle Regulatory Genes in Epstein-Barr Virus-Infected Cell Lines Which Are Refractory to Disruption of Latency by Histone Deacetylase Inhibitors

Countryman¹,

Gradoville

Miller

2008

108

Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.

“…Stimulation and hence activation of latently infected cells appears to be a critical component of the reactivation of gammaherpesviruses. This hypothesis has been further shown by the inhibition of the signaling cascade following stimulation with anti-Ig antibodies with the immunosuppressive drugs cyclosporine A and FK506, which prevents induction of EBV lytic replication (10). These results suggest that the full downstream signaling cascade is necessary to drive reactivation.…”

mentioning

confidence: 83%

Ex Vivo Stimulation of B Cells Latently Infected with Gammaherpesvirus 68 Triggers Reactivation from Latency

Moser

Upton

Gray

et al. 2005

Self Cite

Murine gammaherpesvirus 68 (␥HV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirelyBackground. Gammaherpesviruses are characterized by their ability to establish latency in lymphocytes. The establishment and maintenance of latency is important for persistence in the infected host, while virus reactivation is needed for transmission of the virus to new hosts and may also be required to maintain reservoirs of latently infected cells in the chronically infected host (2,12,15,33,34). The switch between latency and the lytic cycle for the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), has been extensively characterized in established latently infected cell lines in vitro (11,27,42). Initiation of the EBV lytic cycle can be stimulated by several different reagents, including anti-immunoglobulin (anti-Ig), calcium ionophore, sodium butyrate, and the phorbol ester tetradecanoyl phorbol acetate (TPA) (27). KSHV reactivation can also be induced by stimulation with phorbol esters and sodium butyrate (3,19).Murine gammaherpesvirus 68 (␥HV68) is closely related to EBV and KSHV, and infection of mice with ␥HV68 provides a tractable animal model in which to study the pathobiology of