Targeted inhibition of calcineurin signaling blocks calcium-dependent reactivation of Kaposi sarcoma–associated herpesvirus

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants.Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506 IntroductionInfection with the ␥-herpesvirus Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is causally involved in KS, primary effusion lymphoma (PEL), and some forms of multicentric Castleman disease. [1][2][3][4][5] The detection of KSHV in peripheral blood leukocytes is strongly predictive of the future development of KS. [6][7][8][9] Circulating B cells form the major virus reservoir in blood, 10-13 though KSHV has been found in monocytes, CD8 ϩ T cells, and CD34 ϩ cells. 11,[14][15][16] Active replication in blood and subsequent plasma viremia likely disseminate the virus throughout the body, leading to infection of dermal lymphatic endothelial cells, which are believed to be precursor cells for KS. 5 Furthermore, in KS lesions, tumor spindle cells are latently infected by KSHV; a small subpopulation of lytically infected cells produces virus and lytic viral gene products, which may contribute to the maintenance or progression of KS lesions. In general, understanding the pathways that activate virus replication (latent-tolytic switch), in blood as well as in KS lesions, is an important issue in fully understanding KS pathogenesis.In experimental systems to date, KSHV infection of primary B cells in vitro has been inefficient and unstable. 13,17 However, latently infected B cell lines, derived from patients with PEL, and infected immortalized endothelial cells are useful tools to study KSHV reactivation. [18][19][20][21][22][23] Expression of the KSHV lytic cycleassociated immediate-early ORF 50 protein (Rta) is sufficient to induce the entire lytic cycle of active viral replication. 24 Ionomycin, a Ca ϩϩ -ionophore, is known to induce expression of the ORF 50 protein. 25,26 This suggests that calcium-dependent signaling pathways may play a role in virus reactivation.In the immune system, calcium signaling is essential for the expression of many inducible genes encoding cytokines and cell surface receptors. 27,28 One of the enzymes activated by a sustained rise in [Ca ϩϩ ] i is calcineurin, a ubiquitously expressed serinethreonine phosphatase. Activation of the Ca ϩϩ -calcineurin signaling cas...

Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

“…EBV LMP2A and HVS Tip are reported to alter Lyn or Lck activity, a key factor in relaying antigenic receptor activation to calcium mobilization and Ras activation. In the virus infected lymphocytes, calcium mobilization in vitro by chemicals such as ionomycin and thapsigargin is known to initiate lytic viral replication, to produce virus particle (Zoeteweij et al, 2001), necessitating the viral inhibition of the cellular signaling to calcium mobilization.…”

NFATc1 Mediates Vascular Endothelial Growth Factor-induced Proliferation of Human Pulmonary Valve Endothelial Cells

“…Retroviral Transduction-The retroviral expression vectors, retro-EGFP, and retro-EGFP-VIVIT (contains an oligonucleotide coding for MAGPHPVIVITGPHEE at the N terminus of the green fluorescent protein (GFP)), were kindly provided by Dr. A. Blauvelt at NCI, National Institutes of Health and have been previously described (32). For viral production, 293 cells at 70% confluence were transfected in Dulbecco's modified Eagle's medium with 2 g of retroviral expression vector and 2 g of the packaging vector, pCL-10A1 (33), using the Effectene transfection reagent (Qiagen) according to the manufacturer's directions for a 100-mm dish.…”

Castleman's Disease: From Basic Mechanisms to Molecular Therapeutics

“…Exploring the role of the calmodulin-calcineurinnuclear factor of activated T cells pathway in the proliferation of HHV-8 [37] may make calcineurin a potential target for the antiviral proliferation of HHV-8.…”