Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.     

Reactive oxygen species hydrogen peroxide mediates Kaposi's sarcoma-associated herpesvirus reactivation from latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi''s sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman''s disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H₂O₂. Mechanistically, H₂O₂ induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H₂O₂ scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.     

Productively Infected Murine Kaposi's Sarcoma-Like Tumors Define New Animal Models for Studying and Targeting KSHV Oncogenesis and Replication

Kaposi''s sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). KS tumors are composed of KSHV-infected spindle cells of vascular origin with aberrant neovascularization and erythrocyte extravasation. KSHV genes expressed during both latent and lytic replicative cycles play important roles in viral oncogenesis. Animal models able to recapitulate both viral and host biological characteristics of KS are needed to elucidate oncogenic mechanisms, for developing targeted therapies, and to trace cellular components of KS ontogeny. Herein, we describe two new murine models of Kaposi''s sarcoma. We found that murine bone marrow-derived cells, whether established in culture or isolated from fresh murine bone marrow, were infectable with rKSHV.219, formed KS-like tumors in immunocompromised mice and produced mature herpesvirus-like virions in vivo. Further, we show in vivo that the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) enhanced viral lytic reactivation. We propose that these novel models are ideal for studying both viral and host contributions to KSHV-induced oncogenesis as well as for testing virally-targeted antitumor strategies for the treatment of Kaposi''s sarcoma. Furthermore, our isolation of bone marrow-derived cell populations containing a cell type that, when infected with KSHV, renders a tumorigenic KS-like spindle cell, should facilitate systematic identification of KS progenitor cells.     

Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells

Kaposi''s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposi''s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman''s disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.     

Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells

Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.     

A Lytic Viral Long Noncoding RNA Modulates the Function of a Latent Protein

Latent Kaposi''s sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.     

Direct interactions of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed-early promoter and stimulating viral reactivation

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.     

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene

Kaposi''s sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G₁-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G₁-S transition, possibly providing an intracellular milieu favorable for its replication.