Current HIV Research, 2010, 8, 000-000 1 1570-162X/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd. Bryostatin-1 Synergizes with Histone Deacetylase Inhibitors to Reactivate HIV-1 from Latency Moisés Pérez 1 , Amaya García de Vinuesa 1 , Gonzalo Sanchez-Duffhues 1 , Nieves Marquez 2 , M. Luz Bellido 2 , M. Ángeles Muñoz-Fernandez 3 , Santiago Moreno 4 , Trevor P. Castor 5 , Marco A. Calzado 1 and Eduardo Muñoz *,1 1 Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Facultad de Medicina, Avda de Menéndez Pidal s/n, 14004 Córdoba, Spain; 2 Vivacell Biotechnology España, Avda Conde de Vallellano 15, 14004, Córdoba, Spain; 3 Laboratorio de Inmuno-Biología Molecular, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain; 4 Department of Infectious Diseases, Ramon y Cajal Hospital, 28034 Madrid, Spain; 5 Aphios Corporation, 3-E Gill Street, Woburn, MA 01801, USA Abstract: The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication on patients treated with HAART. It has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harboring latent HIV-1 provirus. Using Jurkat-LAT-GFP cells, a tractable model of HIV-1 latency, we have found that bryostatin -1 reactivates HIV-1 through a classical PKC-dependent pathway. Bryostatin-1 also activates MAPKs and NF-B pathways and synergizes with HDAC inhibitors to reactivate HIV-1 from latency. Bryostatin-1 downregulates the expression of the HIV-1 co-receptors CD4 and CXCR4 and prevented de novo HIV-1 infection in susceptible cells. We applied proteomic methods to investigate major changes in protein expression in Jurkat-LAT-GFP under latency and reactivation conditions. We identified up- regulation of proteins that may be involved in the innate anti-HIV-1 response (NKEF-A and MHD2) and in different cell functions (i.e. cofilin-1 and transgelin-2) of the host cells. PKC agonists may represent a valuable pharmacological approach to purge latent HIV from cellular reservoirs and at the moment, the only clinically available PKC agonist is bryostatin-1. This drug has been tested in numerous clinical trials and its pharmacokinetics and toxicity in humans is well known. Moreover, bryostatin-1 potently synergizes with other HDAC inhibitors commonly used in the medical practice such as valproic acid. Therefore, bryostatin-1, alone or in combination with HDAC inhibitors, could be used in HAART treated patients to validate the hypothesis that reactivating HIV-1 from latency could purge HIV-1 reservoirs. Keywords: Bryostatins, HDACs, PKC, HIV-1 latency. INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS). In the absence of highly active anti-retroviral therapy (HAART), HIV-1 replication is active in most of the infected cells and in the majority of patients. However, HIV- 1 establishes long-term infection in a small pool of memory CD4 + T cells, and perhaps other cell types, which contain integrated but transcriptionally silent HIV provirus [1-3]. These latently infected cells, constitute a viral reservoir in which a replication-competent form of the virus persists with more stable kinetics than the main pool of actively replicating virus [4]. It is thought that this reservoir is responsible for the residual viremia below the detection limit in clinical assays that is a common feature in all HAART- treated patients [5, 6]. The extremely long half-life of central and transitional memory CD4 + T cells, combined with a tight control of HIV-1 expression, has been reported to make this reservoir ideally suited to maintain hidden copies of the virus [3, 7]. Although HAART is undoubtedly a life-saving *Address correspondence to this author at the Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Medicina, Avda, de Menendez Pidal s/n, 14004 Córdoba, Spain; Tel: +34 957218267; Fax: +34 957218229; E-mail: fi1muble@uco.es therapy for millions of AIDS patients, the persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication, since latently infected cells remain a permanent source of viral reactivation. As a result, a sudden rebound of the viral load after interruption of HAART is generally observed [8, 9]. For this reason, the eradication of viral reservoirs is at present a new goal for HIV-1 therapeutics [10]. Early introduction and intensification of HAART has been suggested to diminish the frequency of latently infected memory CD4 + T cells [11]. However, a recent report has shown that HAART intensification does not reduce residual viremia in a small cohort of patients [6]. Moreover, it is believed that even a few residual infected cells would be sufficient to produce systemic viremia upon HAART interruption [4], and therefore, it has been hypothesized that reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus [10, 12, 13]. It is thought that viral reactivation would result in lytic cell death of CD4 + T cells because of the cytophatic effect of the virus or through recognition of infected cells by the immune system. In addition, viral reactivation in the presence of HAART would prevent new infection events. In post-integration latency, a number of factors possibly acting simultaneously may contribute to the repression of viral latency, which can be relieved following exposure to