Nuevas estrategias terapéuticas frente a la infección por el VIH-1

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-07-2014The present work is composed of two main goals: “Identification and functional analysis of the HIV-1 Nef PGPG motif, responsible for the interaction with the SH2 domain of Lck, as a new potential therapeutic target” and “Study of the in vitro and ex vivo effects of bryostatin-1 in combination with the antiretrovirals maraviroc or Atripla® on viral reactivation and the inhibition of novel infection issues”. SECTION 1: HIV-1 Nef affects pivotal processes in the infected host cell including T cell receptor signalling. Most of these effects involve the physical association with and alteration of subcellular localization of the host cell kinase Lck. Retargeting of Lck depends on a PxxP motif in Nef that binds to SH3 domains. Moreover, the SH2 domain of Lck was reported to contribute to interactions with Nef, however the identity of the HIV-1 Nef motif responsible for this interaction has remained unknown. To search for the elusive SH2 binding site we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified a PGPG motif encompassing residues 121-137 of HIV-1NL4.3 Nef to be required for the interaction with Lck-SH2. The PGPG motif was critical for Lck dependent Nef functions, including CD4 downregulation, optimal infectivity of viruses produced in T cells as well as intracellular recruitment of Lck. Unexpectedly, we found that the PGPG motif also participated in the binding to the Lck-SH3 domain, indicative of the synergy between PxxP-SH3 and PGPG-SH2 interaction. In accordance, the PGPG motif also participated on Nef dependent inhibition of host cell actin dynamics and MHC-I downregulation, which are PxxP dependent activities of Nef that do not involve Lck. In conclusion, the PGPG motif can mediate interactions of Nef with SH2 domains and supports the activity of the Nef PxxP motif. Thus, we propose the conserved PGPG motif in HIV-1 Nef as a potential pharmacological target for interference with Nef function in HIV-1 infection. SECTION 2: Despite antiretroviral therapy successfully controls HIV-1 viremia in most AIDS patients, virus latency establishment early upon infection impedes HIV-1 eradication in HIV-1+ patients. Bryostatin-1 (BRYO) inhibits in vitro HIV-1 infection of CD4 T lymphocytes (as it downregulates CD4 and CXCR4, viral receptor and coreceptor, respectively) and, at the same time, reactivates virus from latency through PKC/NF-kB pathway activation. Prior to design clinical studies with BRYO to assess its real impact on the size of the HIV-1 latent reservoirs, the potential in vitro effect of BRYO in combination with the antiretrovirals (AR) maraviroc (MVC) and Atripla® (ATP) has been determined. Jurkat-LTRGFP-R5 cell line and two latent and reactivatable HIV-1-infected lymphocytic or monocytic clonal cell lines (J89GFP and THP89GFP respectively) were used as latency models. BRYO reactivated latent HIV-1, reaching levels up to 80% in THP89GFP cells, even in combination with MVC or ATP. Moreover, when AR pre-treated reporter TZM-bl cells were co-cultured with BRYO treated THP89GFP, new infections of reactivated HIV-189.6 were inhibited 50% or 80% for MVC or ATP pre-treated cells, respectively. Remarkably, when AR were combined with BRYO, the combinations maintained the antiviral effect of the drugs with the maximum rate of inhibition by its own. In addition, BRYO-mediated downregulation of surface CD4 and CXCR4 in PBMC was not affected when it was used along with other AR and no hiperactivation or high proliferation effects were observed in these cells. Significantly, we found that BRYO strongly stimulated the viral promoter (LTR) transcription by activating the transcription factor NF-κB in human primary astrocytes and in the astrocytoma cell line U87, suggesting that the reactivation effect of BRYO could be especially important in a cellular reservoir such as astrocytes. Moreover, BRYO was also tested ex vivo for HIV-1 induction in CD4 T cells isolated from infected individuals receiving HAART and was found to exhibit potent induction activity. This work is the first to demonstrate that AR combination with BRYO do not interfere with BRYO activity (in terms of reactivation of latently infected cells and partial inhibition of infection) neither with AR antiviral activity. Thus, we propose BRYO as a viral latency reactivator compound appropriate to be combined with actual antiretroviral treatments in order to purge the viral reservoirs