Manuel Llano - US grants

[unreadable] DESCRIPTION (provided by applicant): Recently we have demonstrated an essential role of the lens epithelium-derived growth factor (LEDGF/p75) in the human immunodeficiency virus type 1 (HIV-1) DNA integration process. T cells lacking this chromatin-bound protein are resistant to HIV-1 infection at the viral integration step suggesting LEDGF/p75 as a target for anti-HIV drug development. Although we demonstrated that LEDGF/p75 chromatin and integrase binding domains are required, its exact role in this process is still unknown. The goal of this proposal is to understand the molecular mechanism of LEDGF/p75 HIV-1 DNA integration. This knowledge will have a direct impact in the development of drugs targeting this process. Our results will help the design of high-throughput screening methods for small-molecule HIV integration inhibitory compounds. [unreadable] [unreadable] Specific Aims [unreadable] (1) To determine if LEDGF/p75 is required for chromatin tethering of HIV pre-integration complexes (PICs). LEDGF/p75 determines chromatin localization of integrase expressed as a sole protein by plasmid transfection in non-infected cells. It has been proposed that LEDGF/p75 acts as a molecular tether between integrase and chromatin. A tethering role of LEDGF/p75 in HIV integration was also postulated because of the requirement of the same LEDGF/p75 domains for integrase localization and HIV integration. However, this model awaits direct evidence for demonstration. Using chimeric LEDGF proteins, subcellular fractionation and DNA damage signaling, we will evaluate this model in HIV infected cells. [unreadable] (2) To evaluate the interaction of LEDGF/p75 with DNA repair proteins. The interaction of LEDGF/p75 with DNA repair proteins will be investigated by GST pull-down experiments. Relevant domains in LEDGF/p75 will be mutated and its relevance in HIV infection evaluated. [unreadable] (3) Development of a high-throughput screening system for small inhibitory compounds of LEDGF/p75-integrase interaction. LEDGF/p75 protects integrase from proteasomal-mediated degradation. Integrase-eGFP fusion protein will be expressed in HeLa cells; drugs interfering with LEDGF/p75-integrase interaction are expected to reduce eGFP expression in these cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): HIV infection requires the integration of a DNA copy of the viral genome into the host DNA. Therefore therapeutic strategies to prevent HIV DNA integration are essential to block HIV infection. The role of cellular factors in this viral process is poorly defined. The Lens Epithelium-Derived Growth Factor p75 (LEDGF/p75) is a well characterized cellular cofactor of HIV DNA integration. However, it remains unexplained why Lentiviruses have evolved dependency of this cellular protein for efficient viral DNA integration. Work from my group and others have determined that LEDGF/p75 tethers the HIV integration complex to the host chromatin. However, this mechanism does not explain the strategies used by the chromatin-bound HIV pre-integration complex to gain access to the host DNA, a necessary step for integration to the host DNA, or to exploit further the cellular DNA repair machinery to promote post-integration DNA repair. New findings from my laboratory provide potential molecular explanations for these essential aspects of the HIV DNA integration process. We have demonstrated for the first time the existence of several protein motifs in LEDGF/p75 necessary for the HIV DNA integration in a chromatin tethering-independent manner. These motifs contain residues that are phosphorylated or SUMOylated, or that are predicted to be part of protein-protein interaction modules. We have postulated that these LEDGF/p75 motifs are involved in recruiting cellular proteins implicated in the HIV DNA integration process. In further support of this model, our laboratory has identified novel LEDGF/p75- interacting proteins known to be involved in chromatin remodeling, DNA repair, and transcriptional elongation. In summary, our model postulate that Lentiviruses have evolved LEDGF/p75-dependency for optimal HIV DNA integration because this cellular protein: (i) tethers the HIV pre-integration complex to subregions of the chromatin engaged in active transcription, and (ii) provides to the virus, through LEDGF/p75-specific protein- protein interactions, access to the host DNA and to DNA repair mechanisms particularly active in actively transcribe DNA. In addition, we postulate that some LEDGF/p75-interacting proteins will impair the HIV cofactor activity of this protein. In this proposal we will demonstrate essential aspects of this novel model. Specifically we will: (i) Define the role in HIV DNA integration of LEDGF/p75-interacting proteins implicated in DNA repair and chromatin remodeling. (ii) Determine the functional implications in HIV DNA integration of LEDGF/p75 motifs involved in post-translational modifications and protein-protein interactions. (iii) Provide further support to our model by determining genome-wide the correlation between LEDGF/p75 location and HIV DNA integration site selection. PUBLIC HEALTH RELEVANCE: The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS). HIV infection requires integrating a DNA copy of the viral genome into our DNA genome. Our laboratory has discovered new promising evidences on how the virus accomplishes this process. In this research proposal we will demonstrate these new mechanisms.

? DESCRIPTION (provided by applicant): Our long-term goal is to contribute to the eradication of HIV infection by finding strategies to eliminate latency. HIV-1 infection escapes eradication by establishing a latent reservoir. Consequently, identification of cellular factors implicated in the establishment or maintenance of latency, which is the focus of this application, could reveal new therapeutic targets for suppression of HIV-1 infection. We have recently discovered a novel role of PARP-1 in silencing HIV-1 gene expression in human CD4+ T cells. Our data indicated that PARP-1 knockdown (KD) CD4+ T cells are up to 90-fold more permissive to HIV-1 replication than control cells. Furthermore, re-expression of PARP-1 in the KD cells substantially diminished their susceptibility to HIV-1. In addition, a small molecule that targets the zinc finge domains of PARP-1, but not inhibitors binding to the active site of this enzyme, also increased viral replication in CD4+ T cells by 60-folds. Importantly, HIV-1 DNA integration or the production phase of the HIV-1 life cycle were not affected by PARP-1 deficiency or pharmacological interference, indicating that PARP-1 affects HIV-1 replication at a post-integration step, more likely at the level of gene expression. Notably, the effect of PARP-1 required CD4/CXCR4-mediated viral entry; i.e. PARP-1 did not affect infection by VSV-G pseudotyped viruses. Based on these results, we envision that PARP-1 negatively regulates HIV Env-induced CD4/CXCR4 signaling, limiting in this manner the availability of specific transcription factors and consequently favoring the establishment of latency. Multiple findings support our central hypothesis: (1) HIV Env-induced CD4/co-receptor signaling increases expression of cellular factors (i.e. NF-ATs, AP-1, and NF-kB) that promote viral replication. (2) PARP-1 negatively regulates the transcription of multiple genes induced by T cell activation, in part through inhibition of NF-AT- or NF-kB-dependent transcription. (3) HIV-1 gene expression is largely influenced by the availability of specific cellular transcription factors and latency is favored in transcription factor-deprived cells. We are well positioned to carry out the proposed studies because of our extensive experience in the characterization of the molecular mechanisms of cellular cofactors in HIV replication, and in particular in PARP-1. Collaborations established with experts in HIV-1 latency and T cell signaling fields will complement and very efficiently synergize with our own expertise in these fields. At the conclusion of this research we expect to have defined the role of PARP-1 in HIV latency and better identified its mechanism of action. The work proposed is important because new therapeutic targets to eradicate HIV infection could be discovered.