Development of strategies to find inhibitors of HIV-1 Nef cellular interaction partners

Abstract

This Ph.D. project has been focused on the study of HIV-1 Nef protein. Nef is a regulatory phosphoprotein and studies on animal models and seropositive patients have demonstrated its importance as virulence factor. Nef, in fact, plays a pivotal role in maintaining high viral load and in progression to AIDS, and is already object of interest of the HIV scientific community in the search of new targets to prevent or block HIV disease. Nef exerts three main functions in HIV-1 target cells (i.e.CD4+ T-helper lymhpocytes, monocytes/macrophages, dendritic cells, microglial cells): 1) the modulation of cellular signalling pathways; 2) the enhancement of viral infectivity; 3) the regulation of expression of cell surface receptors, including the well-studied down-regulation of CD4 and certain class I major histocompatibility complex (MCH-I) antigens. Altogether these effects increase viral infectivity and spreading contributing to render the virus able to escape the immune system response. Nef presents a two-domains structure: a structurally flexible N-terminal membrane anchor domain and a well conserved and folded C-terminal core domain. In addition it is post-translationally modified by phosphorylation and by myristoylation which is important for the Nef signalling activity. Nef exerts its functions working as a molecular adaptor, interacting and influencing the activity of more than 30 intracellular partners and several Nef interaction sites have been identified. As a consequence of Nef ability to bind multiple targets certain Nef effects dominate over others in time- or cell type- dependent manner. Macrophages are the first cells to be infected by HIV and represent the main reservoir of the virus. In these cells Nef induces the synthesis and the release of a specific sub-set of chemokines and cytokines able to recruit Tcells on the infectious site rendering them susceptible to HIV infection. Moreover, recent studies, carried out by the research group where it has been performed this Ph.D. project, have demonstrated that Nef is efficiently internalized by human non infected monocytes derived macrophages (MDMs) in in vitro cultures and mediates the NF-kB-mediated synthesis of specific cytokines and chemokines that in turn lead to the activation by phosphorylation of STAT (Signal Transducer and Activator of Transcription)-1, -2 and -3. Importantly, it has been demonstrated that the Nef interaction site designated as acidic cluster (A60QEEEE65) is required for this process. In addiction, modelling analysis and silencing experiments indicate that this region represents a putative binding motif for specific TNF receptor associated factor (TRAF) adaptor family members. It has been proposed, in fact, that in macrophages Nef intersects the CD40 signalling pathway and TRAF are involved in the signalling events downstream this receptor. Therefore, the study of the Nef interactome, of the molecular basis underlying the Nef acidic cluster/TRAFs interactions, and the finding of molecules able to block the main Nef interaction sites is certainly of great interest both to clarify the so far enigmatic biology of Nef and to open new perspectives in the field of HIV-AIDS therapy. In detail, this project has been centred on the study of Nef anchor domain and of its acidic cluster with the aim to further investigate on the role of these regions in the Nef-mediated signalling. To achieve these goals, the first part of the project has been focused on the production of a Nef target region designated as N-Term76-Nef that has been used first to carry out experiments on cellular systems, and secondly as target for phage display experiments in order to identify Nef binding peptides. Thus, an efficient protocol for the expression and the purification of the N-Term76-Nef has been setup and as result the production of a high level of purity of the target protein has been obtained. During the second part of the work N-Term76-Nef has been used for the treatment of THP-1 cells in order to evaluate the role of the Nef anchor domain in the Nef signalling effects. It has been demonstrated that NTerm76- Nef region alone is able to affect the macrophages cell signalling as the Nef full-length protein does. This finding highlights on the importance of the Nef anchor domain in the Nef functions and opens new intriguing perspectives regarding the biology of Nef. Finally, phage display of random peptide experiments have been performed to identify peptide to be used both for the characterization of the Nef interactome and for the search of potential Nef binding inhibitors. To this purpose, a 50 amino acid long phage random peptide library has been designed and produced and N-Term76-Nef has been used as target for the affinity selection cycles that has been performed through bio-panning methodology. The selection cycles have been carried out until the obtainment of a final phage sub-library enriched for the interested phage. Sequencing analysis of the selected phage genomes have been performed while modelling analysis and pull-down experiments are currently under planning and design with the final goal to isolate peptides displaying the best binding affinity for Nef. Once found, the more suitable peptides will be tested in cell culture systems to verify their potential Nef inhibitory activity.