AIDS INFRASTRUCTURE AND RESEARCH DEVELOPMENT

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective I. Extend our current research team recruiting postdoctoral candidates, and enhancing their careers through a comprehensive developmental program AIM 1. Identify and recruit three suitable candidates. We developed a job description and published an advertisement both internally, in a local journal, and nationally in Science for the first position in July 2006. We got several candidates, and picked the best one (which was an internal candidate) to give a seminar at our Lab Meeting. Dr Syed Ali, who worked on bacterial pathogenesis, although not an HIV/AIDS researcher, had many of the qualifications (highly experienced in molecular techniques, and had animal handling experience) we were looking for including we knew him previously and knew he was a hard worker. Thus, we decided to hire him and had him in the lab by September 2006. We are currently in the process of repeating this process to identify and recruit a second postdoctoral candidate. AIM 2. Develop a research project and plan for each candidate from the current needs of the research group with consultation between the candidate and the PIs. An ongoing project between Dr Bond and Dr Powells laboratories is to study Nef-induced vesicle secretion. Our hypothesis suggests that Nef-induced pathogenesis is driven by secretion of Nef into the serum (which is secreted in exosome-like vesicles) that then induces pathogenic effects in several organ systems including the immunological system. Thus, understanding Nef secretion will be important in fully developing an understanding of Nef-induced pathogenesis. A senior graduate student is currently working on her PhD project which involves showing Nef protein is physically in the membrane of the secreted vesicles and doing the initial analysis of the mechanics of Nef secretion. A second topic is to begin an analysis of the genetics of Nef-induced secretion, or identifying the epitopes on Nef protein that drive extracellular secretion of Nef containing exosomes. After discussions on the various aspects of our research, the committee and Dr Ali decided to begin his work on the topic: "Genetics of Nef-induced Secretion". This will complement the work ongoing on the mechanics of secretion. Dr Ali, working with a junior graduate student, has made significant progress in this project already. From the literature, the N-terminus of Nef protein has been shown to contain several epitopes that are involved in endosomal transport. Thus, we made the assumption that extracellular secretion may require the N-terminus of Nef as well as possibly some other sequences to get extracellular exosome secretion. Using truncation mutagenesis of a nefGFP (green fluorescent protein) vector construct, Dr Ali and Patrick Campbell developed a series of nested C-terminal truncations of the nef gene attached at the C-terminus to the full length GFP gene. Using transfection procedures they introduced these constructs into HEK293 cells and screened for intracellular GFP as well as extracellular GFP. The truncation construct(s) that contain all the epitopes necessary for extracellular exosome secretion will give us a phenotype of both intracellular and extracellular fluorescence due to GFP in both compartments. Those construct(s) that do not contain all the epitopes necessary for extracellular exosome secretion will give us a phenotype of only intracellular fluorescence due to GFP only inside the cells. They identified that the constructs containing 50 N-terminal amino acids or less gave only intracellular fluorescence. Those constructs containing 70 N-terminal amino acids or more gave both intracellular and extracellular fluorescence. This clearly suggested that a key epitope allowing secretion exists between HIV-1 Nef amino acids 50 and 70. Within that region of the Nef protein lay one known epitope, the phosphofurin acidic cluster sequence (PACS) comprised of a stretch of highly acidic amino acids (EEEE). Dr Ali and Patrick Campbell made a construct replacing the glutamic acid residues (E) with alanine residues (A). Assuming the PACS epitope was the key motif involved in secretion, this NefPACS_4A construct would generate a NefGFP fusion protein that would not be secreted extracellularly, with fluorescence only observed in the intracellular compartment. In fact, this is what they observed clearly indicating that they have identified the PACS domain as one key epitope involved in Nef-induced exosome secretion. Currently, they are (1) screening the motifs N-terminal of the PACS domain to identify other epitopes key in Nef-induced exosome secretion. The hope is to identify ALL the sequences of Nef required for Nef-induced exosome secretion. Additionally, (2) using confocal microscopy, they are looking at the intracellular location(s) of the NefGFP protein expressed from all those constructs. This should shed some light on the intracellular pathway leading to exosome secretion.