AIDS INFRASTRUCTURE AND RESEARCH DEVELOPMENT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目标一 扩大我们目前的研究团队，招募博士后候选人，并通过全面的发展计划提高他们的职业生涯 AIM 1. 确定并招募三名合适的候选人。 2006年7月，我们制定了一份工作描述，并在一份当地杂志和全国性的《科学》杂志上刊登了招聘广告。 我们得到了几个候选人，并挑选了最好的一个（这是一个内部候选人）在我们的实验室会议上举行研讨会。 赛义德·阿里博士致力于细菌发病机理的研究，虽然不是艾滋病毒/艾滋病研究人员，但他拥有我们正在寻找的许多资格（在分子技术方面经验丰富，并有动物处理经验），包括我们以前认识他，知道他是一个勤奋的工作者。 因此，我们决定雇用他，并在2006年9月之前让他进入实验室。 我们目前正在重复这一过程，以确定和招募第二名博士后候选人。 AIM 2. 在候选人和PI之间进行协商的情况下，根据研究小组的当前需求为每位候选人制定研究项目和计划。 邦德博士和鲍威尔博士的实验室正在进行的一个项目是研究Nef诱导的囊泡分泌。 我们的假设表明Nef诱导的发病机制是由Nef分泌到血清中（分泌在外泌体样囊泡中）驱动的，然后在包括免疫系统在内的几个器官系统中诱导致病作用。 因此，了解Nef分泌将是重要的，在充分发展的理解Nef诱导的发病机制。 一名大四的研究生目前正在进行她的博士项目，该项目涉及显示Nef蛋白在分泌囊泡的膜中的物理位置，并对Nef分泌的机制进行初步分析。 第二个主题是开始Nef诱导分泌的遗传学分析，或鉴定驱动含有Nef的外来体的细胞外分泌的Nef蛋白上的表位。 在对我们研究的各个方面进行讨论后，委员会和Ali博士决定开始他关于“Nef诱导分泌的遗传学”这一主题的工作。 这将补充正在进行的分泌机制的工作。 阿里博士与一名大三的研究生一起工作，已经在这个项目上取得了重大进展。 从文献中，已显示Nef蛋白的N-末端含有参与内体转运的几个表位。 因此，我们假设胞外分泌可能需要Nef的N-末端以及可能的一些其他序列来获得胞外外泌体分泌。 利用nefGFP（绿色荧光蛋白）载体构建体的截短诱变，Ali博士和帕特里克坎贝尔开发了一系列nefGFP基因的嵌套式C末端截短，将其连接在全长GFP基因的C末端。 使用转染程序，他们将这些构建体引入HEK 293细胞中，并筛选细胞内GFP以及细胞外GFP。 含有细胞外外来体分泌所必需的所有表位的截短构建体将由于两个区室中的GFP而给予我们细胞内和细胞外荧光的表型。 那些不含有细胞外外来体分泌所必需的所有表位的构建体将由于GFP仅在细胞内而给予我们仅细胞内荧光的表型。 他们发现含有50个或更少N-末端氨基酸的构建体仅产生细胞内荧光。 含有70个或更多N-末端氨基酸的那些构建体给出细胞内和细胞外荧光。 这清楚地表明，允许分泌的关键表位存在于HIV-1 Nef氨基酸50和70之间。 在Nef蛋白的该区域内存在一个已知的表位，磷酸弗林酸性簇序列（PACS），其由一段高度酸性的氨基酸（EEEE）组成。 阿里博士和帕特里克坎贝尔制造了一个用丙氨酸残基（A）取代谷氨酸残基（E）的结构。 假设PACS表位是参与分泌的关键基序，该NefPACS_4A构建体将产生不会分泌到细胞外的NefGFP融合蛋白，仅在细胞内区室中观察到荧光。 事实上，他们观察到的结果清楚地表明，他们已经将PACS结构域确定为参与Nef诱导的外泌体分泌的一个关键表位。 目前，他们（1）筛选PACS结构域的N-末端基序，以鉴定Nef诱导的外泌体分泌中的其他关键表位。 希望能够鉴定Nef诱导的外泌体分泌所需的所有Nef序列。 此外，（2）使用共聚焦显微镜，他们正在观察从所有这些构建体表达的NefGFP蛋白的细胞内位置。 这将有助于阐明导致外泌体分泌的细胞内途径。

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 Powell's 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.