AIDS INFRASTRUCTURE AND RESEARCH DEVELOPMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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 目标1.确定并招聘三名合适的候选人。 2006年7月，我们制定了一份工作说明，并在一家当地杂志和全国范围内的《科学》杂志上刊登了一则招聘广告。我们得到了几个候选人，并挑选了最好的一个(内部候选人)在我们的实验室会议上举行了一次研讨会。赛义德·阿里博士致力于细菌发病机制的研究，尽管他不是艾滋病毒/艾滋病研究人员，但他拥有我们寻找的许多资质(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，知道他是一名勤奋的工作人员。因此，我们决定聘用他，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进程，以确定和招募第二名博士后候选人。 目标2.从研究小组的当前需求出发，与候选人和绩效指标协商，为每个候选人制定一个研究项目和计划。 邦德博士和鲍威尔博士实验室之间正在进行的一个项目是研究Nef诱导的囊泡分泌。我们的假设认为，Nef诱导的致病机制是通过将Nef分泌到血清中(在外周体样囊泡中分泌)，然后在包括免疫系统在内的几个器官系统中诱导致病效应。因此，了解Nef的分泌对于全面了解Nef诱导的发病机制将是重要的。一名大四的研究生目前正在进行她的博士项目，该项目涉及展示Nef蛋白质实际存在于分泌小泡的膜中，并对Nef分泌机制进行初步分析。第二个主题是开始分析Nef诱导分泌的遗传学，或确定Nef蛋白上驱动含有外切体的Nef细胞外分泌的表位。在讨论了我们研究的各个方面之后，委员会和Ali博士决定开始他的工作，主题是“Nef诱导的分泌物的遗传学”。这将补充正在进行的分泌机制方面的工作。 阿里博士与一名三年级研究生一起工作，已经在这个项目上取得了重大进展。从文献中可以看出，Nef蛋白的N端含有几个与内体转运有关的表位。因此，我们假设胞外分泌可能需要Nef的N端以及可能的其他一些序列才能获得胞外外体分泌。利用截断突变构建的nefGFP(绿色荧光蛋白)载体，Ali博士和Patrick Campbell博士开发了一系列NEF基因的嵌套C末端截断，该基因连接在全长GFP基因的C末端。利用转基因程序，他们将这些构建体引入HEK293细胞，并筛选细胞内和细胞外的GFP。截短结构(S)包含胞外外体分泌所需的所有表位，由于绿色荧光蛋白在两个隔室中都有细胞内和细胞外荧光的表型。那些不包含细胞外外切小体分泌所需的所有表位的构建体(S)将给我们一个仅细胞内荧光的表型，因为只有细胞内的绿色荧光蛋白。他们发现，含有50个或更少N-末端氨基酸的构建体只发出细胞内的荧光。那些含有70个或更多N末端氨基酸的构建体既有细胞内的荧光，也有细胞外的荧光。这清楚地表明，在HIV-1Nef氨基酸50和70之间存在一个允许分泌的关键表位。 在Nef蛋白区域内有一个已知表位，即由一段高酸性氨基酸(EEEE)组成的磷酸呋喃酸性簇序列(PACS)。Ali博士和Patrick Campbell博士构建了一个结构，用丙氨酸残基(A)取代谷氨酸残基(E)。假设PACS表位是参与分泌的关键基序，该NefPACS4A构建将产生一个不会在细胞外分泌的NefGFP融合蛋白，荧光只在细胞内观察到。事实上，这是他们观察到的，清楚地表明他们已经确定PACS域是参与Nef诱导的外切体分泌的一个关键表位。 目前，他们正在(1)筛选PACS域的N末端基序，以确定在Nef诱导的外切体分泌中关键的其他表位。希望能鉴定出Nef诱导的外切体分泌所需的所有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结构表达的NefGFP蛋白的细胞内位置(S)。这应该会为细胞内导致外切体分泌的途径提供一些线索。

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.