Dynamic of Retroviruses Assembly and Budding

描述(申请人提供)：我在英国动物卫生研究所病毒学和免疫学系三年内完成了我的博士学位，在那里我研究了非洲猪瘟病毒的细胞内传播，这是一种大型DNA病毒。我的工作促成了3篇第一作者论文的发表。我于2004年9月毕业。 2005年1月，我加入了位于美国纽约的艾伦·戴蒙德艾滋病研究中心的Paul Bieniasz小组。作为一名博士后，我正在继续探索病毒感染的细胞生物学。我目前的工作重点是人类免疫缺陷病毒I型(HIV-1)的组装和萌发。我首先确定HIV-1组装的产生部位是质膜。最近，我们与洛克菲勒大学细胞生物物理实验室负责人桑迪·西蒙合作，开发了创新的微观方法，在单个颗粒的尺度上实时研究HIV-1颗粒组装。这些方法为了解活细胞中单个病毒颗粒的起源和确定常规技术无法获得的参数提供了前所未有的视角。到目前为止，我的博士后工作共发表论文5篇，其中第一作者论文3篇。 我的直接目标是利用这些方法来了解HIV-1 Gag蛋白与颗粒组装和释放部位的关键细胞因子之间相互作用的动态。在K99阶段，我将花更多的时间在桑迪·西蒙实验室，在那里我将接受开发新的精细显微镜工具的培训。我将利用现有工具和正在开发的工具，继续我关于艾滋病毒-1组装和释放动态的工作。 一旦完成了赠款的培训阶段，我打算成立我自己的小组。作为一名独立的研究员，我将继续利用我在博士后培训期间开发的技术和工具来继续我对HIV-1感染的细胞生物学的研究。首先，在R00阶段，我将专注于HIV-1复制的一个特定方面：基因组包装的细胞生物学。在这个领域肯定需要新的方法来确定，例如，HIV-1基因组在细胞中的哪里二聚化，什么机制管理着基因组的细胞质运输到组装位置，在细胞RNA中的哪里首先与Gag结合，等等。这也是Paul Bieniasz实验室没有研究的一个研究领域，所以我作为独立研究员的工作不应该与他的工作重叠。R00阶段将允许我创建自己的小组，而不需要在第一年编写拨款的负担，并允许我立即雇用一些员工。在这段写赠款的空闲时间之后，我和我的工作人员应该已经收集了足够的初步数据来提交一份R01。 第一个R01应该是建立一个成功的研究HIV-1细胞生物学的实验室的垫脚石。我的长期目标是扩展到我感兴趣的其他鲜为人知的艾滋病毒-1生物学领域，例如进入后的事件。更具体地说，我将把我的工作重点放在部分解体的病毒颗粒的形成、细胞内运输和核输入上，这些颗粒被称为前整合复合体。HIV-1生命周期这些关键步骤的所有细胞生物学方面都是未知的；了解它们可能会揭示新的和基本的细胞途径。 我决心在令人兴奋的HIV-1细胞生物学领域追求我的职业生涯。收到竞争性的K99/R00奖助金肯定会帮助我实现这个目标。 项目简介：更好地了解HIV-1感染对于开发新类别的抗病毒药物至关重要。为了剖析HIV-1组装的机制，我们开发了一种检测方法，使我们能够实时地、在单个颗粒的尺度上可视化病毒颗粒的形成。我们建议使用这一创新的方法来研究HIV-1组装和释放过程中关键病毒和细胞成分之间的动态相互作用。

DESCRIPTION (provided by applicant): I completed my PhD in three years in the Department of Virology and Immunology of the Institute for Animal Health, England, where I studied the intracellular transport of African swine fever virus, a large DNA virus. My work led to the publication of 3 first-author papers. I graduated in September 2004. In January 2005, I joined Paul Bieniasz group at the Aaron Diamond AIDS Research Center, in New York, USA. As a post-doctoral fellow, I am continuing to explore the cell biology of virus infection. My current work focuses on the assembly and budding of Human immunodeficiency virus type I (HIV-1). I first established that the productive site of HIV-1 assembly was the plasma membrane. More recently, in collaboration with Sandy Simon, the head of the Cellular Biophysics laboratory at the Rockefeller University, we have developed innovative microscopic approaches to study HIV-1 particles assembly in real time, at the scale of individual particles. These approaches have allowed an unprecedented view of the genesis of individual virus particles in live cells and the determination of parameters that were inaccessible with conventional techniques. So far, my post-doctoral work led to the publication of 5 papers, including 3 first author papers. My immediate objectives are to exploit these approaches to understand the dynamic of the interaction between HIV-1 Gag proteins and key cellular factors at the site of particle assembly and release. During the K99 phase, I will spend more time in Sandy Simon laboratory, where I will be trained to develop new elaborate microscopic tools. I will pursue my work on the dynamic of HIV-1 assembly and release, using existing tools, as well as tools under development. Once the training phase of the grant will be completed, I intend to start my own group. As an independent investigator, I will continue exploiting the techniques and tools that I have developed during my post-doctoral training to pursue my studies on the cell biology of HIV-1 infection. Initially, during the R00 phase, I will focus on a particular aspect of HIV-1 replication: the cell biology of genome packaging. New approaches are definitively warranted in this field to determine, for instance, where in the cell the HIV-1 genome dimerizes, what mechanisms govern the cytoplasmic trafficking of the genome to assembly sites, where in the cell RNA first binds Gag, etc. This is also an area of research that is not studied in Paul Bieniasz laboratory, so my work as an independent investigator should not overlap with his. The R00 phase will allow me to start my own group without the burden of writing a grant in the first year and allow me to hire some staff immediately. After this grant-writing free time, my staff and I should have collected enough preliminary data to submit an R01. This first R01 should be a stepping-stone toward establishing a successful laboratory studying the cell biology of HIV-1. My long-term goal is to expand into other poorly understood areas of HIV-1 biology that interest me, such as on post-entry events. More specifically, I will focus my work on the formation, intracellular transport and nuclear import of partially disassembled viral particles, named pre- integration complexes. All cell biological aspects of these crucial steps of the HIV-1 life cycle are unknown; understanding them is likely to reveal new and fundamental cellular pathways. I am determined and highly motivated to pursue my career in the exciting field of HIV-1 cell biology. Receiving the competitive K99/R00 grant will certainly help me reach this goal. Project narrative: A better understanding of HIV-1 infection is crucial for the development of new classes of antiviral drugs. In order to dissect the mechanisms of HIV-1 assembly, we have developed an assay that allows us to visualize the formation of viral particles in real time and at the scale of single particles. We propose to use this innovative assay to study the dynamic interactions between key viral and cellular components during HIV-1 assembly and release.