Single Molecule Studies of HIV Envelope Properties

描述(由申请人提供)：拟议的导师定量研究职业发展奖(K25)的总体目标是提供一段时间的导师教学和研究培训，使Krishanu Ray博士以他的物理背景过渡到独立的生物医学研究人员的职业生涯。雷博士的近期目标是扩大他目前的研究项目，应用先进的荧光光谱技术，在艾滋病毒感染的早期阶段询问病毒与宿主的相互作用。具体地说，Ray博士将采用以前没有描述过的基于荧光的新方法来进行病毒进入过程。这项为期四年的培训计划包括以艾滋病毒相关研究为重点的课程安排和研讨会。该项目将侧重于获得艾滋病毒生物学方面的专业知识，特别是在马里兰大学医学院(UMSM)的约瑟夫·R·拉科维奇博士和安东尼·L·德维科博士这两位杰出科学家的指导下，使用基于光学成像和光谱的方法进行艾滋病毒研究。由于Ray博士位于荧光光谱中心(CFS)，并且毗邻UMSM的人类病毒学研究所(IHV)，因此有可能研究HIV蛋白的生物物理性质和与HIV-1膜结合的中和抗体。长期目标是让雷博士获得先进的生物医学研究技能和知识，以补充他作为光谱学家的技能。他的最终目标是发展成为一名独立的、跨学科的生物医学研究人员。这项K25奖项将使Ray博士能够利用CFS和IHV现有的杰出资源，并在这一独特的多学科环境中扩大他的专业知识和经验。Ray博士将开发应用荧光相关光谱(FCS)和单分子检测(SMD)的方法，在分子水平上直接探索HIV-1被膜与细胞表面受体和/或抗被膜抗体相互作用的性质。假设在受体结合之前和期间影响HIV包膜的构象动力学的详细了解将解释观察到的对各种抑制剂的敏感性。因此，雷博士将专注于以下目标：1)开发在单分子水平上研究HIV-1 gp120-CD4相互作用的方法。2)用单分子光谱学方法研究HIV-1Env三聚体的构象动力学。3)用FCS和SMD比较不同中和效价的人单抗与HIV-1包膜三聚体的结合情况。AIMS的成功完成将在个体分子水平上提供新的定量方法，以了解病毒进入的早期步骤。这一计划将提供一个极好的环境，申请者在密切的指导下，可以深入参与将先进的荧光光谱和成像方法应用于艾滋病毒研究。随着候选人在生物医学光谱和成像研究方面走向独立，导师将更多地面向生物问题，以提供全面的投资组合。人类免疫缺陷病毒1型(HIV-1)一直是深入研究的主题。大多数抗逆转录病毒药物作用于感染后步骤，不能治愈艾滋病毒-1感染。因此，继续强调开发药物或疫苗，通过靶向能够与细胞表面辅助受体结合的HIV-1表面三聚体来阻止进入前事件。在这一应用中，将在单分子水平上确定HIV-1被膜与细胞表面受体和/或抗被膜抗体相互作用的性质。这种基于单分子荧光的方法的新应用有望提供与疫苗和药物开发相关的独特信息。

DESCRIPTION (provided by applicant): The overall objective of the proposed Mentored Quantitative Research Career Development Award (K25) is to provide a period of mentored didactic and research training that will allow Dr. Krishanu Ray with his physics background to make a career transition into an independent biomedical researcher. The immediate goal is for Dr. Ray to expand his current research program in applying advanced fluorescence spectroscopy techniques to interrogate virus-host interactions in early stages of HIV infection. Specifically, Dr. Ray will employ novel fluorescence based approaches not previously described for viral entry process. The four-year training plan involves a structured curriculum of coursework and seminars focusing on HIV related research. This program will be heavily weighted toward gaining expertise regarding the HIV biology, particularly using optical imaging and spectroscopy based approaches to HIV research under the mentorship of two exceptional scientists, Dr. Joseph R. Lakowicz and Dr. Anthony L. DeVico at the University of Maryland School of Medicine (UMSM). Because of Dr. Ray's location in the Center for Fluorescence Spectroscopy (CFS) and adjacent to the Institute of Human Virology (IHV) at the UMSM, it is possible to study the biophysical properties of HIV proteins and neutralizing antibody binding against HIV-1 envelope. The long-term goal is for Dr. Ray to acquire advanced biomedical research skills and knowledge that complement his skills as a spectroscopist. The ultimate goal is for him to develop into an independent, interdisciplinary biomedical researcher. This K25 award will enable Dr. Ray to take advantage of the outstanding resources available at CFS and IHV and expand his expertise and experience in this unique multidisciplinary setting. Dr. Ray will develop methods applying fluorescence correlation spectroscopy (FCS) and single molecule detection (SMD) to directly probe the nature of HIV-1 envelope interactions with cell surface receptors and/or anti-envelope antibodies at the molecular level. It is hypothesized that a detailed understanding of the conformational dynamics that impact the HIV envelope before and during receptor engagement will explain the observed susceptibility to various inhibitors. Accordingly, Dr. Ray will focus on the following aims: 1) To develop methods for examining HIV-1 gp120-CD4 interactions at the single molecule level. 2) To characterize HIV-1 Env trimer conformational dynamics using single molecule spectroscopy. 3) To compare the binding of human monoclonal antibodies with different neutralizing potencies to HIV-1 envelope trimers by FCS and SMD. Successful completion of the Aims will provide new quantitative approaches at the individual molecular level towards understanding early steps in viral entry. This program will provide an excellent milieu in which the applicant - under close mentorship - can become deeply involved in applying advanced fluorescence spectroscopic and imaging approaches to HIV research. Mentorship will be geared more toward biological issues to provide a well-rounded portfolio as the candidate progresses toward independence in biomedical spectroscopic and imaging research. Human-immunodeficiency virus type 1 (HIV-1) has been the subject of intensive research. The majority of antiretroviral drugs acts at a post-infection step and cannot cure HIV-1 infection. Consequently, there is a continued emphasis on developing either drugs or vaccines that block pre-entry events by targeting the HIV-1 surface trimers which enable binding to cell surface coreceptors. In this application, the nature of HIV-1 envelope interactions with cell surface receptors and/or anti-envelope antibodies will be determined at the single molecule level. This novel application of single molecule fluorescence based approaches is expected to provide unique information relevant to vaccine and drug development.