Single Molecule Studies of HIV Envelope Properties

拟议的指导定量研究职业发展奖（K25）的总体目标是提供一段时间的指导教学和研究培训，这将使Krishanu Ray博士与他的物理学背景，使职业过渡到一个独立的生物医学研究人员。雷博士的近期目标是扩大他目前的研究计划，应用先进的荧光光谱技术来询问艾滋病毒感染早期阶段的病毒-宿主相互作用。具体来说，Ray博士将采用以前没有描述过的基于荧光的新方法用于病毒进入过程。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和研讨会的结构化课程。该计划将在很大程度上侧重于获得有关艾滋病毒生物学的专业知识，特别是在两位杰出的科学家约瑟夫·R博士的指导下使用基于光学成像和光谱学的方法进行艾滋病毒研究。Lakowicz和Anthony L. DeVico在马里兰州医学院（UMSM）。由于Ray博士在荧光光谱中心（CFS）的位置，并且毗邻UMSM的人类病毒学研究所（IHV），因此可以研究HIV蛋白的生物物理特性和中和抗体结合HIV-1包膜。雷博士的长期目标是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。他的最终目标是成为一名独立的跨学科生物医学研究人员。这个K25奖项将使Ray博士能够利用CFS和IHV的优秀资源，并在这个独特的多学科环境中扩展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，以直接探测HIV-1包膜与细胞表面受体和/或抗包膜抗体在分子水平上相互作用的性质。据推测，详细了解的构象动力学，影响HIV包膜之前和期间的受体接合将解释所观察到的各种抑制剂的敏感性。因此，Ray博士将专注于以下目标：1）开发在单分子水平上检测HIV-1 gp 120-CD 4相互作用的方法。2)使用单分子光谱表征HIV-1 Env三聚体构象动力学。3)通过FCS和SMD比较具有不同中和效力的人单克隆抗体与HIV-1包膜三聚体的结合。目标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。该计划将提供一个很好的环境，其中申请人-在密切的指导下-可以深入参与应用先进的荧光光谱和成像方法进行艾滋病毒研究。导师制将更多地面向生物学问题，以提供一个全面的投资组合，因为候选人在生物医学光谱和成像研究方面的独立性。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在感染后阶段起作用，不能治愈HIV-1感染。因此，持续强调开发药物或疫苗，通过靶向HIV-1表面三聚体（其能够结合细胞表面辅助受体）来阻断进入前事件。在本申请中，将在单分子水平上确定HIV-1包膜与细胞表面受体和/或抗包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.