Mechanisms of Viral Fusion and Inactivation

该项目的目的和范围分为两个具体目标， 具体目标1：开发定点光敏 标记/交联作为研究膜蛋白相互作用的工具 涉及光活化探针与膜蛋白和脂质反应的方法 在通过来自各种供体的能量转移原位激活这些探针之后 发色团在目前的研究中，我们使用了膜双层特异性探针 碘萘叠氮（INA）。我们已经用这种方法来确定病毒的哪些蛋白质 包膜在感染过程中穿透靶细胞膜，从而识别 参与病毒融合的蛋白质和膜区室。我们监测了 正粘病毒、杆状病毒、牛痘病毒和 慢病毒在融合过程中进入靶细胞膜。这些研究揭示了 融合蛋白的一部分插入到病毒和靶细胞膜之前， 在融合期间和之后。并建立了中间阶段的动力学参数。 融合反应，并可能解决病毒进入的途径（质膜与 内体）。我们还在寻求一种光亲和交联策略来识别蛋白质 参与HIV/SIV通过树突状细胞之间的病毒突触传播的机制 细胞和CD 4+淋巴细胞特异性目标2：靶向病原体的疏水结构域， 灭活和疫苗开发我们的实验室开发了一种新技术， 有许多不同的包膜病原体。我们的方法使用光活化 疏水性化合物，其在暴露于UV光时形成氮烯自由基， 与膜蛋白和脂质共价结合。这种共价修饰具有以下能力： 损害多种膜蛋白的功能。我们关注的是 由病毒包膜糖蛋白介导的膜融合导致病毒的抑制 感染我们已经能够证明这种灭活技术的广泛适用性， HIV、流感病毒、埃博拉病毒、马尔堡病毒和VEE病毒，使用化合物如碘萘叠氮 （INA）.通过排他地靶向胞内结构域，暴露的表位得以保留，使得表位的表位可以被保留。 灭活病原体的优秀疫苗候选者。小鼠研究表明， 用INA灭活的流感病毒的小鼠的抗流感病毒抗体具有异亚型保护作用， 这种疾病我们正在扩大这一免疫战略的应用范围， 透过与香港卫生署合作， 格鲁吉亚大学。我们正在研究进一步改进我们的技术， 新型疏水交联剂的开发。使用1，5-二叠氮萘的结果 (DAN)表明交联增强了灭活病原体的结构， 一些洗涤剂抗性，可能允许清除任何残留的感染性 同时保持失活剂的完整性。这种选择性膜 稳定化后进行去污剂处理提供了正交灭活的方法， 这是从整个灭活病原体生产安全有效疫苗的关键。病毒 类颗粒（VLP）是疫苗生产的另一种方法， 稳定化技术，以延长其保质期。该技术广泛应用于 任何包膜病原体在一个非常有效的方式，这是至关重要的情况下， 新的威胁。

The purpose and scope of this project is subdivided into two specific aims that are detailed below: Specific Aim 1: Development of Site-Directed Photosensitized Labeling/crosslinking as a Tool to Study Membrane Protein Interactions We have developed a methodology that involves reaction of photo-activable probes with membrane proteins and lipids following activation of these probes in situ by energy transfer from a variety of donor chromophores. In the current studies we have used the membrane bilayer specific probe iodonaphthylazide (INA). We have used this method to establish which proteins of the viral envelope penetrate the target cell membrane in the course of infection and thus identify proteins and membrane compartments that participate in viral fusion. We monitored the insertion and redistribution of viral envelope proteins of orthomyxo, rhabdo, vaccinia and lentiviruses into the target cell membrane in the course of fusion. These studies shed light on portions of fusogenic proteins that insert into the viral and target membranes before, during and after fusion. We also established kinetic parameters of the intermediate stages in the fusion reaction and potentially resolve the route of viral entry (plasma membrane versus endosomal). We are also pursuing a photo affinity cross linking strategy to identify proteins involved in the mechanisms of HIV/SIV transmission through the viral synapse between dendritic cells and CD4+ lymphocytes Specific Aim 2: Targeting the Hydrophobic Domain of Pathogens for Inactivation and Vaccine Development Our laboratory has developed a novel technology to inactivate a number of diverse enveloped pathogens. Our method uses photoactivatable hydrophobic compounds that, upon exposure to UV light, form nitrene radicals which react covalently with membrane proteins and lipids. This covalent modification has the ability to impair the function of multiple membrane proteins. We have focused on the impairment of membrane fusion mediated by viral envelope glycoproteins leading to inhibition of viral infection. We have been able to show the wide applicability of this inactivation technique to HIV, Influenza, Ebola, Marburg and VEE viruses, using compounds such as Iodonaphthyl azide (INA). By exclusively targeting the lipidic domain, exposed epitopes are preserved making the inactivated pathogens excellent vaccine candidates. Mouse studies have shown that immunization of mice with influenza virus inactivated with INA conferred heterosubtypic protection against the disease. We are extending the application of this immunization strategy to provide protection against pandemic viruses like avian influenza through our collaboration with the University of Georgia. We are investigating further improvements of our technology through the development of novel hydrophobic crosslinking agents. Results using 1,5-diazidonaphthalene (DAN) show that crosslinking reinforces the structure of the inactivated pathogens and confers some detergent resistance, potentially allowing for the purging of any residual infectious agents while preserving the integrity of the inactivated ones. This selective membrane stabilization followed by detergent treatment provides a method for orthogonal inactivation, which is key in producing safe, effective vaccines from whole inactivated pathogens. Viruses like particles (VLPs) are another approach to vaccine production, and would benefit from stabilization techniques to prolong their shelf-life. This technology is widely applicable to any enveloped pathogen in a very time efficient manner, which is critical in the case of emerging threats.