Mechanisms of Viral Fusion and Inactivation

本项目的目的和范围细分为两个具体目标，具体如下：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我们已经开发了一种方法，该方法涉及光活化探针与膜蛋白和脂质的反应，这些探针通过各种供体发色团的能量转移原位活化。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）。我们已经使用这种方法来确定病毒包膜的哪些蛋白质在感染过程中穿透靶细胞膜，从而鉴定参与病毒融合的蛋白质和膜隔室。在融合过程中，我们监测了正粘病毒、弹状病毒、牛痘病毒和慢病毒的包膜蛋白插入靶细胞膜并重新分布。这些研究揭示了融合蛋白在融合之前、期间和之后插入病毒和靶膜的部分。我们还建立了融合反应中间阶段的动力学参数，并可能解析病毒进入途径（质膜与内体）。我们还在寻求一种光亲和交联策略，以确定参与HIV/SIV通过树突状细胞和CD 4+淋巴细胞之间的病毒突触传播机制的蛋白质。具体目标2：靶向病原体的疏水结构域用于灭活和疫苗开发我们的实验室已经开发出一种新技术，以鉴定许多不同的包膜病原体。我们的方法使用可光活化的疏水化合物，在暴露于紫外线时，形成氮宾自由基，与膜蛋白和脂质共价反应。这种共价修饰能够削弱多种膜蛋白的功能。我们一直专注于由病毒包膜糖蛋白介导的膜融合的损害，导致病毒感染的抑制。我们已经能够证明这种灭活技术对HIV、流感、埃博拉、马尔堡和VEE病毒的广泛适用性，使用化合物如碘萘叠氮（INA）。通过专门靶向噬菌体结构域，暴露的表位得以保留，使得灭活的病原体成为优异的疫苗候选物。小鼠研究表明，用INA灭活的流感病毒免疫小鼠可提供针对该疾病的异亚型保护。我们正在通过与格鲁吉亚大学的合作，扩大这一免疫战略的应用，以提供对禽流感等大流行病毒的保护。我们正在研究通过开发新型疏水交联剂来进一步改进我们的技术。使用1，5-二叠氮萘（DAN）的结果表明，交联增强了灭活病原体的结构，并赋予了一定的耐洗涤剂性，可能允许清除任何残留的感染因子，同时保持灭活病原体的完整性。这种选择性膜稳定化，然后进行去污剂处理，提供了一种正交灭活方法，这是从完整灭活病原体生产安全有效疫苗的关键。病毒样颗粒（VLP）是疫苗生产的另一种方法，并将受益于稳定技术，以延长其保质期。该技术以非常省时的方式广泛适用于任何有包膜的病原体，这在出现威胁的情况下至关重要。

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.