Mechanisms of Viral Fusion and Inactivation

该项目的目的和范围可分为以下两个具体目标：具体目标1：开发位点导向光敏标记/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，该方法涉及光激活探针与膜蛋白和脂质的反应，这些探针通过来自各种供体发色团的能量转移在原位激活。在目前的研究中，我们使用了膜双分子层特异性探针碘萘酰肼（INA）。我们利用这种方法确定了病毒包膜的哪些蛋白在感染过程中穿透靶细胞膜，从而确定了参与病毒融合的蛋白和膜区室。在融合过程中，我们监测了正乳突病毒、横纹肌病毒、牛痘病毒和慢病毒包膜蛋白在靶细胞膜上的插入和重分布。这些研究揭示了融合蛋白在融合前、融合中和融合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，并可能解决病毒进入途径（质膜与内体）。我们还在寻求一种光亲和交联策略，以确定通过树突状细胞和CD4+淋巴细胞之间的病毒突触参与HIV/SIV传播机制的蛋白质。特异性目标2：靶向病原体的疏水结构域进行灭活和疫苗开发。我们的实验室已经开发出一种新技术来灭活许多不同的包膜病原体。我们的方法使用光活化的疏水化合物，暴露在紫外线下，形成与膜蛋白和脂质共价反应的亚硝基。这种共价修饰能够破坏多种膜蛋白的功能。我们重点研究了病毒包膜糖蛋白介导的膜融合损伤导致病毒感染的抑制。我们已经能够证明这种灭活技术广泛适用于艾滋病毒、流感病毒、埃博拉病毒、马尔堡病毒和VEE病毒，使用的化合物如碘多萘酰叠氮（INA）。通过专门靶向脂质结构域，暴露的抗原表位得以保存，使灭活的病原体成为极好的候选疫苗。小鼠研究表明，用INA灭活流感病毒的小鼠免疫可获得异亚型的疾病保护。我们正在扩大这一免疫战略的应用范围，通过我们与佐治亚大学的合作，为防范禽流感等大流行性病毒提供保护。我们正在研究通过开发新型疏水交联剂进一步改进我们的技术。使用1,5-二叠氮多萘（DAN）的结果表明，交联增强了灭活病原体的结构，并赋予了一定的洗涤剂抗性，潜在地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。这种选择性膜稳定和洗涤剂处理提供了一种正交灭活方法，这是用整个灭活病原体生产安全有效疫苗的关键。病毒颗粒（vlp）是生产疫苗的另一种方法，将受益于稳定技术以延长其保质期。该技术广泛适用于任何包膜病原体，而且非常省时，这对于新出现的威胁至关重要。

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.