Assembly of Live Nipah Virus with Complement Factors

描述（由申请人提供）：补体系统是大多数动物病毒在自然感染中遇到的先天免疫反应的关键部分。虽然很明显，补体（C′）是中和一些RNA病毒的重要因素，但关于C′如何调节副粘病毒感染的机制细节知之甚少。在这里，我们试图填补补体（C′）与新出现的高致病性副粘病毒尼帕病毒（NiV）相互作用的理解空白。这个项目源于我们最近发表的发现：1)补体在体外被含有NiV F和G糖蛋白的假型激活，但重要的是，与我们迄今为止测试过的任何其他副粘病毒不同，补体不会导致中和。我们的初步数据还表明：2)细胞C'抑制因子I蛋白酶与NiV F相关，而与HN蛋白无关；3)与NiV假型相关的因子I可以通过裂解iC3b使C3b失活。第二和第三个U发现的新颖之处在于，迄今为止还没有其他病原体被报道招募因子I作为逃避补体的机制。此外，对我们的假设的支持将显示副粘病毒fuu蛋白在逃避先天免疫中的新功能。在目标1中，我们将使用生化方法来检验功能性人因子I与NiV F蛋白特异性相互作用的假设。目标2的工作将涉及在欧盟生物安全等级4 （BSL4）条件下的活体NiV感染研究。我们将确定活的NiV在体外激活C′的程度以及C′中和NiV传染性的能力。除了募集可溶性因子外，我们假设NiV还捕获细胞表面的C'抑制剂，如CD46和CD55。这将使用活的NiV感染表达不同水平的组织培养细胞进行测试

DESCRIPTION (provided by applicant): The complement system is a critical part of innate immune responses that most animal viruses encounter during natural infections. While it is clear that complement (C') is an important factor in neutralization of some RNA viruses, very few mechanistic details are known about how C' regulates paramyxovirus infections. Here, we seek to fill gaps in understanding of interactions of complement (C') with the emerging highly pathogenic paramyxovirus Nipah virus (NiV). This project emerged from our recent published finding that: 1) complement is activated in vitro by pseudotypes containing the NiV F and G glycoproteins, but importantly, unlike any other paramyxovirus we have tested so far this does not result in neutralization. Our preliminary data also demonstrate that: 2) the cellular C' inhibior Factor I protease associates with the NiV F but not HN protein and 3) Factor I associated with NiV pseudotypes can inactivate C3b by cleavage into iC3b. The novelty of the second and third U findings is that no other pathogen has been reported so far to recruit Factor I as a mechanism to evade complement. In addition, support for our hypothesis would show a new function for the paramyxovirus F U U protein in evading innate immunity. U In Aim 1, we will use biochemical approaches to test the hypothesis that functional human Factor I U U interacts specifically with the NiV F protein. Work in Aim 2 will involve studies with live NiV infection under U U Biosafety Level 4 (BSL4) conditions. We will determine the extent to which live NiV activates C' in vitro and the capacity of C' to neutralize NiV infectivity. In addition to recruitment of soluble Factor , we hypothesize that NiV also captures cell surface inhibitors of C' such as CD46 and CD55. This will be tested using live NiV infection of tissue culture cells that express varying levels of inhibitors. This pilot project seeks to extend our novel findings from studies with NiV pseudotypes into live NiV under BSL-4 conditions, and to gain further supporting data for a novel mechanism of C' evasion. Both are necessary steps to establish a secure foundation for more mechanistic detailed studies with live virus and into experiments in animal models. PUBLIC HEALTH RELEVANCE: There is intense interest in mechanisms that modulate the interplay between viruses and the host innate immune system. The role of complement in neutralizing emerging paramyxoviruses such as Nipah virus and how these viruses counteract complement pathways are unknown. To our knowledge, our novel finding on how Nipah virus evades neutralization has not been described for any other pathogen. Insights into inhibition of complement will have implications for virus pathogenesis and rational approaches to vaccine development.