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期间遇到的先天免疫应答的关键部分。虽然很明显补体（C '）是中和某些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，我们试图填补空白的理解补体（C '）与新兴的高致病性副粘病毒尼帕病毒（NiV）的相互作用。该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的任何其他副粘病毒不同，这不会导致中和。我们的初步数据还证明：2）细胞C'抑制剂因子I蛋白酶与NiV F蛋白缔合，但不与HN蛋白缔合，和3）与NiV假型缔合的因子I可通过切割成iC 3b来阻断C3 b。第二个和第三个U发现的新奇在于，迄今为止还没有报道其他病原体招募因子I作为逃避补体的机制。此外，支持我们的假设将显示副粘病毒F U U蛋白在逃避先天免疫中的新功能。在目的1中，我们将使用生物化学方法来测试功能性人因子I U U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U U生物安全4级（BSL 4）条件下对活NiV感染的研究。我们将确定活NiV在体外激活C'的程度和C'中和NiV感染性的能力。除了可溶性因子的募集之外，我们假设NiV还捕获C'的细胞表面抑制剂，例如CD 46和CD 55。这将使用表达不同水平的NiV的组织培养细胞的活NiV感染来测试。 抑制剂的该试点项目旨在将我们的新发现从NiV假型研究扩展到BSL-4条件下的活NiV，并为C'逃避的新机制获得进一步的支持数据。这两个步骤都是必要的，以建立一个安全的基础，更详细的机制研究与活病毒和动物模型的实验。

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.