Assembly of Live Nipah Virus with Complement Factors

补体系统是大多数动物病毒遇到的先天免疫反应的关键部分 在自然感染中。虽然很明显补体（C '）是中和补体的重要因素， 对于一些RNA病毒，关于C'如何调节副粘病毒的机制细节知之甚少 感染.在这里，我们试图填补理解补体（C '）与新出现的 高致病性副粘病毒尼帕病毒（NiV）。 该项目源于我们最近发表的发现：1）补体在体外被激活， 含有NiV F和G糖蛋白的假型，但重要的是，与任何其他副粘病毒不同， 到目前为止已经测试过，这不会导致中和。我们的初步数据还表明：2） 细胞C'抑制剂因子I蛋白酶与NiV F蛋白结合，但不与HN蛋白结合，和3）因子I相关 与NiV假型可以通过切割成iC 3b来切割C3 b。第二个和第三个的新奇 研究结果是，到目前为止，还没有其他病原体报道招募因子I作为逃避的机制。 补体此外，支持我们的假设将显示一个新的功能，副粘病毒F 避免先天免疫的蛋白质。 在目标1中，我们将使用生物化学方法来检验功能性人I因子 与NiV F蛋白特异性相互作用。目标2的工作将涉及在以下条件下对活NiV感染的研究： 生物安全4级（BSL 4）条件。我们将确定活NiV在体外激活C'的程度， C'中和NiV感染性的能力。除了可溶性因子I的募集，我们假设 NiV还捕获C'的细胞表面抑制剂，如CD 46和CD 55。这将使用实时测试 表达不同水平抑制剂的组织培养细胞的NiV感染。 这个试点项目旨在将我们的新发现从NiV假型研究扩展到活NiV 在BSL-4条件下，获得进一步的支持数据，为一种新的C'逃避机制。两者都是 采取必要步骤，为用活病毒进行更详细的机制研究奠定安全基础， 动物模型的实验。

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' inhibitor 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 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 protein in evading innate immunity. In Aim 1, we will use biochemical approaches to test the hypothesis that functional human Factor I interacts specifically with the NiV F protein. Work in Aim 2 will involve studies with live NiV infection under 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 I, 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.