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。 在自然感染期间。虽然补体(C‘)显然是中和的一个重要因素 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，我们试图填补补语(C‘)与新兴动词相互作用的理解空白 高致病性副粘病毒尼帕病毒(Niv)。 这个项目源于我们最近发表的发现：1)补体在体外被激活，通过 含有新城疫病毒F和G糖蛋白的伪型，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了，这不会导致中和。我们的初步数据还表明：2) 细胞C‘抑制因子I与NIV F相关，而与HN蛋白无关；3)因子I相关 对于NIV，伪型可以通过裂解成ICC3b来灭活C3b。第二部和第三部的新颖性 结果是，到目前为止，还没有其他病原体报告招募凝血因子I作为一种逃避机制 互补性。此外，支持我们的假设将显示副粘病毒F的新功能 逃避先天免疫的蛋白质。 在目标1中，我们将使用生化方法来检验以下假设：功能性人类因子I 与Niv F蛋白特异地相互作用。AIM 2的工作将涉及在以下情况下对活的新城疫病毒感染的研究 生物安全级别4(BSL4)条件。我们将确定活的Niv在体外激活C‘的程度和 C‘中和新城疫病毒感染性的能力。除了可溶性因子I的招募外，我们还假设 NIV还捕获C‘的细胞表面抑制物，如CD46和CD55。这将使用LIVE进行测试 新城疫病毒感染组织培养细胞，表达不同程度的抑制物。 这个试点项目试图将我们的新发现从对Niv假型的研究扩展到活的Niv 在BSL-4条件下，获得了C‘逃避新机制的进一步支持数据。两者都是 为更机械化的活病毒详细研究奠定安全基础的必要步骤 在动物模型上进行实验。

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.