Assembly of Live Nipah Virus with Complement Factors

说明(申请人提供)：补体系统是大多数动物病毒在自然感染过程中遇到的先天免疫反应的关键部分。虽然补体(C‘)显然是一些RNA病毒中和的一个重要因素，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糖蛋白的伪型激活，但重要的是，与我们迄今测试的任何其他副粘病毒不同，这不会导致中和。我们的初步数据还表明：2)细胞内的C‘抑制因子I酶与NIV F结合，而不与HN蛋白结合；3)与NIV伪型相关的因子I可以通过裂解iC3b来灭活C3b。第二个和第三个U发现的新奇之处在于，到目前为止还没有其他病原体报道招募凝血因子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蛋白在逃避天然免疫中的新功能。U在目标1中，我们将使用生化方法来验证功能性人类因子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生物安全级别4(BSL4)条件下对活的新城疫病毒感染的研究。我们将测定活的新城疫病毒在体外激活C‘的程度以及C’中和新城疫病毒感染性的能力。除了可溶性因子的募集，我们推测NIV还捕获了C‘的细胞表面抑制物，如CD46和CD55。这将通过组织培养细胞的活Niv感染进行测试，组织培养细胞表达不同水平的 抑制剂。这个试点项目试图将我们从NIV伪型研究中得到的新发现推广到BSL-4条件下的活体NIV，并为C‘逃避的新机制获得进一步的支持数据。这两个步骤都是为更机械化的活体病毒详细研究和动物模型实验奠定安全基础的必要步骤。 与公共卫生相关：人们对调节病毒和宿主先天免疫系统之间相互作用的机制非常感兴趣。补体在中和如尼帕病毒等新出现的副粘病毒中的作用以及这些病毒如何中和补体途径尚不清楚。据我们所知，我们关于尼帕病毒如何逃避中和的新发现还没有针对任何其他病原体进行描述。对补体抑制的深入了解将对病毒的发病机制和疫苗开发的合理方法产生影响。

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.