INNATE IMMUNE RECEPTOR ANTAGONISM BY ORTHOPOXVIRUS PROTEINS

描述（由申请人提供）：我们已经鉴定了一种分泌型正痘病毒编码的蛋白，称为OMCP，其结合先天免疫效应细胞上的两种受体。第一个是NKG 2D-NK和T细胞的活化受体。第二种尚未确定，在单核细胞/巨噬细胞和B细胞上表达。这些结合活性在人类和所有测试的啮齿动物物种中是保守的。 我们假设OMCP是由正痘病毒寄生的细胞分泌的，以竞争性拮抗NKG 2D和巨噬细胞受体。这阻止了每个受体结合其各自的配体并触发针对感染细胞的免疫功能。我们进一步假设，OMCP的作用使这些病原体在感染组织中达到更高的滴度，特别是在适应性免疫开始之前的粘膜表面。 我们的目标是：[1]了解这是如何发生的生物物理细节 包括热力学结合参数和每个相关蛋白质的原子尺度结构;[2]以确定为什么阻断这些受体增强病毒适应性。 因此，我们将确定和克隆巨噬细胞OMCP受体，生产重组蛋白，并使用结合试验和X射线晶体学表征各自的分子相互作用。我们还将产生表达无效OMCP等位基因的重组病毒以及表达单特异性OMCP的病毒，以便可以单独研究每种结合活性的影响。 这项工作与公共卫生的相关性源于新出现的啮齿动物传播的正痘病毒所构成的威胁，这些病毒偶尔会导致人们患上严重的疾病。与天花不同，这些病毒不能通过疫苗接种根除;它们的动物载体与人类持续接触;并且它们在野生环境中可被恶意实体接近，用于工程化获得免疫逃避和/或耐药表型。令人惊讶的是，人们对免疫系统如何检测这些因子以及它们如何反过来对抗这种检测知之甚少。对OMCP的研究将为这个问题提供信息，因为它与两种分子结合，几乎根据定义，这两种分子对这些病毒在宿主中的生命周期很重要。这项工作也将提供这些受体的功能免疫学的见解 这可能是可扩展到其他感染的洞察力。

DESCRIPTION (provided by applicant): We have identified a secreted orthopoxvirus-encoded protein, termed OMCP, which binds two receptors on innate immune effector cells. The first is NKG2D - an activating receptor of NK and T cells. The second, as yet undefined, is expressed on monocytes/macrophages and B cells. These binding activities are conserved in humans and all tested rodent species. We hypothesize that OMCP is secreted by orthopoxvirus-parasitized cells to competitively antagonize both NKG2D and the macrophage receptor. This prevents each receptor from binding its respective ligand and triggering immune function against the infected cell. We further hypothesize that actions of OMCP allow these pathogens to reach higher titers in infected tissues, particularly mucosal surfaces, prior to the onset of adaptive immunity. Our objectives are: [1] to understand the biophysical details of how this happens including the thermodynamic binding parameters and atomic-scale structures of each involved protein; and [2] to establish why blockade of these receptors enhances viral fitness. Accordingly, we will identify and clone the macrophage OMCP receptor, produce recombinant proteins, and characterize the respective molecular interactions using binding assays and X-ray crystallography. We will also generate recombinant viruses expressing a null OMCP allele as well as viruses expressing monospecific OMCPs so that the effects of each binding activity can be studied in isolation. The relevance of this work to public health stems from the threat posed by emerging rodent-borne orthopoxviruses that occasionally cause severe disease in people. Unlike smallpox, these viruses cannot be eradicated by vaccination; their animal vectors have ongoing contact with humans; and they are accessible in the wild to malicious entities for engineered acquisition of immunoevasive and/or drug- resistant phenotypes. Surprisingly little is known about how the immune system detects these agents, and how they in turn counter that detection. Study of OMCP will inform this issue since it binds to two molecules that, almost by definition, are important to these viruses' life cycles in their hosts. This work will also provide immunologic insight into the function of these receptors insight that likely is extensible to other infections.