INNATE IMMUNE RECEPTOR ANTAGONISM BY ORTHOPOXVIRUS PROTEINS

描述(申请人提供)：我们已经确定了一种分泌型正痘病毒编码蛋白，称为OMCP，它结合了天然免疫效应细胞上的两个受体。第一种是NKG2D--一种激活NK和T细胞的受体。第二种尚未确定，表达于单核/巨噬细胞和B细胞。这些结合活性在人类和所有受试啮齿动物中都是保守的。 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NKG2D和巨噬细胞受体。这阻止了每个受体与其各自的配体结合，并触发针对受感染细胞的免疫功能。我们进一步假设，在适应性免疫开始之前，OMCP的作用允许这些病原体在感染组织中达到更高的滴度，特别是粘膜表面。 我们的目标是：[1]了解这一过程的生物物理细节，包括每个相关蛋白质的热力学结合参数和原子级结构；以及[2]确定为什么阻断这些受体可以增强病毒的适应性。 因此，我们将鉴定和克隆巨噬细胞OMCP受体，生产重组蛋白，并使用结合分析和X射线结晶学来表征各自的分子相互作用。我们还将产生表达空OMCP等位基因的重组病毒以及表达单一特异性OMCP的病毒，以便可以分离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构成的威胁，这些病毒偶尔会导致人类患上严重疾病。与天花不同，这些病毒不能通过接种疫苗来根除；它们的动物媒介与人类持续接触；它们可以在野外被恶意实体接触，通过工程获得免疫逃避和/或抗药性表型。令人惊讶的是，关于免疫系统如何检测这些病原体，以及它们如何反过来对抗这种检测，人们知之甚少。对OMCP的研究将为这个问题提供信息，因为它与两个分子结合，根据定义，这两个分子对这些病毒在宿主中的生命周期非常重要。这项工作还将提供对这些受体功能的免疫学洞察力，这一洞察力可能扩展到其他感染。

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.