Molecular mechanism of immune evasion by the E3-19K protein of Adenovirus

描述（由申请人提供）：人类腺病毒（Ads）是一种大型DNA病毒，可引起从胃肠道和上呼吸道感染到眼睛感染的疾病。像许多大型DNA病毒一样，ad已经进化出一种特殊的机制来逃避免疫系统的检测。广告编码一种基因产物E3-19K，它结合并保留感染细胞内质网中的MHC I类分子，抑制它们向细胞表面的出口，从而抑制抗原呈递。有大量证据表明E3-19K/MHC I关联在ad建立和维持病毒持久性的能力中起作用。本提案的目标是利用分子、结构和功能方法深入了解E3-19K蛋白靶向MHC I类分子在内质网中保留并调节细胞免疫反应的机制。我们最近对重组可溶性E3-19K与MHC I类分子的相互作用进行了一些关键观察。我们发现Ad血清型2 (Ad2) E3-19K与未成熟（无肽）和成熟（充满肽）MHC I类分子相关。我们还发现E3-19K以等位基因和位点特异性的方式靶向MHC I类分子。此外，我们认为ad2e3 - 19k保守区域的残基89至93形成了一个溶剂暴露环，这对其免疫调节功能至关重要。这与我们结合HLA-A2的Ad2 E3-19K的第一个x射线晶体结构的初步数据一致。在本课题中，我们将(1)完成Ad2 E3-19K/HLA-A2配合物的高分辨率结构测定。我们将通过确定其他E3-19K/MHC I对的三维结构来询问我们的结构观察在多大程度上可以推广到不同Ad血清型和亚群的E3-19K蛋白。总之，这些结构对于识别与E3-19K的MHC I等位基因和位点特异性相关的特征具有价值。(2)通过免疫测定E3-19K与感染细胞中不同构象形式的MHC I类分子和肽负载复合物组分相关联的能力，检查E3-19K与I类抗原呈递途径之间的功能关系。我们还将尝试结晶并确定E3-19K与无肽MHC I类分子结合的三维结构，以及(3)确定E3-19K的MHC I位点特异性是否导致感染细胞上MHC I类分子的选择性下调，以及它是否对自然杀伤（NK）细胞的裂解提供保护。我们还将确定涉及MHC I类分子的跨膜和细胞质结构域的相互作用是否在E3-19K的位点特异性中起作用。这些研究有可能揭示E3-19K在抑制nk细胞介导的ad感染细胞的识别和裂解中的新功能。总之，通过剖析E3-19K如何与MHC I类分子相互作用的分子基础，我们将更好地理解E3-19K如何调节细胞免疫反应。我们的研究结果也有望有助于理解健康个体中Ad感染的建立和维持，以及免疫功能低下宿主的疾病发展。

DESCRIPTION (provided by applicant): Human Adenoviruses (Ads) are large DNA viruses that cause diseases ranging from gastrointestinal and upper respiratory tract infections to eye infections. Like many large DNA viruses, Ads have evolved a specific mechanism to escape detection by the immune system. Ads encode a gene product, E3-19K, that binds to and retains MHC class I molecules in the endoplasmic reticulum of infected cells, inhibiting their egress to the cell surface and consequently suppressing antigen presentation. There is substantial evidence suggesting that the E3-19K/MHC I association plays a role in the ability of Ads to establish and maintain viral persistence. The goal of this proposal is to use molecular, structural and functional approaches to gain in-depth knowledge of the mechanism by which the E3-19K protein targets MHC class I molecules for retention in the ER and modulates cellular immune responses. We recently made a number of key observations on recombinant, soluble E3-19K interaction with MHC class I molecules. We showed that Ad serotype 2 (Ad2) E3-19K associates with immature (peptide-free) and mature (peptide-filled) MHC class I molecules. We also showed that E3-19K targets MHC class I molecules in an allele- and locus-specific manner. Moreover, we suggested that residues 89 to 93 in the conserved region of Ad2 E3-19K form a solvent-exposed loop that is critical for its immunomodulatory function. This is consistent with our preliminary data of the first x-ray crystal structure of Ad2 E3-19K bound to HLA-A2. In this proposal, we will (1) complete the high-resolution structure determination of the Ad2 E3-19K/HLA-A2 complex. We will ask to what extent our structural observations can be generalized across E3-19K proteins of different Ad serotypes and subgroups by determining the three-dimensional structures of other E3-19K/MHC I pairs. Together, these structures will be valuable for identifying features responsible for the MHC I allele- and locus-specificity of E3-19K, (2) examine the functional relationship between E3-19K and the class I antigen presentation pathway by immunoassaying for the ability of E3-19K to associate with different conformational forms of MHC class I molecules and components of the peptide-loading complex in infected cells. We will also attempt to crystallize and determine the three-dimensional structure of E3-19K bound to a peptide-free MHC class I molecule, and (3) determine if the MHC I locus-specificity of E3- 19K leads to the selective downregulation of MHC class I molecules on infected cells and if it confers protection to lysis by natural killer (NK) cells. We will also determine whether interaction involving the transmembrane and cytosolic domain of MHC class I molecules plays a role in the locus-specificity of E3-19K. These studies have the potential to reveal a novel function for E3-19K in suppressing NK-cell mediated recognition and lysis of Ad-infected cells. Overall, in dissecting the molecular basis of how E3-19K interacts with MHC class I molecules, we will provide a better understanding of how E3-19K modulates cellular immune responses. Our findings are also expected to be relevant to understand establishment and maintenance of Ad infection in healthy individuals, and disease development in immunocompromised hosts.