Structure and Function of Viral Immunoevasins

病毒免疫球蛋白的结构和功能

基本信息

批准号：
7732678
负责人：
David Margulies
金额：
$ 46.26万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7732678
关键词：
Accession Number Acute Alanine Antibodies Bacteria Baculoviruses Basic Science Binding Biochemical Biological Biological Process Calorimetry Cell Line Cell physiology Cell surface Cells Characteristics Chronic Coculture Techniques Collaborations Communicable Diseases Complex Cytomegalovirus DNA Viruses Data Collection Databases Dendritic Cells Deposition Dimerization Drosophila genus Electrophoretic Mobility Shift Assay Employee Strikes Engineering Escherichia coli Extracellular Domain Family Family member Fibroblasts Fractionation Gene Family Genes Glycoproteins Goals Green Fluorescent Proteins Head Herpesviridae Human Hybridomas Hydrogen Bonding ITGAX gene Immune response Immune system Immunoprecipitation In Vitro Inclusion Bodies Infection Insecta Label Lead Ligands Light Major Histocompatibility Complex Manuscripts Methodology Modeling Molecular Molecular Sieve Chromatography Monoclonal Antibodies Mouse Strains Murid herpesvirus 1 Mus Mutagenesis Mutation Natural Killer Cells Peptides Population Production Property Proteins Publishing Recombinants Reporter Resolution Rodent Role Screening procedure Simplexvirus Solutions Spleen Splenocyte Stress Structure T-Cell Receptor T-Lymphocyte Tail Time Titrations Transfection Universities Viral Viral Physiology Virus Virus Diseases Work X ray diffraction analysis X-Ray Diffraction analytical ultracentrifugation base beta-2 Microglobulin cell type crosslink dimer disulfide bond insight interest killer T cell monomer mutant pathogen receptor research study sedimentation equilibrium sedimentation velocity tool

项目摘要

The focus of this work has been to understand the molecular details that control initial steps in the recognition of cells infected with pathogens such as viruses by cells of the innate and adaptive immune systems. In particular, we study the large family of major histocompatibility complex (MHC)-encoded molecules from a biophysical and structural perspective. Thus, we are interested in how MHC-I molecules interact with receptors on natural killer (NK) cells or on T lymphocytes through their NK and T cell receptors, respectively. These studies are dependent upon structural, functional, and biophysical analysis of the interaction of the molecules in question, and attempts are made to correlate binding properties with function and structure. Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy, and we have directed our effort to understand the function, cellular expression, and structure of a set of these MHC-I molecules encoded by the mouse cytomegalovirus (mCMV). We have analyzed the expression of several of these genes by transfection in different cell types, and have established that, unlike the classical MHC-I molecules, the viral MHC-I molecules (referred to as MHC-Iv) do not require either beta-2 microglobulin or self-peptide for expression. In addition, MHC-Iv molecules m152 and m155 are not expressed at the cell surface under any circumstances. By engineering the m144 molecule for expression in E. coli, we produced X-ray diffraction quality crystals of m144 in complex with its beta-2 microglobulin light chain, and have completed data collection, molecular replacement solution, and refinement of the m144 structure at 1.9 Angstrom resolution. This study reveals, for the first time, that one of the mCMV MHC-Iv molecules indeed preserves the fundamental molecular fold characteristic of classical MHC-I molecules. The structure reveals a molecule that lacks bound peptide, and has unique structural features that contribute to thermal stability. Mutagenesis experiments confirm the importance of a unique disulfide bond of this molecule. In addition, we have examined the cellular expression of additional cytomegalovirus MHC-Iv immunoevasins, M37, m151, and m153. Each of these has unique cell biological features. In collaboration with Stipan Jonjic, University of Rijeka, we have generated monoclonal antibodies to m153, and have recently demonstrated that m153 forms stable non-covalent homodimers. Diffraction quality crystals of selenomethionyl m153, expressed in Drosophila S2 cells, have been obtained. Using single anomalous dispersion (SAD) methodology, we have solved the structure of m153, and refined the structure to 2.4 Angstrom resolution. This crystallographic model, coordinates and structure factors, have been deposited in the Protein Data Bank under accession number 2O5N, and a manuscript describing the structure in detail has been published. The most striking feature of this completely new MHC-like structure is that the molecule forms a stable head to tail homodimer. To confirm the dimerization interface observed in the crystal structure of m153 we have introduced alanine mutations at four residues involved in hydrogen bonds at the interface. The mutant m153 and wild type m153 was expressed transiently in baculovirus insect cells and purified. Analysis of the wild type and mutant m153 proteins by size exclusion chromatography and analytical ultracentrifugation experiments indicated that the mutations destabilized the dimer and that the mutant protein existed as a monomer. The biological function of m153 is unknown. To gain insight into its role during viral infection we have developed several strategies to identify the putative ligand of m153. Initial immunoprecipitation experiments using metabolically labelled mouse fibroblasts, infected with MCMV or transfected with m153, failed to identify any binding partners in the mouse fibroblasts. In a second approach, we constructed an m153 reporter cell as a tool to screen cell lines and primary cells for ligands. The indicator cell line 43.1 is a T cell hybridoma, derived from 2B4 cells, in which an NFAT-driven GFP construct was stably introduced. Upon triggering of the TCR by antibody cross-linking these cells produce GFP. To generate an m153-specific GFP reporter cell line a fusion construct consisting of the extracellular domain of m153 and the transmembrane and intracellular portions of the human zeta protein was generated. The m153-Zeta construct was introduced in the 43.1 cell line by retroviral infection. The resulting m153-Zeta/43.1 cells produce GFP after overnight culture on plates coated with the anti-m153 monoclonal antibody m153.16 and thus provide a valuable ligand screening tool. Ten murine cell lines from various origins were screened with the m153-Zeta reporter cells, but none stimulated the production of GFP. Recently we have found that freshly isolated spleen cells from several different mouse strains stimulate the m153-Zeta reporter cells to produce GFP, whereas the parental 43.1 cells and a control cell line, which expresses another viral glycoprotein fused to human zeta, were unaffected by coculture with the splenocytes. Further fractionation of the spleen cell populations indicate that CD11c+ dendritic cells are the most potent in activating the indicator cells. As a complementary study of mCMV-encoded immunoevasins, we have examined the direct interaction of m152, a known mCMV immunoevasin, with the stress-induced MHC-I-like molecule Rae-1beta;. We have expressed the extracellular domains of RAE-1beta; and H60 (as a control) as inclusion bodies in bacteria, and refolded them in vitro. A soluble recombinant form of the extracellular domain of m152 has been purified from insect cells. Results from native gel shift assay and size exclusion chromatography suggested a direct interaction between m152 and RAE-1 β. Both sedimentation equilibrium and sedimentation velocity analysis as well as isothermal titration calorimetry revealed that m152 binds RAE-1beta; tightly (KD< 5 microM) and at a 1:1 ratio. These studies clearly indicate a direct interaction of m152 with RAE-1β and lead to further experiments to define the molecular details of this interaction. Additional biochemical and structural studies of the mCMV immunovasin m04, which can block NK and T cell recognition of cytomegalovirus-infected cells, are underway. In particular, m04 has been studied by multidimensional NMR, and a preliminary structure of its basic fold, unique in the structural database has been determined. Further refinement is underway.

这项工作的重点是了解控制先天免疫系统和适应性免疫系统的细胞识别感染病原体（例如病毒）的细胞的初始步骤的分子细节。特别是，我们从生物物理和结构的角度研究主要组织相容性复合体（MHC）编码分子的大家族。因此，我们感兴趣的是 MHC-I 分子如何分别通过其 NK 和 T 细胞受体与自然杀伤 (NK) 细胞或 T 淋巴细胞上的受体相互作用。这些研究依赖于对相关分子相互作用的结构、功能和生物物理分析，并尝试将结合特性与功能和结构联系起来。疱疹病毒家族的大型 DNA 病毒产生模仿宿主 MHC-I 分子的蛋白质，作为其免疫逃避策略的一部分，我们致力于了解由 MHC-I 分子编码的一组 MHC-I 分子的功能、细胞表达和结构。小鼠巨细胞病毒（mCMV）。我们通过转染不同细胞类型分析了其中几个基因的表达，并确定，与经典的 MHC-I 分子不同，病毒 MHC-I 分子（称为 MHC-Iv）不需要 β- 2.用于表达的微球蛋白或自肽。此外，MHC-Iv分子m152和m155在任何情况下都不在细胞表面表达。通过改造 m144 分子在大肠杆菌中表达，我们生产了 m144 与其 β-2 微球蛋白轻链复合物的 X 射线衍射质量晶体，并已完成数据收集、分子替换解决方案和 m144 结构的精修1.9 埃分辨率。这项研究首次揭示，其中一个 mCMV MHC-Iv 分子确实保留了经典 MHC-I 分子的基本分子折叠特征。该结构揭示了一种缺乏结合肽的分子，并且具有有助于热稳定性的独特结构特征。诱变实验证实了该分子独特的二硫键的重要性。此外，我们还检查了其他巨细胞病毒 MHC-Iv 免疫逃避素、M37、m151 和 m153 的细胞表达。其中每一种都具有独特的细胞生物学特征。我们与里耶卡大学的 Stipan Jonjic 合作，生成了 m153 的单克隆抗体，并且最近证明 m153 形成稳定的非共价同二聚体。已获得在果蝇 S2 细胞中表达的硒代甲硫氨酰 m153 的衍射质量晶体。使用单反常色散（SAD）方法，我们解析了m153的结构，并将结构细化至2.4埃分辨率。该晶体学模型、坐标和结构因子已存入蛋白质数据库，登录号为 2O5N，并且详细描述该结构的手稿已发表。这种全新的 MHC 样结构最显着的特征是该分子形成稳定的头尾同二聚体。为了确认在 m153 晶体结构中观察到的二聚界面，我们在界面处涉及氢键的四个残基处引入了丙氨酸突变。突变型m153和野生型m153在杆状病毒昆虫细胞中瞬时表达并纯化。通过尺寸排阻色谱法和分析超速离心实验对野生型和突变型 m153 蛋白进行的分析表明，突变使二聚体不稳定，并且突变型蛋白作为单体存在。 m153 的生物学功能尚不清楚。为了深入了解其在病毒感染期间的作用，我们开发了几种策略来识别 m153 的假定配体。使用代谢标记的、感染 MCMV 或转染 m153 的小鼠成纤维细胞进行的初始免疫沉淀实验未能鉴定出小鼠成纤维细胞中的任何结合伴侣。在第二种方法中，我们构建了 m153 报告细胞作为筛选细胞系和原代细胞中配体的工具。指示细胞系 43.1 是源自 2B4 细胞的 T 细胞杂交瘤，其中稳定引入了 NFAT 驱动的 GFP 构建体。当抗体交联触发 TCR 时，这些细胞会产生 GFP。生成 m153 特异性 GFP 报告细胞系融合生成了由 m153 的胞外结构域和人 zeta 蛋白的跨膜和胞内部分组成的构建体。通过逆转录病毒感染将 m153-Zeta 构建体引入 43.1 细胞系中。所得的 m153-Zeta/43.1 细胞在涂有抗 m153 单克隆抗体 m153.16 的平板上培养过夜后产生 GFP，从而提供有价值的配体筛选工具。使用 m153-Zeta 报告细胞筛选了来自不同来源的 10 种小鼠细胞系，但没有一个细胞系刺激 GFP 的产生。最近，我们发现来自几种不同小鼠品系的新鲜分离的脾细胞刺激 m153-Zeta 报告细胞产生 GFP，而亲本 43.1 细胞和表达另一种与人 zeta 融合的病毒糖蛋白的对照细胞系不受共培养的影响与脾细胞。脾细胞群的进一步分离表明 CD11c+ 树突状细胞在激活指示细胞方面最有效。作为 mCMV 编码的免疫逃避素的补充研究，我们检查了 m152（一种已知的 mCMV 免疫逃避素）与应激诱导的 MHC-I 样分子 Rae-1beta 的直接相互作用。我们已经表达了RAE-1beta的胞外结构域；和H60（作为对照）作为细菌中的包涵体，并在体外重新折叠它们。 m152 胞外结构域的可溶性重组形式已从昆虫细胞中纯化出来。天然凝胶位移测定和尺寸排阻色谱的结果表明 m152 和 RAE-1 β 之间存在直接相互作用。沉降平衡和沉降速度分析以及等温滴定量热法均表明 m152 结合 RAE-1beta；紧密（KD < 5 microM）且比例为 1:1。这些研究清楚地表明 m152 与 RAE-1β 的直接相互作用，并导致进一步的实验来确定这种相互作用的分子细节。 mCMV 免疫血管蛋白 m04 的其他生化和结构研究正在进行中，该药物可以阻断 NK 和 T 细胞对巨细胞病毒感染细胞的识别。特别是，通过多维核磁共振研究了m04，并确定了结构数据库中唯一的其基本折叠的初步结构。进一步完善工作正在进行中。