Structure and Function of Viral Immunoevasins

病毒免疫球蛋白的结构和功能

• 批准号: 9354827
• 负责人: David Margulies
• 金额: $ 46.34万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/9354827
• 关键词: Activated Natural Killer Cell; Affinity; Attention; Bacteria; Binding; Binding Proteins; Binding Sites; Biochemistry; CD8B1 gene; Cell Communication; Cell physiology; Cell surface; Cells; Collaborations; Communicable Diseases; Complement; Complex; Cytomegalovirus; Cytomegalovirus Infections; DNA Viruses; Data; Dimerization; Employee Strikes; Engineering; Escherichia coli; Evolution; Family; Genes; Goals; Head; Herpesviridae; Homologous Gene; Human; Immune response; Immune system; Immunocompromised Host; Immunodeficiency and Cancer; Infection; Intervention; Label; Lead; Ligands; Ligation; Light; Major Histocompatibility Complex; Mammals; Maps; Methodology; Methods; Modeling; Molecular; Molecular Structure; Murid herpesvirus 1; Mus; Natural Killer Cells; Neoplasms; Pathway interactions; Peptides; Protein Family; Proteins; Publishing; Recombinants; Reporter; Resolution; Roentgen Rays; Site; Stress; Structure; Surface; System; T-Cell Receptor; T-Lymphocyte; Tail; Transfection; Viral; Viral Physiology; Viral Proteins; Virus; Virus Diseases; Work; base; beta-2 Microglobulin; cell type; human disease; improved; insight; interest; killer T cell; member; molecular dynamics; mutant; new technology; novel; pathogen; protein folding; receptor; research study; restraint

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. Understanding the function, mechanism, structure, and evolution of the interaction of virus-encoded molecules recognized by the immune system can lead not only to a deeper understanding of molecular interactions in general and of cell-cell interactions in the immune system, but also may lead to rational approaches to intervention in virus infection and neoplasia. In particular, we study representative members of the large family of major histocompatibility complex (MHC)-encoded molecules from a biophysical and structural perspective. We are interested in how MHC-I molecules interact with receptors on natural killer (NK) cells and on T lymphocytes through their NK and T cell receptors, respectively. 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 efforts to understand the function, cellular expression, and structure of a set of these MHC-I (referred to as MHC-Iv) molecules encoded by the mouse cytomegalovirus (mCMV). We have analyzed the expression of several of these genes after transfection in different cell types, and have established that, unlike the classical MHC-I molecules, the viral MHC-I molecules do not require either the light chain component of the classical MHC-I molecule, beta-2 microglobulin, or self-peptide for expression. Although several of these MHC-Iv molecules are expressed at the surface of virus-infected cells early after infection, several others, including m152 and m155 are not expressed well at the cell surface, suggesting that their functions result from intracellular activities. In earlier studies, we determined the structures of the MHC-Iv molecules, m144, m152, and m153. Each of these molecules represents a different mode of immunoevsive action. m152 down-regulates host molecules crucial for recognition by either T cells or NK cells -- specifically it down-regulates host MHC-I molecules to elude CD8 T cell recognition. In addition, m152 down-regulates ligands of the NKG2D NK cell activating receptor, in particular the RAE-1 family of stress induced molecules. m153 has an unknown function, but is highly conserved in sequence among a number of mCMV strains that derive from wild mice, suggesting a conserved function. Our studies of m152 demonstrated the direct interaction of this mCMV encoded MHC-Iv protein both with host MHC-I and the stress-induced RAE-1 molecules, and we have determined the X-ray crystallographic structure of m152 in complex with its RAE-1 ligand. This structure reveals a novel adaptation of the MHC-I protein fold for binding to RAE-1, another member of the MHC-I family. Since the NK activating receptor, NKG2D also interacts with RAE-1, it was important to compare the interaction of m152 with RAE-1 with the interaction of NKG2D. Surprisingly, the sites of interaction are the same, and competition experiments confirm this. The details of the interaction of m152 with RAE-1 have been confirmed by examination of the binding of some 18 site directed mutants of RAE-1. Thus m152 provides a novel example of an mCMV-encoded MHC-I-like protein that binds two different classes of MHC-I-like proteins. The mCMV protein m153 provides another unique example of the varied functions of mCMV MHC-Iv molecules. Although the precise function of m153 is not known, we have explored this extensively using a reporter cell system, in which m153 expressing cells become fluorescent on ligation of their surface expressed m153 by cells bearing an m153 ligand. Our studies of the function of m153 have been complemented by the determination of the crystallographic structure of this molecule, which has been solved and refined to 2.4 Angstrom resolution. The most striking feature of this 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 analyzed a number of interface mutants, confirming the site of dimerization. In addition to the m145 family of mouse CMV molecules, we have directed considerable attention to another novel family of putative immunoevasins of the MCMV, the m04 family. These molecules, that include m02, m04, and m06 seem to have distinct functions. m04 accompanies the MHC-I molecule to the cell surface, and m06 directs MHC-I molecules to an endosomal/lysomal pathway. This is a distinct protein family, and efforts to explore the structure of any of its members crystallographically have been difficult. We have successfully engineered m04 and examined its binding to MHC-I to quantify this interaction. We have determined the structure of m04 in solution using NMR in collaboration with Drs.Nik Sgourakis and Ad Bax. NMR structure determination is based on determination of multidimensional spectra that allow assignment of molecular restraints to various interatomic distances within the molecule. Because we expressed the recombinant molecule in bacteria, we were able to label it with a variety of isotopic precursors (13C, 15N, 2D, in various combinations) permitting gathering of spectra allowing interpretation of different interatomic distances. To reduce the amount of data needed, allowing us to employ sparse NMR data, Drs. Sgourakis and Bax exploited a new technology using Rosetta modeling to determine the solution structure of the m04 protein. This structure is a unique beta-fold, based distantly on the Ig-fold, that is representative of the full m02-m06 family of viral proteins. Thus, we not only determined a completely new structure in solution, indicative of a previously elusive protein family, but have applied a novel methodology was recently published in Structure. We have extended these findings on m04 to the related MCMV immunoevasin, m06. This molecule has been engineered for expression in E. coli, and binding studies indicated that it interacts with MHC-I molecules with low but detectable affinity. Using a recombinant MHC-I molecule that gives an outstanding multidimensional NMR spectrum, we have recently been able to map the MHC-I binding site of m06. This work has recently been published.

这项工作的重点是了解控制先天和适应性免疫系统细胞识别感染病原体（如病毒）的细胞的初始步骤的分子细节。了解免疫系统识别的病毒编码分子相互作用的功能、机制、结构和进化，不仅可以更深入地了解免疫系统中一般的分子相互作用和细胞-细胞相互作用，而且可以为病毒感染和肿瘤的干预提供合理的方法。特别是，我们从生物物理和结构的角度研究了主要组织相容性复合体（MHC）编码分子大家族的代表成员。我们对MHC-I分子如何分别通过NK细胞和T细胞受体与自然杀伤细胞（NK）和T淋巴细胞上的受体相互作用感兴趣。疱疹病毒家族的大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，我们已经指导我们的努力，以了解由小鼠巨细胞病毒（mCMV）编码的一组MHC-I（称为MHC-Iv）分子的功能，细胞表达和结构。我们分析了这些基因在不同细胞类型中转染后的表达，并确定，与经典MHC-I分子不同，病毒MHC-I分子不需要经典MHC-I分子的轻链成分、β -2微球蛋白或自肽来表达。尽管这些MHC-Iv分子中有几个在感染后早期在病毒感染的细胞表面表达，但其他几个，包括m152和m155，在细胞表面不表达，这表明它们的功能是由细胞内活动引起的。在早期的研究中，我们确定了MHC-Iv分子m144、m152和m153的结构。这些分子中的每一种都代表一种不同的免疫反应模式。m152下调对T细胞或NK细胞识别至关重要的宿主分子，特别是下调宿主mhc - 1分子以逃避CD8 T细胞的识别。此外，m152下调NKG2D NK细胞激活受体的配体，特别是应激诱导分子RAE-1家族。m153具有未知的功能，但在许多源自野生小鼠的mCMV菌株中序列高度保守，表明其具有保守功能。我们对m152的研究表明，这种mCMV编码的MHC-Iv蛋白与宿主MHC-I和应力诱导的RAE-1分子直接相互作用，我们已经确定了m152与其RAE-1配体复合物的x射线晶体结构。这种结构揭示了MHC-I蛋白折叠与MHC-I家族的另一成员RAE-1结合的新适应性。由于NK激活受体NKG2D也与RAE-1相互作用，因此比较m152与RAE-1的相互作用与NKG2D的相互作用是很重要的。令人惊讶的是，相互作用的地点是相同的，竞争实验也证实了这一点。m152与RAE-1相互作用的细节已经通过检查RAE-1的18个位点定向突变体的结合得到证实。因此，m152提供了mcmv编码的mhc - i样蛋白结合两种不同类型mhc - i样蛋白的新例子。mCMV蛋白m153提供了mCMV MHC-Iv分子不同功能的另一个独特例子。虽然m153的确切功能尚不清楚，但我们已经使用报告细胞系统广泛地探索了这一点，在报告细胞系统中，表达m153的细胞在携带m153配体的细胞连接其表达m153的表面时变得荧光。我们对m153功能的研究得到了该分子晶体结构测定的补充，该分子的晶体结构已被求解并细化到2.4埃的分辨率。这种新的类mhc结构最显著的特征是分子形成了一个稳定的头尾同源二聚体。为了证实在m153晶体结构中观察到的二聚化界面，我们分析了一些界面突变体，确定了二聚化的位置。除了小鼠巨细胞病毒分子的m145家族外，我们还将相当多的注意力投向了另一个新的假定的MCMV免疫规避家族，即m04家族。这些分子，包括m02， m04和m06似乎有不同的功能。m04伴随着MHC-I分子到达细胞表面，m06引导MHC-I分子到达内体/溶体途径。这是一个独特的蛋白质家族，从晶体学上探索其任何成员的结构一直很困难。我们已经成功地设计了m04，并检测了它与mhc - 1的结合，以量化这种相互作用。我们与博士合作，利用核磁共振确定了溶液中m04的结构。Nik Sgourakis和Ad Bax。核磁共振结构的确定是基于多维光谱的确定，允许分子约束分配到分子内不同的原子间距离。因为我们在细菌中表达了重组分子，我们能够用各种同位素前体（13C, 15N, 2D，各种组合）标记它，允许收集光谱，从而解释不同的原子间距离。为了减少所需的数据量，允许我们使用稀疏的核磁共振数据。Sgourakis和Bax利用Rosetta模型开发了一种新技术来确定m04蛋白的溶液结构。这种结构是一种独特的β折叠，与igg折叠有很大的关系，它代表了病毒蛋白的整个m02-m06家族。因此，我们不仅在溶液中确定了一个全新的结构，表明了一个以前难以捉摸的蛋白质家族，而且应用了一种新的方法，最近发表在《结构》杂志上。我们将m04的这些发现扩展到相关的MCMV免疫逃逸蛋白m06。该分子已被设计用于在大肠杆菌中表达，结合研究表明它与mhc - 1分子相互作用，具有低但可检测的亲和力。利用重组mhc - 1分子，我们最近能够绘制出m06的mhc - 1结合位点。这项工作最近已发表。