Structure and Function of Viral Immunoevasins

病毒免疫球蛋白的结构和功能

• 批准号: 8555951
• 负责人: David Margulies
• 金额: $ 51.83万
• 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/8555951
• 关键词: Activated Natural Killer Cell; Address; Affinity; Binding; Biochemistry; Biological Assay; Biological Process; Blocking Antibodies; C57BL/6 Mouse; CD8B1 gene; Cell Communication; Cell Line; Cell physiology; Cell surface; Cells; Communicable Diseases; Complement; Complex; Confocal Microscopy; Crystallization; Cytomegalovirus; Cytomegalovirus Infections; DNA Viruses; Dendritic Cells; Dimerization; Employee Strikes; Engineering; Epithelial Cells; Evolution; Expression Library; Family; Flow Cytometry; Fractionation; Genes; Glycoproteins; Goals; Head; Herpesviridae; Homologous Gene; Human; ITGAX gene; Immune response; Immune system; Immunocompromised Host; Immunologic Deficiency Syndromes; Inbred BALB C Mice; Infection; Insecta; Intervention; Laboratories; Lead; Lectin; Ligands; Ligation; Light; Major Histocompatibility Complex; Mammals; Methods; Modeling; Molecular; Molecular Structure; Murid herpesvirus 1; Mus; NK Cell Activation; Natural Killer Cells; Peptides; Population; Predisposition; Production; Protein Binding; Proteins; Recombinants; Reporter; Reporting; Resistance; Resolution; Roentgen Rays; Screening procedure; Site; Spleen; Staining method; Stains; Stress; Structure; Surface; Surface Plasmon Resonance; System; T-Cell Receptor; T-Lymphocyte; Tail; Time; Transfection; Viral; Viral Physiology; Virion; Virus; Virus Diseases; Work; base; beta-2 Microglobulin; cDNA Expression; cell type; human disease; improved; insight; interest; killer T cell; killer inhibitory receptor; member; molecular dynamics; mutant; novel; pathogen; protein folding; receptor; research study; three dimensional structure

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. It is our contention that 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. In particular, we study representative members of 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. 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 structure of the MHC-Iv molecule, m144, and showed that it preserved an MHC-I like fold, though it was devoid of bound peptide. In the past year we have extended our studies to include expression, binding and structural studies of m152, m153, and m157. Each of these molecules represents a different mode of 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. and m157 has been shown to be a primary ligand for NK cell receptors, Ly49I, an NK cell inhibitory receptor expressed in BALB/c (virus-susceptible) mice, and Ly49H, an NK cell activation receptor expressed in resistant C57BL/6 mice. 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. A number of murine cell lines from various origins were screened with these reporter cells, but none stimulated the production of GFP. Freshly isolated spleen cells and in particular CD11c+ dendritic cells (DC) are particularly potent in activation the indicator cells. Further fractionation of the spleen cell populations indicate that CD11c+ dendritic cells (DC) are the most potent in activating the indicator cells. Several complementary approaches are now underway to identify the ligand(s) of m153 expressed on DC: staining and competition for staining of known DC markers with an m153 tetramer both by flow cytometry and by confocal microscopy; antibody blocking of the reporter cell assay; subfractionation of the DC population that carries the stimulatory ligand; mass spectrometric identification of molecules that pull down from DC lysates with the m153 tetramer; and screening of a cDNA expression library generated from DC. The consistent finding, that m153, an early mCMV encoded cell surface molecule, engages a molecule expressed at the surface of DCs, is itself a provocative finding. mCMV is known to replicate in epithelial cells as well as DC, and we hypothesize that one of the functions of m153 is to promote the infectious spreading of mCMV virus particles to DCs as a further site for secondary replication. 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. The biological function of m153 is unknown. Another member of the mCMV MHC-Iv family of molecules is m157, a glycoprotein expressed at the cell surface early in mCMV infection. Previous studies in other laboratories have identified both the NK cell lectin-like receptors Ly49I and Ly49H as ligands for m157. More importantly, Ly49H (expressed inC57BL/6 mice) has been shown to provide the basis of resistance to viral infection. Ly49I (the NK inhibitory receptor expressed in BALB/c may also contribute to viral susceptibility. Although the three-dimensional structure of m157 has been determined by the Garcia group, no report of m157 in complex with either Ly49I or Ly49H has been reported. This is an important and relevant problem because it addresses the issues of MHC-Iv evolution and function. We have addressed this question by engineering Ly49H and Ly49I and purifying m157 expressed in an insect cell system Surface Plasmon Resonance binding studies confirm that this recombinant m157 binds specifically to both Ly49H and Ly49I with modest affinity. Based on these binding studies, we have set up crystallization screens and have identified condition under which m157/Ly49I complexes form crystals. At the present time the best crystals have diffracted to 8 , not yet adequate for a molecular structure determination. However, additional conditions are being explored and we expect to obtain crystals of better quality. Efforts are also underway to explore the structural basis of the interaction of additional viral MHC-I-like molecules with their respective cellular ligands. mCMV m145 and m155 and ligands MULT1 and H60 have been expressed and purified, and we have determined the structure of MULT1 and expect to have the m145/MULT1 complex.

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。我们的论点是，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，不仅可以更深入地理解一般分子相互作用以及免疫系统中细胞与细胞的相互作用。系统，还可能导致干预病毒感染的合理方法。 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）编码分子大家族的代表性成员。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。 疱疹病毒家族的大型 DNA 病毒会产生模仿宿主 MHC-I 分子的蛋白质，作为其免疫逃避策略的一部分，我们已努力了解一组这些 MHC-I（称为小鼠巨细胞病毒 (mCMV) 编码的 MHC-Iv）分子。我们分析了这些基因中的几个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 的轻链成分分子、β-2微球蛋白或用于表达的自肽。 尽管这些MHC-Iv分子中的一些分子在感染后早期就在病毒感染的细胞表面表达，但包括m152和m155在内的其他一些分子在细胞表面表达不佳，表明它们的功能是细胞内活动的结果。在早期的研究中，我们确定了 MHC-Iv 分子 m144 的结构，并表明它保留了类似 MHC-I 的折叠，尽管它没有结合肽。 去年，我们扩展了研究范围，包括 m152、m153 和 m157 的表达、结合和结构研究。 这些分子中的每一个都代表不同的作用模式。 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-I 分子以逃避 CD8 T 细胞识别。 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的 RAE-1 家族。 m153 的功能未知，但在许多源自野生小鼠的 mCMV 毒株中序列高度保守，表明其功能保守。 m157 已被证明是 NK 细胞受体、Ly49I（在 BALB/c（病毒敏感）小鼠中表达的 NK 细胞抑制性受体）和 Ly49H（在耐药 C57BL/6 小鼠中表达的 NK 细胞激活受体）的主要配体。 我们对 m152 的研究证明了这种 mCMV 编码的 MHC-Iv 蛋白与宿主 MHC-I 和应激诱导的 RAE-1 分子的直接相互作用，并且我们已经确定了 m152 与其 RAE-1 复合物的 X 射线晶体结构。 1个配体。 该结构揭示了 MHC-I 蛋白折叠与 RAE-1（MHC-I 家族的另一个成员）结合的新适应。 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 的相互作用非常重要。 令人惊讶的是，相互作用的位点是相同的，竞争实验证实了这一点。 m152 与 RAE-1 相互作用的细节已通过检查 RAE-1 的 18 个定点突变体的结合得到证实。因此，m152 提供了 mCMV 编码的 MHC-I 样蛋白的新例子，该蛋白结合两种不同类别的 MHC-I 样蛋白。 mCMV 蛋白 m153 提供了 mCMV MHC-Iv 分子多种功能的另一个独特例子。 尽管 m153 的精确功能尚不清楚，但我们已经使用报告细胞系统对其进行了广泛的探索，其中表达 m153 的细胞在其表面表达的 m153 与带有 m153 配体的细胞连接时发出荧光。用这些报告细胞筛选了来自不同来源的许多小鼠细胞系，但没有一个细胞系刺激 GFP 的产生。新鲜分离的脾细胞，特别是 CD11c+ 树突状细胞 (DC) 在激活指示细胞方面特别有效。 脾细胞群的进一步分级表明 CD11c+ 树突状细胞 (DC) 在激活指示细胞方面最有效。 目前正在采用几种互补方法来鉴定 DC 上表达的 m153 配体：通过流式细胞术和共聚焦显微镜对已知 DC 标记物与 m153 四聚体进行染色和竞争染色；报告细胞测定的抗体阻断；携带刺激配体的 DC 群体的亚分级；对使用 m153 四聚体从 DC 裂解物中拉出的分子进行质谱鉴定；以及筛选由DC产生的cDNA表达文库。 m153（一种早期 mCMV 编码的细胞表面分子）与 DC 表面表达的分子结合这一一致发现本身就是一个令人兴奋的发现。 已知 mCMV 在上皮细胞和 DC 中复制，我们假设 m153 的功能之一是促进 mCMV 病毒颗粒向 DC 的感染性传播，作为二次复制的进一步位点。 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解析并细化至 2.4 埃分辨率。这种新的 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 m153 晶体结构中观察到的二聚化界面，我们分析了许多界面突变体，确认了二聚化位点。 m153 的生物学功能尚不清楚。 mCMV MHC-Iv 分子家族的另一个成员是 m157，一种在 mCMV 感染早期在细胞表面表达的糖蛋白。 其他实验室之前的研究已经确定 NK 细胞凝集素样受体 Ly49I 和 Ly49H 都是 m157 的配体。 更重要的是，Ly49H（在C57BL/6小鼠中表达）已被证明可以提供抵抗病毒感染的基础。 Ly49I（BALB/c中表达的NK抑制性受体）也可能与病毒易感性有关。尽管Garcia小组已经确定了m157的三维结构，但尚未报道m157与Ly49I或Ly49H形成复合物。是一个重要且相关的问题，因为它解决了 MHC-Iv 进化和功能的问题。我们通过改造 Ly49H 和 Ly49I 并纯化 m157 解决了这个问题。在昆虫细胞系统中表达 表面等离子共振结合研究证实，这种重组 m157 以适度的亲和力与 Ly49H 和 Ly49I 特异性结合 基于这些结合研究，我们建立了结晶筛选并确定了 m157/Ly49I 复合物形成的条件。目前最好的晶体已经衍射到 8 ，还不足以确定分子结构，但是，正在探索其他条件，我们期望获得 8 晶体。更好的质量。 人们还在努力探索其他病毒 MHC-I 样分子与其各自细胞配体相互作用的结构基础。 mCMV m145和m155以及配体MULT1和H60已经表达和纯化，并且我们已经确定了MULT1的结构并期望得到m145/MULT1复合物。