Structure and Function of Viral Immunoevasins

这项工作的重点是了解控制先天性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。了解由免疫系统识别的病毒编码分子的相互作用的功能、机制、结构和进化不仅可以导致对一般分子相互作用和免疫系统中细胞-细胞相互作用的更深入理解，而且还可能导致对病毒感染和肿瘤进行干预的合理方法。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）编码分子的大家族的代表性成员。我们感兴趣的是MHC-I分子如何与自然杀伤（NK）细胞和T淋巴细胞上的受体相互作用，分别通过它们的NK和T细胞受体。疱疹病毒家族的大DNA病毒产生模拟宿主MHC-I分子的蛋白质作为其免疫逃避策略的一部分，并且我们已经指导我们的努力来理解由小鼠巨细胞病毒（mCMV）编码的一组这些MHC-I（称为MHC-Iv）分子的功能、细胞表达和结构。我们已经分析了MHC-IV家族的几个成员的表达和结合，特别是分子m144，m152和m153。在早期的研究中，我们确定了MHC-IV分子m144、m152和m153的结构。在最近与多伦多大学的Oscar阿吉拉尔博士和James卡莱尔博士的合作（手稿已提交）中，我们已经证明了重组m153，多聚化作为染色剂，特异性识别树突状细胞系DC 2.4表面表达的分子，以及ILC 3样NK细胞MNK-3。这一发现补充了我们的合作者发现m153调节NK细胞配体Clr-b的细胞表面水平。除了我们对病毒MHC样分子的研究外，我们最近还研究了另一组推定的mCMV免疫evasins，即m04家族。这个家族，包括m02，m04和m06，似乎有不同的作用机制。m04伴随MHC-I分子到达细胞表面，m06引导MHC-I分子进入内体/溶酶体途径。我们已经成功地改造了m04并检查了其与MHC-I的结合以量化这种相互作用。我们先前与Nik Sgourakis和Ad Bax博士合作使用NMR确定了m04在溶液中的结构。NMR结构测定基于多维光谱的测定，多维光谱允许将分子限制分配到分子内的各种原子间距离。 从这些早期的研究出发，我们现在正在用细胞成像实验来补充它们，这些实验旨在检查在细胞内进行的MHC-I折叠的精确中间体。 我们已经确定m06将MHC-I分子路由到内体/溶酶体途径，从而有助于细胞表面MHC-I的下调。结合于MHC分子H2-Ld的m06蛋白的分子复合物的初步筛选表明，衍射质量的晶体应该是可实现的，以允许m06逃避功能的独特机制的确定性结构测定。对MHC分子、宿主MHC样分子和病毒MHC样分子的比较分子结构的两项大规模综述（Natarajan et al，2018;和Jiang et al，2019，出版中）表明，MHC家族分子表现出特异性和简并性的一般能力在于与肽或配体相互作用位点相关的分子柔性区域。 最近来自其他实验室的实验已经鉴定出由传染性软疣病毒MC 80编码的新型免疫evasin，其通过与蛋白质加载复合物中的tapasin相互作用来阻碍抗原呈递，并将tapasin引导至内质网降解途径。我们目前正在表达的MC 80蛋白的结构和结合研究，并期望这一基础知识将提高我们的理解的MHC肽加载途径，以及病毒免疫逃避的许多途径。

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 and binding of several members of the MHC-Iv family, in particular the molecules, m144, m152, and m153. In earlier studies, we determined the structures of the MHC-Iv molecules, m144, m152, and m153. In a recent collaboration with Drs. Oscar Aguilar and James Carlyle (manuscript submitted) at the University of Toronto, we have demonstrated that recombinant m153, multimerized as a staining agent, specifically recognizes molecules expressed on the surface of a dendritic cell line, DC2.4, as well as on an ILC3-like NK cell, MNK-3. This finding complements our collaborators finding that m153 regulates the cell surface level of an NK cell ligand, Clr-b. In addition to our studies of the viral MHC-like molecules, we have recently addressed another set of the putative mCMV immunoevasins, the m04 family. This family, that includes m02, m04, and m06 seems to have distinct mechanisms of action. m04 accompanies the MHC-I molecule to the cell surface, and m06 directs MHC-I molecules to an endosomal/lysosomal pathway. We have successfully engineered m04 and examined its binding to MHC-I to quantify this interaction. We previously 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. Proceeding from these earlier studies, we are now complementing them with cellular imaging experiments designed to examine the precise intermediates of MHC-I folding that proceed within the cell. We have established that m06 routes MHC-I molecules to the endosomal/lysosomal pathway, thus contributing to the downregulation of cell surface MHC-I. Preliminary screening of molecular complexes of the m06 protein bound to the MHC molecule H2-Ld indicate that diffraction quality crystals should be achievable to allow definitive structural determination of the unique mechanism of m06 evasive function. Two large scale reviews of the comparative molecular structures of MHC molecules, host MHC-like molecules, and viral MHC-like molecules (Natarajan et al, 2018; and Jiang et al, 2019, in press) indicate that the general ability of molecules of the MHC family to exhibit both specificity and degeneracy lies in regions of molecular flexibility, related to either peptide or ligand interaction sites. Recent experiments from other labs have identified a novel immunoevasin encoded by the Molluscum contagiosum virus, MC80, that impedes antigen presentation by interacting with tapasin in the protein loading complex, and directs tapasin to an endoplasmic reticulum degradative pathway. We are currently expressing the MC80 protein for structural and binding studies, and expect that this basic knowledge will both enhance our understanding of the MHC peptide loading pathway as well as of the many avenues of viral immunoevasion.