Structure and Function of Viral Immunoevasins

这项工作的重点是了解控制先天免疫系统和获得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的分子相互作用和细胞-细胞相互作用，而且可能导致合理的干预病毒感染和肿瘤的方法。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)编码的分子大家族的代表性成员。我们感兴趣的是MHC-I分子如何分别通过自然杀伤细胞(NK)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。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，作为其免疫规避策略的一部分，我们致力于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分子的结构，m144，m152和m153。在最近与多伦多大学的Oscar Aguar博士和James Carlyle博士的合作(提交的手稿)中，我们证明了作为着色剂的重组m153能够特异性识别表达在树突状细胞系DC2.4和类似ILC3的NK细胞MNK-3表面的分子。这一发现补充了我们的合作者的发现，即m153调节NK细胞配体Clr-b的细胞表面水平。除了我们对病毒类MHC分子的研究外，我们最近还研究了另一组可能的MCMV免疫转运蛋白，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伴随着MHC-I分子到达细胞表面，而M06引导MHC-I分子进入内切/溶酶体途径。我们已经成功地设计了M04，并检测了它与MHC-I的结合，以定量这种相互作用。我们之前与Nik Sgourakis博士和Ad Bax博士合作，使用核磁共振确定了m04在溶液中的结构。核磁共振结构的确定是基于多维光谱的确定，该多维光谱允许将分子约束分配给分子内不同的原子间距离。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，这些实验旨在检查细胞内进行的MHC-I折叠的精确中间产物。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。对M06蛋白与MHC分子H2-LD结合的分子络合物的初步筛选表明，应该可以获得衍射质量的晶体，以便最终确定M06逃避功能的独特机制。对MHC分子、宿主MHC类分子和病毒MHC类分子的比较分子结构的两次大规模综述(Natarajan等人，2018；酱等人，2019)表明，MHC家族分子表现出特异性和简并性的一般能力存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发现了一种由传染性软疣病毒MC80编码的新的免疫EVASIN，它通过与蛋白质负载复合体中的Tapasin相互作用来阻碍抗原递送，并将Tapasin引导到内质网降解途径。我们目前正在表达MC80蛋白，用于结构和结合研究，预计这一基础知识将增强我们对MHC多肽装载途径以及病毒免疫逃避的许多途径的理解。

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.