Analyzing HCMV infection and immune evasion by mass spectrometry

巨细胞病毒（CMV）表达多种免疫逃避因子，并以各种方式操纵感染细胞中的MHC肽呈递以逃避免疫系统的清除。虽然已经鉴定了CMV免疫evasins的几个靶点，但免疫evasins如何调节感染细胞的免疫肽组以抑制病毒抗原的呈递在很大程度上仍然是难以捉摸的。我们将结合联合收割机基于质谱的免疫肽组学与同位素标记氨基酸（pSILAC）的代谢脉冲标记，以准确定量HCMV免疫evasins对MHC-I抗原加工途径的干扰。首先，我们将研究单个免疫evasins异位表达对成纤维细胞免疫肽组的影响。这类实验不仅使我们能够研究免疫evasins对抗原呈递的抑制，而且还能够鉴定与免疫evasins的作用可能诱导的受损肽加工（TEIPPs）相关的T细胞表位。接下来，我们将研究在感染Δ US 2 -11 HCMV感染的成纤维细胞的成纤维细胞中单个免疫evasins对免疫肽组的调节。这将揭示个体免疫evasins如何抑制病毒T细胞表位的呈递。最后，我们将用野生型和Δ US 2 -11 HCMV感染的成纤维细胞进行定量免疫肽组和蛋白质组实验。这些实验将通过与项目P01合作对不同细胞类型（如巨噬细胞和内皮细胞）进行定量蛋白质组分析来补充。与项目P03一起，我们将进行定量免疫肽组和相互作用组分析，以探索免疫evasins US 10的功能。此外，我们将描述HCMV感染细胞中各种HLA-C等位基因和HLA-E的配体组的特征。与项目P02合作，我们将确定MCMV感染的成纤维细胞的免疫肽组及其通过病毒m04和MATp 1蛋白的操纵。这些研究将为CMV免疫逃逸的分子和细胞功能提供深刻的见解，并可能从长远来看，能够开发针对HCMV免疫逃逸的治疗干预的新策略。

Cytomegaloviruses (CMV) express a variety of immunoevasive factors and manipulate MHC peptide presentation in infected cells in various ways to escape elimination by the immune system. Although several targets of CMV immunoevasins have been identified, it remains largely elusive how immunoevasins modulate the immunopeptidome of the infected cells to suppress the presentation of viral antigens. We will combine mass spectrometry-based immunopeptidomics with metabolic pulse-labeling by isotopically labeled amino acids (pSILAC) to accurately quantify the perturbation of the MHC-I antigen processing pathway by HCMV immunoevasins. First, we will study the effect of ectopic expression of individual immunoevasins on the immunopeptidome of fibroblasts. This kind of experiments will not only enable us to study the suppression of antigen presentation by immunoevasins, but also to identify T-cell epitopes associated with impaired peptide processing (TEIPPs) that might be induced by the action of immunoevasins. Next, we will study the modulation of the immunopeptidome by individual immunoevasins in fibroblasts infected with ΔUS2-11 HCMV-infected fibroblasts. This should reveal how individual immunoevasins suppress the presentation of viral T-cell epitopes. Finally, we will perform quantitative immunopeptidome and proteome experiments with wild type and ΔUS2-11 HCMV-infected fibroblasts. These experiments will be complemented by quantitative proteome analyses of different cell types, such as macrophages and endothelial cells, in collaboration with project P01. Together with project P03, we will conduct quantitative immunopeptidome as well as interactome analyses to explore the function of the immunoevasins US10. In addition, we will characterize the ligandomes of various HLA-C alleles and of HLA E in HCMV-infected cells. In collaboration with project P02, we will determine the immunopeptidome of MCMV-infected fibroblasts and its manipulation by the viral m04 and MATp1 proteins. These studies will provide profound insights in the molecular and cellular function of CMV immunoevasins, and potentially in the long run enable the development of new strategies for therapeutic intervention against the immune evasion of HCMV.