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的MHC多肽递呈，以逃避免疫系统的清除。虽然已经确定了CMV免疫转移酶的几个靶点，但免疫转移酶如何调节感染细胞的免疫肽段以抑制病毒抗原的提呈在很大程度上仍不清楚。我们将结合基于质谱学的免疫表位学和同位素标记氨基酸(PSILAC)的代谢脉冲标记(PSILAC)来准确量化HCMV免疫抑制素对MHC-I抗原处理途径的干扰。首先，我们将研究个体免疫抑制素的异位表达对成纤维细胞免疫表位的影响。这类实验不仅使我们能够研究免疫抑制素对抗原提呈的抑制作用，而且还可以识别可能由免疫抑制素诱导的与肽加工受损(TEIPPs)相关的T细胞表位。接下来，我们将研究单个免疫球蛋白对感染ΔUS2-11型巨细胞病毒感染的成纤维细胞免疫表位的调节作用。这应该揭示了单个免疫转运蛋白如何抑制病毒T细胞表位的呈递。最后，我们将对野生型和ΔUS2-11型人巨细胞病毒感染的成纤维细胞进行定量免疫肽组和蛋白质组实验。与P01项目合作，这些实验将得到不同细胞类型的定量蛋白质组分析的补充，例如巨噬细胞和内皮细胞。与P03项目一起，我们将进行免疫多肽定量分析以及相互作用组分析，以探索免疫血管蛋白US10的功能。此外，我们还将描述各种人类白细胞抗原C等位基因和人类巨细胞病毒感染细胞中人类白细胞抗原E的配基组。与P02项目合作，我们将确定MCMV感染的成纤维细胞的免疫肽组以及病毒m04和MATp1蛋白对其的调控。这些研究将对巨细胞病毒免疫因子的分子和细胞功能提供深刻的见解，并有可能从长远来看，为针对巨细胞病毒免疫逃避的治疗干预开发新的策略。

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.