Control of CD8+ T cells through interference of Cytomegalovirus with the "peptide loading complex" (PLC)

MHC I类抗原递呈给CD8+T细胞是抵抗病毒感染细胞的主要免疫防御机制。内源性抗原通过多肽负载复合体(PLC)被负载到内质网中的MHC I上，PLC由MHC I、多肽转运体TAP和包括Tapasin在内的伴侣组成。Tapasin使MHC I和TAP紧密相连，并与氧化还原酶ERp57一起调节MHC多肽结合槽的多肽接受状态。巨细胞病毒可导致免疫受损的人患上严重疾病，反映了免疫系统对感染作出反应和病毒本身逃避感染之间的微妙平衡。为了应对MHC I类抗原的呈递，HCMV编码了几种翻译后策略，这些策略在转基因细胞中得到了广泛的研究。我们分析了自然感染HCMV的细胞中的PLC，并监测了整个HCMV复制过程中PLC的组成。代谢标记实验表明，不仅在感染早期没有MHC I掺入PLC，而且在感染早期开始缺乏Tapasin，但在感染后期最为明显。相反，Western印迹分析显示，感染细胞中Tapasin的稳定水平仅缓慢下降，表明合成受阻，而不是降解。我们发现，在8小时P.I.的初始诱导后，在24小时P.I.对Tapasin的转录有强烈的抑制。这种情况在整个复制周期中持续存在。此外，与MHC I转录水平升高相比，TAP1和TAP2转录水平也降低。该项目现在遵循两个进一步的目标。首先，我们将分析巨细胞病毒感染过程中Tapasin从MHC I类分子中解离的分子基础。这种作用发生在抑制Tapasin转录之前，并且不依赖于已知的MHC-I类抗原呈递途径的HCMV抑制剂US2、US3和US6。我们的目标是确定在HCMV感染过程中对PLC的破坏起关键作用的负责基因。第二个目标是在修复PLC的组装和功能后，分析在HCMV感染过程中MHC I提呈的抗原。这将为评价CD8+T细胞识别HCMV抗原的实际逃逸能力和设计HCMV疫苗提供参考。

MHC I antigen presentation to CD8+ T-cells is a major mechanism of immune defense against virally infected cells. The endogenous antigens are loaded onto MHC I in the ER by the peptide loading complex (PLC), consisting of MHC I, the peptide transporter TAP, and chaperones including tapasin. Tapasin brings MHC I and TAP in close vicinity and together with the oxidoreductase ERp57 the peptide-receptive state of the MHC peptide-binding groove is regulated. HCMV can cause severe disease in immunocompromised persons, reflecting the delicate balance between the immune system reacting on the infection and the evasion of it by the virus itself. To cope with MHC I antigen presentation HCMV encodes for several post-translational strategies which have been extensively studied in transfected cells. We have analysed the PLC in naturally HCMV-infected cells and monitored the composition of the PLC throughout HCMV replication. Metabolic labeling experiments have revealed not only the absence of MHC I incorporation into the PLC during early time points of infection, but also lack of tapasin starting at early but being most pronounced at late times of infection. In contrast, Western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. We found, after an initial induction at 8 hrs p.i., a strong inhibition of tapasin transcription at 24 hrs p.i. that persisted throughout the replication cycle. Furthermore, also reduction of TAP1 and TAP2 transcription was observed contrasting the elevated levels of MHC I transcripts. The project now follows two further objectives. First, the molecular basis of tapasin dissociation from MHC class I during HCMV infection will be analysed. This effect precedes the repression of tapasin transcription and occurs independently from the known HCMV inhibitors of the MHC class I pathway of antigen presentation, US2, US3 and US6. The goal is to identify the responsible HCMV gene that is supposed to exert a key function in the disruption of the PLC during HCMV infection. Identification of PLC regulating factors will allow for the second objective which is the analysis of MHC I presented antigens during HCMV infection after repairing PLC assembly and function. This will be informative for the evaluation of the actual escape of CD8+ T cell recognition of HCMV antigens and HCMV vaccine design.