The Mechanisms of cross-presentation of a long-lived viral protein

一些病毒衍生的蛋白在感染细胞中稳定且长期存活，但仍能产生细胞毒性T淋巴细胞(CTL)的免疫优势T细胞表位。最近，我们解决了淋巴细胞性脉络膜脑膜炎病毒(LCMV)核蛋白(NP)的这个谜团，并证明NP118表位只能呈现在H-2LD I类分子上，只要NP是从头合成的，而不能通过预先存在的、长期存在的、成熟的NP的降解来维持。在这个项目中，我们想要研究像LCMV-NP这样的长寿蛋白如何被巨噬细胞和树突状细胞交叉呈递。我们最近发现，对于交叉呈现，LCMV-NP需要成熟的NP蛋白以及HSP90家族伴侣的活性。我们将调查，在交叉呈现的哪个步骤需要HSP90活性，HSP90和NP是否相互作用，以及哪些其他伴侣和蛋白酶参与了LCMV-NP的交叉呈现。有趣的是，坏死性抗原供体细胞的上清液中含有NP交叉呈递所必需的不稳定因子。该因子将通过质谱学进行纯化、表征和鉴定。通过对包被LCMV-NP的微粒进行紫外光交联，我们将试图在巨噬细胞和树突状细胞吞噬LCMV-NP后，寻找参与LCMV-NP加工的新蛋白质。

Several virus-derived proteins are stable and long-lived in infected cells and nevertheless give rise to immunodominant T cell epitopes of cytotoxic T lymphocytes (CTL). Recently we have addressed this enigma for the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) and demonstrated that the NP118 epitope can only be presented on H-2Ld class I molecules as long as the NP is synthesized de novo but can not be sustained from degradation of pre-existing and long-lived, mature NP. In this project we would like to investigate how a very long-lived protein like LCMV-NP can be cross-presented by macrophages and dendritic cells. We have recently shown that for cross-presentation the mature NP protein as well as the activity of HSP90 family chaperones are required for LCMV-NP cross-presentation. We will investigate, at what step in cross-presentation HSP90 activity is required, whether HSP90 and NP interact, and what other chaperones and proteases are involved in the cross-presentation of LCMV-NP. Interestingly, the supernatant of necrotic antigen donor cells contains a heat labile factor that is required for NP cross-presentation. This factor will be purified, characterized, and identified by mass-spectrometry. By UV-based cross-linking of LCMV-NP coated microparticles, we will try to identify new proteins that are involved in LCMV-NP processing after endocytosis by macrophages and dendritic cells.