Human endogenous retrovirus superantigen HERV-K18

描述(申请人提供)：人类内源性逆转录病毒(HERV)K18被发现编码超抗原(SAG)，其特征是能够激活带有特定TCRVP元件的T细胞。与具有良好特征的细菌下垂相比，对病毒下垂的了解较少。HERV凹陷可能是建立EB病毒长期持续感染和L糖尿病发病机制的重要因素。K18SAG代表了TCRs的一种独特的配体类型，既不同于传统的多肽抗原，也不同于其他已知的SAG。这些HERV凹陷通常处于休眠状态，可以被干扰素-α和EBV转录激活。与其他仅激活特定T细胞亚群的SAGS相比，EBV激活的Herv-K18 SAGS最初以Vp13特异性方式刺激T细胞，但随后快速多克隆激活，其分子机制尚不清楚。细菌SAG-受体复合体的晶体结构表明，每个SAG与宿主受体有明显的结合。我们假设Herv SAG与细菌凹陷的相互作用不同，和/或具有不同的宿主受体结合动力学。该应用的长期目标是确定Herv凹陷的三维结构及其与TCR和MHC分子的相互作用。这将需要获得相对较高数量的可溶性病毒唾液。然而，生产可溶的和功能性的病毒凹陷一直是一个具有挑战性的问题。因此，我们建议进行以下研究：目标1：建立一种使用GFP折叠报告系统高效表达HERV-K18 SAGS的策略，其中GFP荧光提供关于融合靶蛋白折叠成功或失败的信息；目标2.HERV-K18 SAGS及其与TCR和II类MHC分子的可溶变异体的功能特性和结晶。第一轮突变已经产生了几个具有增强荧光的K18 SAG变体，这表明溶解性有所改善。此R21应用程序旨在为后续的R01应用程序奠定基础。当前应用程序的探索/开发性质与R21机制一致。对这些HERV下垂及其与宿主受体的复合体的详细结构和功能特征将有助于我们了解病毒下垂是如何发挥其生物学功能的，并可能揭示我们如何设法在不受伤害的情况下与敌人睡觉。

DESCRIPTION (provided by applicant): Human endogenous retrovirus (HERV) K18 was found to encode superantigens (SAgs) which are characterized by the ability to activate T-cells bearing particular TCR Vp elements. Compared to the well characterized bacterial SAgs, viral SAgs are less understood. The HERV SAgs may be essential for establishing the long-term persistent infection of Epstein-Barr virus (EBV) and for the pathogenesis of type-l diabetes. The K18 SAg represents a unique type of ligand for TCRs, distinct from both conventional peptide antigens and other known SAgs. These HERV SAgs are normally dormant and can be transcriptionally activated by interferon-alpha and by EBV. Compared to other SAgs which only activate specific subsets of T cells, the EBV-activated HERV-K18 SAgs stimulates T cells initially in a Vp13-specific manner, but followed by a rapid polyclonal activation, for which the molecular mechanism is unknown. The crystal structures of bacterial SAgs-receptor complexes revealed that each SAg binds distinctly to the host receptors. We hypothesize that the HERV SAg interacts differently than, and/or has host receptor binding kinetics different from those of, the bacterial SAgs. The long-term objective of the application is to determine the three-dimensional structures of the HERV SAgs and their interactions with TCR and MHC molecules. This will require the availability of relatively high quantities of soluble viral SAgs. However, production of soluble and functional viral SAgs has been a challenging problem. Therefore, we propose to perform the following studies: Aim 1: Development of a highly efficient strategy to express soluble HERV-K18 SAgs using a GFP folding reporter system, in which the GFP fluorescence provides information regarding the folding success or failure of the fusion target protein; Aim 2. Functional characterization and crystallization of soluble variants of the HERV-K18 SAgs and their complexes with TCR and class II MHC molecules. A first round of mutagenesis has generated several K18 SAg variants with enhanced fluorescence, suggesting improved solubility. This R21 application is intended to lay the ground work for a subsequent R01 application. The exploratory/development nature of the current application is consistent with the R21 mechanism. A detailed structural and functional characterization of these HERV SAgs and their complexes with the host receptors will help us understand how viral SAgs perform their biological functions and may shed light on how we manage to sleep with the enemy without getting hurt.