Mechanisms of Inactivation of T Cells by HSV

描述（由申请方提供）：疱疹病毒已经进化出多种机制来应对宿主免疫反应造成的威胁。其中之一，我们称之为“失活”，与HSV感染的细胞物理接触的T细胞在其通过T细胞受体（TcR）对刺激做出反应的能力方面出现了严重缺陷。失活的T细胞不执行颗粒介导的细胞毒性，也不分泌细胞因子后TcR刺激。我们最近已经证明了失活的T细胞内的两个信号传导缺陷，一个与HSP 90的磷酸化有关，一个与用于活化T细胞的连接子（LAT）的去磷酸化有关。然而，HSV感染的细胞诱导失活的机制尚不清楚。为解决这一问题，我们提出以下建议。具体目标1。确定缺陷HSV颗粒是否需要递送病毒蛋白以使病毒失活。基于我们的初步数据，最可能的假设是通过缺乏DNA的缺陷病毒颗粒将蛋白质递送至T细胞而发生失活。为了评估这一点，我们将使用由大量包装在缺陷颗粒内的病毒反式激活因子驱动的报告基因构建体转染的T细胞，以确定病毒蛋白向T细胞中的递送是否与失活相关。接下来，我们将使用纯化的颗粒来确定这些颗粒的结合和融合是否足以诱导失活。具体目标2。确定灭活所需和足够的病毒蛋白。我们将使用HSV被膜和包膜蛋白的缺失突变体来确定哪些病毒蛋白是发生失活所必需的。一旦确定了必要的蛋白质，我们将在T细胞中单独和组合表达这些蛋白质，以确定是否有任何足以介导失活。具体目标3。确定失活刺激的细胞靶点。为了开始了解导致HSP 90磷酸化和LAT去磷酸化的细胞内事件，我们将确定失活信号的初始细胞靶点。我们将通过鉴定与发现在目标2中足够的病毒蛋白相互作用的细胞蛋白，并通过评估对已知与HSP 90和LAT相互作用或修饰HSP 90和LAT的细胞蛋白的灭活效果来实现这一点。阐明HSV灭活T细胞的机制将为T细胞的调节提供新的见解，并可能为药物操作提供靶点。

DESCRIPTION (provided by applicant): The herpesviruses have evolved a variety of mechanisms to deal with the threat posed by the host immune response. In one of these, which we refer to as "inactivation", T cells that come in physical contact with HSV-infected cells develop a profound defect in their ability to respond to stimulation via the T cell receptor (TcR). Inactivated T cells do not perform granule-mediated cytotoxity, nor do they secrete cytokines after TcR stimulation. We have recently demonstrated two signaling defects within inactivated T cells, one related to the phosphorylation of HSP90, and one related to the dephosphoryation of linker for activation of T cells (LAT). However, the mechanism by which HSV-infected cells induce inactivation is not clear. To address this, we propose the following. Specific Aim 1. Determine whether delivery of viral protein by defective HSV particles is necessary for inactivation to occur. Based on our preliminary data, the most likely hypothesis is that inactivation occurs via delivery of protein to the T cells by defective viral particles lacking DNA. To evaluate this, we will use T cells transfected with a reporter construct driven by a viral transactivator abundantly packaged within the defective particles, to determine whether delivery of viral protein into T cells correlates with inactivation. Next, we will use purified particles to determine whether binding and fusion of these particles are sufficient to induce inactivation. Specific Aim 2. Determine the viral proteins necessary and sufficient for inactivation. We will use deletion mutants of HSV tegument and envelope proteins to determine which viral proteins are necessary for inactivation to occur. Once the necessary proteins are identified, we will express these proteins individually and in combination in T cells to determine whether any are sufficient to mediate inactivation. Specific Aim 3. Determine the cellular target for the inactivating stimulus. To begin to understand the intracellular events leading to phosphorylation of HSP90 and dephosphorylation of LAT, we will determine the initial cellular target for the inactivating signal. We will do this by identifying the cellular proteins interacting with the viral proteins found to be sufficient in aim 2, and by evaluating the effect of inactivation on cellular proteins known to interact with or modify HSP90 and LAT. The elucidaton of the mechanism by which HSV inactivates T cells will provide new insight into the regulation of T cells, and may suggest targets for pharmacologic manipulation.