LAT-HVEM Interactions Effect HSV-1 Latency/Reactivation

描述（由申请方提供）：在美国，单纯疱疹病毒1型（HSV-1）是由感染因子引起的角膜失明的最常见原因，也是免疫功能正常个体中散发性致死性脑炎的最常见原因。大多数HSV-1疾病是由于病毒再激活，而不是原发性急性感染。因此，了解HSV-1潜伏-再激活对于开发对抗HSV-1疾病的方法是重要的。HSV-1使用疱疹病毒进入介质（HVEM）作为感染细胞的一种途径。HVEM是TNF受体超家族的成员，可以通过充当促炎和抑制信号之间的分子开关来调节免疫应答。当神经元上的HVEM与T细胞上的BTLA结合时，双向途径被激活。激活的HVEM激活NF-kB，其通过减少凋亡促进神经元细胞存活。活化的BTLA限制T细胞活化。因此，增加HVEM应该阻断细胞凋亡并降低T细胞功能-与HSV-1 LAT基因的活性相同，并且被认为参与LAT如何增强潜伏期/再激活（尽管LAT阻断细胞凋亡并降低T细胞应答的机制尚不清楚）。我们的初步和最近发表的结果表明：1）HSV-1潜伏期和再激活在HVEM-/-小鼠中均显著降低，2）两种小的非编码LAT RNA（sncRNA）可以上调神经元上的HVEM。基于这些发现，我们假设LAT增强潜伏期和再激活的一种机制是通过上调HVEM，HVEM反过来通过减少细胞凋亡和局部T细胞应答来促进HSV-1潜伏期和再激活。我们的具体目标是：1。构建HSV-1双敲除（KO）突变体（DsncRNA 1和2），其对于两种LAT sncRNA（sncRNA 1和sncRNA 2）都是KO的。我们预计该突变体将不再增加HVEM表达，并且与LAT（-）突变体类似，潜伏期/再激活将减少。这将强烈支持以下假设：两种LAT sncRNA通常上调HVEM，这进而降低细胞凋亡和T细胞应答，导致增加的潜伏期/再活化。2.构建并分析在LAT（-）基因组背景上表达由LAT启动子驱动的HVEM的HSV-1突变体（DLAT-HVEM）。我们期望由该突变体表达的HVEM恢复LAT（-）病毒的wt LAT（+）样潜伏/再激活。这将强烈支持这样的假设，即增加HVEM表达是LAT增强潜伏期/再激活的重要机制。我们预计成功完成这项探索性R21申请将获得RO 1资助，涉及HVEM LAT上调机制和开发治疗干预措施以阻断这种上调或阻断双向HVEM通路的组成部分。

DESCRIPTION (provided by applicant): In the US, herpes simplex virus type 1 (HSV-1) is the most common cause of corneal blindness due to an infectious agent and the most common cause of sporadic lethal encephalitis in immune competent individuals. Most HSV-1 disease is due to viral reactivations rather than the primary acute infection. Thus understanding HSV-1 latency-reactivation is important for developing methods to combat HSV-1 diseases. HSV-1 uses herpes virus entry mediator, or HVEM, as one route of infecting cells. HVEM is a member of the TNF receptor superfamily and can regulate immune responses by acting as a molecular switch between proinflammatory and inhibitory signaling. When HVEM on a neuron binds to BTLA on a T cell, a bidirectional pathway is activated. Activated HVEM activates NF-kB which promotes neuronal cell survival by decreasing apoptosis. Activated BTLA limits T cell activation. Thus, increasing HVEM should block apoptosis and decrease T cell function - the same activities attributed to the HSV-1 LAT gene and that are thought to be involved in how LAT enhances latency/reactivation (although the mechanism(s) by which LAT blocks apoptosis and decreases T cell response are not known). Our preliminary and recently published results indicate that: 1)HSV-1 latency and reactivation are both significantly reduced in HVEM-/- mice and 2)Two small non-coding LAT RNAs (sncRNAs) can upregulate HVEM on neurons. Based on these findings we hypothesize that one mechanism by which LAT enhances latency and reactivation is by upregulating HVEM which in turn promotes HSV-1 latency and reactivation by decreasing both apoptosis and the local T cell response. Our Specific Aims are: 1. Construct an HSV-1 double knockout (KO) mutant (DsncRNA1&2) that is KO'd for both LAT sncRNAs (sncRNA1 & sncRNA2). We expect that this mutant will no longer increase HVEM expression and will have reduced latency/reactivation similar to LAT(-) mutants. This would strongly support the hypothesis that the two LAT sncRNAs normally upregulate HVEM which in turn decreases apoptosis and T cell responses resulting in increased latency/reactivation. 2. Construct and analyze an HSV-1 mutant (DLAT-HVEM) that expresses HVEM driven by the LAT promoter on a LAT(-) genomic background. We expect the HVEM expressed by this mutant to restore wt LAT(+)- like latency/reactivation to the LAT(-) virus. This would strongly support the hypothesis that increasing HVEM expression is an important mechanism by which LAT enhances latency/reactivation. We expect successful completion of this exploratory R21 application to lead to an RO1 grant involving the mechanism of LAT upregulation of HVEM and the development of therapeutic interventions to block this upregulation or block components of the bidirectional HVEM pathway.