LAT-HVEM Interactions Effect HSV-1 Latency/Reactivation

描述(申请人提供)：在美国，单纯疱疹病毒1型(HSV-1)是由感染性因素引起的最常见的角膜失明原因，也是免疫能力正常的人中最常见的散发性致死性脑炎的原因。大多数单纯疱疹病毒1型疾病是由于病毒重新激活，而不是原发的急性感染。因此，了解HSV-1潜伏期再激活对于开发抗击HSV-1疾病的方法具有重要意义。HSV-1使用疱疹病毒进入介质或HSEM作为感染细胞的一种途径。HVEM是肿瘤坏死因子受体超家族的一员，通过在促炎信号和抑制信号之间发挥分子开关的作用来调节免疫反应。当神经元上的HVEM与T细胞上的BTLA结合时，双向通路被激活。激活的HVEM激活了核因子-kB，而核因子-kB通过减少凋亡来促进神经细胞的存活。激活的BTLA限制T细胞的激活。因此，增加HVEM应该会阻止细胞凋亡并降低T细胞功能--这些活动归因于HSV-1 LAT基因，并被认为与LAT如何增强潜伏期/再激活有关(尽管LAT阻止细胞凋亡和降低T细胞反应的机制(S)尚不清楚)。我们的初步和最近发表的结果表明：1)HSV-1潜伏期和重新激活在hvem-/-小鼠中都显著减少；2)两个小的非编码LAT RNA(SNcRNAs)可以上调hvem在神经元上的表达。基于这些发现，我们推测LAT增强潜伏期和重新激活的一个机制是通过上调HSV-1潜伏期和重新激活，从而通过减少细胞凋亡和局部T细胞反应来促进HSV-1潜伏期和重新激活。我们的具体目标是：1.构建一个对两个LAT SNcRNAs(SNcRNA1和SncRNA2)都具有KO的HSV-1双基因敲除突变体(DsncRNA1&2)。我们预计该突变体将不再增加hvem的表达，并将具有类似于LAT(-)突变体的缩短潜伏期/重新激活。这将有力地支持这样的假设，即两个LAT SNcRNAs通常上调hvem，进而减少细胞凋亡和T细胞反应，导致潜伏期/重新激活增加。2.构建并分析了在LAT(-)基因组背景上表达由LAT启动子驱动的HSV-1突变体(DLAT-HVEM)。我们期望这个突变体表达的hvem能够恢复wt LAT(+)样的潜伏期/对LAT(-)病毒的重新激活。这将有力地支持这样的假设，即增加hvem的表达是LAT增强潜伏期/重新激活的重要机制。我们期望成功完成这一探索性的R21应用将导致RO1拨款，涉及LAT上调HVEM的机制，并开发治疗干预措施来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.