LAT-HVEM Interactions Effect HSV-1 Latency/Reactivation

描述（由申请人提供）：在美国，单纯疱疹病毒1型（HSV-1）是由感染性病原体引起的角膜失明的最常见原因，也是免疫正常个体中散发致命性脑炎的最常见原因。大多数1型单纯疱疹病毒疾病是由于病毒再激活而不是原发急性感染。因此，了解1型单纯疱疹病毒的潜伏-再激活对于开发对抗1型单纯疱疹病毒疾病的方法非常重要。HSV-1使用疱疹病毒进入介质（HVEM）作为感染细胞的途径之一。HVEM是TNF受体超家族的一员，可以作为促炎和抑制信号传导之间的分子开关来调节免疫反应。当神经元上的HVEM与T细胞上的BTLA结合时，一个双向通路被激活。活化的HVEM激活NF-kB，通过减少细胞凋亡促进神经元细胞存活。活化的BTLA限制T细胞的活化。因此，HVEM的增加应该会阻止细胞凋亡并降低T细胞的功能——与HSV-1 LAT基因相同的活性，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细胞反应的机制尚不清楚）。我们的初步和最近发表的研究结果表明：1)HSV-1的潜伏期和再激活在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反过来通过减少凋亡和局部T细胞反应来促进HSV-1的潜伏期和再激活。我们的具体目标是：1；构建HSV-1双敲除（KO）突变体（DsncRNA1&2），该突变体对两个LAT sncrna （sncRNA1 & sncRNA2）均KO。我们预计该突变体将不再增加HVEM的表达，并且将减少与LAT（-）突变体相似的潜伏期/再激活。这有力地支持了两个LAT sncrna正常上调HVEM的假设，这反过来又减少了细胞凋亡和T细胞反应，从而增加了潜伏期/再激活。2. 构建并分析在LAT（-）基因组背景下表达由LAT启动子驱动的HVEM的HSV-1突变体（DLAT-HVEM）。我们期望该突变体表达的HVEM能够恢复LAT（+）样的潜伏期/ LAT（-）病毒的再激活。这将有力地支持增加HVEM表达是LAT增强延迟/再激活的重要机制的假设。我们期望成功完成这一探索性R21申请，从而获得涉及LAT上调HVEM机制的RO1资助，并开发治疗性干预措施来阻断这种上调或阻断HVEM双向通路的成分。

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.