HSV-1 Encoded MicroRNAs in the Pathogenesis and Treatment of Ocular Herpes

单纯疱疹病毒-1（HSV-1）是一种高度传播的感染，在世界各地很常见和流行。类似 与其他人类疱疹病毒（HHV）相比，HSV-1在宿主体内维持终身潜伏期，需要通过以下途径进行免疫逃避： 各种复杂的机制。尽管HSV-1具有大量（>80）的蛋白质编码基因， 潜伏期病毒基因表达的普遍接受的表现是非编码区的积累， 转录物和一组microRNA（miR）。HSV-1编码的病毒miRNAs（v-miRs）被证明可以控制 病毒和宿主转录物，并调节病毒的向性，裂解开关，免疫颠覆等，而多项研究 尽管已经检查了各种细胞系中的HSV-1谱，但这些v-miR的关键生物学功能仍然未知。因此，我们认为， 我们建议评估：（1）疾病进展和再激活过程中v-miR的系统表达动力学，（2） 通过干扰免疫细胞功能在发病机制中的综合作用，和（3）通过免疫细胞功能调节v-miR的治疗靶向， 合成寡核苷酸以减轻HSV-1介导的眼部疱疹。使用我们建立的小鼠眼 为了预防疱疹，我们将比较原发和再活化小鼠角膜组织和血液中的HSV-1 miRNA谱。识别 正和负调节v-miR可以产生对宿主-病毒相互作用的新见解。获得的信息将 用于设计治疗性v-miR抑制剂以沉默候选v-miR功能。合成的影响 靶向候选v-miR的寡核苷酸（v-miR抑制剂）将在小鼠中评估眼部疾病进展。 模型V-miR抑制剂（单独或组合）将被局部递送，并且病毒释放，病毒转录物/基因组， 并测量疾病严重程度评分。接下来，我们将评估v-miR如何使宿主免疫系统 功能障碍，HSV-1持久性所需的整体特征。使用v-miR抑制剂，我们将评估免疫 可在体内渗透和恢复功能。免疫细胞亚群将在病毒感染的 使用流式细胞术和单细胞RNA测序使用v-miR抑制剂治疗的动物。此外，成绩单 与抗原加工/呈递途径相关的基因的表达谱，对有效的抗病毒反应至关重要， 将被量化。这将鉴定v-miR可通过其促进眼部免疫逃避的体内机制。 组织中接下来，我们将剖析v-miR介导的抗原调节异常的潜在机制， 通过巨噬细胞和树突细胞的加工/呈递以及T细胞的活化。表达V-miR的骨髓细胞将 评估病毒抗原的摄取和加工以及自体T辅助细胞（CD 4+）和T细胞毒性细胞的活化 (CD8+）细胞。此外，我们将评估v-miR对CD 4 + T细胞极化的影响。生成的数据将 为现有的知识差距提供重要信息。总的来说，拟议的翻译研究的重点是 鉴定v-miR在眼部疾病发病机制中的治疗和机制方面， 免疫细胞反应。

Herpes Simplex Virus-1 (HSV-1), a highly transmissible infection, is common and endemic throughout the world. Similar to other human herpesviruses (HHV), HSV-1 maintains lifelong latency inside host that requires immune evasion through various sophisticated mechanisms. Although HSV-1 is equipped with large repertoire (>80) of protein coding genes, the commonly accepted manifestations of viral gene expression during latency are the accumulation of a noncoding transcript and a set of microRNAs (miR). HSV-1 encoded viral miRNAs (v-miRs) are demonstrated to control expression of both viral and host transcripts and regulate viral tropism, lytic switching, immune subversion, etc. While multiple studies have examined HSV-1 profiles in various cell lines, key biological functions of these v-miRs remain unknown. Therefore, we propose to evaluate: (1) systematic expression dynamics of v-miRs during disease progression and reactivation, (2) comprehensive role in the pathogenesis by perturbing immune cells functions and (3) therapeutic targeting of v-miR by synthetic oligonucleotides to mitigate HSV-1-mediated ocular herpes. Using our established mouse model of ocular herpes, we will compare HSV-1 miRNA profiles in primary and reactivated mice corneal tissues and blood. Identifying positive and negative regulatory v-miRs can yield novel insights into host-virus interaction. The information gained will be used in designing therapeutic v-miR Inhibitors to silence candidate v-miRs functions. Effect of synthetic oligonucleotides (v-miR Inhibitors) targeting candidate v-miRs will be assessed on ocular disease progression in a mice model. V-miR inhibitors (alone or in combination) will be topically delivered and virus release, viral transcript/genome, and disease severity score will be measured. Next, we will evaluate how v-miRs can render host immune system dysfunctional, an integral feature required for HSV-1 persistence. Using v-miR inhibitors, we will assess whether immune infiltration and functions can be restored in vivo. Immune cell subsets will be comprehensively profiled in virus-infected animals treated with v-miR inhibitor using flow cytometry and single cell RNA sequencing. In addition, transcript expression profiles of genes related to antigen processing/presentation pathway, critical for potent antiviral response, will be quantified. This will identify the in vivo mechanisms through which v-miRs can facilitate immune evasion in ocular tissues. Next, we will dissect underlying mechanisms of v-miR-mediated dysregulation of antigen processing/presentation by macrophages and dendritic cells and activation of T cells. V-miR expressing myeloid cells will be assessed for uptake and processing of viral antigens and activation of autologous T helper (CD4+) and T cytotoxic (CD8+) cells. In addition, we will assess the impact of v-miRs on the polarization of CD4+ T cells. The data generated will provide significant information to existing knowledge gaps. Overall, the proposed translational study focuses on identifying the therapeutic and mechanistic aspect of v-miRs in ocular disease pathogenesis through modulation of immune cell responses.