Targeting Nuclear HSF1 as a Novel Anti-HCMV Strategy

项目摘要 人巨细胞病毒（HCMV）感染世界上大多数人，并可导致严重的疾病， 免疫功能低下的患者和新生儿。该病毒在骨髓细胞中建立终身潜伏期， 在静止感染的单核细胞中传播到外周器官。抗病毒治疗延缓病毒复制， 但不能清除感染的细胞。病毒反弹、耐药性和药物毒性使治疗复杂化， 产生了对改进的治疗方法的强烈需求。我们主张抑制HCMV复制必须 与杀死受感染的单核细胞相结合。我们发现HCMV感染的成纤维细胞和 单核细胞迅速刺激热休克因子（HSF）1，一种应激反应转录因子， 与热休克（HS）诱导的典型激活不同。使用一种新型的工具化合物， DTHIB，已被证实选择性抑制HSF 1活性，我们发现DTHIB抑制HSF 1 减弱HCMV裂解性复制并刺激潜伏感染的单核细胞死亡。这些研究提供 一项概念验证研究的开始，即HSF 1拮抗剂可能有能力提供双重的 “命中”抑制HCMV复制和消除潜伏感染的骨髓细胞所必需的单一药物。因此，在本发明中， 我们的中心假设是用工具化合物DTHIB抑制HSF 1将限制两种感染 并在受感染的宿主中通过伴随减弱HCMV裂解性复制而传播， 细胞类型和消除潜伏感染的单核细胞。第一个目标将继续评估抗病毒药物 通过检查DTHIB对不同HCMV的药物效力，确定DTHIB作为HCMV裂解性复制抑制剂的潜力 允许的细胞类型、病毒株和感染多样性（MOI）。我们还将进行转录组（RNA 测序）分析和功能研究，以鉴定依赖于HCMV诱导的HSF 1的基因 负责促进裂解性复制的活性和DTHIB对这种HCMV表达的影响- 诱导的，HSF 1依赖的基因谱。第二个目标将继续评估DTHIB刺激 通过测试对感染不同病毒的单核细胞的选择性药物毒性， 病毒株和不同MOI。同时，我们将进行translatome（多聚体分析）分析， 使用DTHIB的功能研究，以确定HSF 1依赖的基因，负责促进生存的 潜伏感染的单核细胞。第三个目的是评估DTHIB对裂解性复制的体内抗病毒活性， 病毒传播和发病机制，使用一种新的小鼠移植模型与人类皮肤器官， 同时监测人组织中HCMV复制以及监测单核细胞介导的HCMV传播 远端部位。

Project Summary Human cytomegalovirus (HCMV) infects the majority of people in the world and can cause serious disease in immunocompromised patients and neonates. The virus establishes life-long latency in bone marrow cells and disseminates to peripheral organs in quiescently infected monocytes. Antiviral therapy delays virus replication, but does not eliminate infected cells. Virus rebound, resistance, and drug toxicity complicate treatment and create a strong demand for improved therapeutics. We advocate that the suppression of HCMV replication must be in combination with the killing of infected monocytes. We found that HCMV infection of fibroblasts and monocytes rapidly stimulated the activity of heat shock factor (HSF) 1, a stress-responsive transcription factor, in a distinct fashion from canonical activation induced by heat shock (HS). Using a novel tool compound called DTHIB, which has been validated to selectively inhibit HSF1 activity, we found inhibition of HSF1 with DTHIB attenuated HCMV lytic replication and stimulated death of latently infected monocytes. These studies provide the beginnings of a proof-of-concept study that HSF1 antagonists may have the capacity to provide the double “hit” necessary to suppress HCMV replication and eliminate latently infected myeloid cells in a single drug. Thus, our central hypothesis is that inhibition of HSF1 with the tool compound DTHIB will limit both infection and spread within an infected host by concomitantly attenuating HCMV lytic replication in permissive cell types and eliminating latently infected monocytes. The first aim will continue to evaluate the antiviral potential of DTHIB as an inhibitor of HCMV lytic replication by examining the drug efficacy on different HCMV permissive cell types, viral strains, and multiplicities of infection (MOIs). We will also conduct transcriptome (RNA sequencing) analyses and functional studies using DTHIB to identify genes dependent on HCMV-induced HSF1 activity responsible for promoting lytic replication and the impact of DTHIB on the expression of this HCMV- induced, HSF1-dependent gene profile. The second aim will continue to assess the ability of DTHIB to stimulate the death of latently infected monocytes by testing the selective drug toxicity on monocytes infected with different viral strains and at different MOIs. In conjunction, we will perform translatome (polysomal profiling) analyses and functional studies using DTHIB to identify HSF1-dependent genes responsible for promoting the survival of latently infected monocytes. The third aim will assess the in vivo antiviral activity of DTHIB on lytic replication, viral spread, and pathogenesis using a novel murine transplant model with human skin organ, which can simultaneously monitor HCMV replication in human tissue as well as monitor monocyte-mediated HCMV spread to distal sites.