Targeting Nuclear HSF1 as a Novel Anti-HCMV Strategy

项目摘要 人类巨细胞病毒(HCMV)感染世界上大多数人，并可在2004年导致严重疾病 免疫功能低下的患者和新生儿。该病毒在骨髓细胞中建立了终身潜伏期，并 在静止期感染的单核细胞中传播到外周器官。抗病毒治疗延缓了病毒复制， 但不能消除受感染的细胞。病毒反弹、耐药性和药物毒性使治疗和 创造了对改进疗法的强烈需求。我们主张抑制人巨细胞病毒复制必须 与杀灭受感染的单核细胞相结合。我们发现，人巨细胞病毒感染成纤维细胞和 单核细胞迅速刺激热休克因子(HSF)1的活性，HSF 1是一种应激反应转录因子， 与热休克(HS)诱导的典型激活方式截然不同。使用一种名为 DTHIB已被证实可以选择性地抑制HSF1的活性，我们发现DTHIB对HSF1有抑制作用 抑制HCMV裂解复制，刺激潜伏感染单核细胞死亡。这些研究提供了 一项概念验证研究的开始，即HSF1拮抗剂可能有能力提供双重 在单一药物中抑制HCMV复制和消除潜伏感染的髓系细胞所必需的“HIT”。因此， 我们的中心假设是，用工具化合物DTHIB抑制HSF1将限制这两种感染 并通过同时减弱允许的HCMV裂解复制在受感染的宿主内传播 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 DTHIB对不同类型人巨细胞病毒裂解复制的抑制作用 允许细胞类型、病毒株和感染的多样性(MOI)。我们还将进行转录组(RNA 利用DTHIB分析和功能研究以确定依赖于HCMV诱导的HSF1的基因 促进裂解复制的活性及DTHIB对该HCMV-1表达的影响 诱导的、依赖于HSF1的基因图谱。第二个目标将继续评估DTHIB的刺激能力 潜伏感染单核细胞对不同病毒感染单核细胞的选择性毒性试验 病毒株和不同的Mois。同时，我们将进行翻译组(多体谱分析)分析和 应用DTHIB进行的功能研究以确定HSF1依赖的基因，这些基因负责促进肿瘤的存活 潜伏感染单核细胞。第三个目标将评估DTHIB在体内对裂解复制的抗病毒活性， 病毒的传播和发病机制使用了一种新的带有人类皮肤器官的小鼠移植模型，它可以 同时监测人巨细胞病毒在人体组织中的复制以及单核细胞介导的人巨细胞病毒传播 到远端部位。

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.