Virus-host interactions regulating innate signaling for human cytomegalovirus latency

项目摘要 我们的研究目标是阐明HCMV调节宿主的分子机制 CD 34+造血祖细胞（HPC）中用于建立和维持病毒免疫的信号传导 潜伏期和从潜伏期再激活。HCMV仍然是固体疫苗接种后发病和死亡的重要原因。 器官和造血干细胞移植，尽管在诊断和治疗方面取得了进展。HCMV 潜伏期是复杂的，调节HCMV的建立和维持的信号机制 潜伏期以及HCMV的再活化知之甚少。我们已经确定了一个病毒基因位点， 基因组的ULb'区，其协调四种基因UL 133、UL 135、UL 136和UL 137的表达， UL 138，来自多顺反子转录物。在人CD 34 + HPC和体内使用最先进的体外模型 在人源化小鼠模型中，我们将定义病毒-宿主相互作用调节宿主信号传导， 建立潜伏期。我们的初步数据表明，潜伏期决定因子UL 138与宿主相互作用， 含有WD重复序列的蛋白48（WDR 48），其充当活化泛素特异性蛋白酶的支架， USP 1、USP 12和USP 46。WDR 48-USP 1复合物调节STAT 1和AKT信号传导，我们证明了 UL 138通过介导WDR 48-USP 1的活性诱导STAT 1的活化。我们进一步表明，USP 1 活性对于潜伏感染的建立很重要，因此当USP 1被抑制时，病毒 在没有复制刺激的情况下复制。我们假设UL 138相互作用指导WDR 48-USP 复合物调节先天信号传导以抑制病毒复制以防止再激活。在目标1中，我们 确定UL 138如何影响WDR 48/USP复合物和功能。我们将绘制UL 138中的氨基酸 与WDR 48/USP复合物相互作用所需的，并产生缺陷的重组病毒， 这些互动。我们将区分UL 138-WDR 48/USP 1相互作用与UL 138-EGFR相互作用的作用 交互.此外，我们已经表明，UL 138维持AKT信号转导，至少部分通过相互作用 与EGFR的相互作用，但与USP 12和USP 46的相互作用可能为AKT的调节提供额外的途径。 目标2将确定UL 138-WDR 48/USP相互作用如何影响延迟和再激活，以及 UL 138在使用重组病毒激活STAT 1和AKT和敲低宿主因子中的作用。这个项目 将提供全面的和机制的见解，多方面的调节宿主信号， 控制HCMV潜伏期。我们已经确定的病毒和宿主因子网络使我们能够 为控制HCMV潜伏期或再激活的抗病毒策略定义新的宿主和病毒靶点。

PROJECT SUMMARY The goal of our research program is to elucidate molecular mechanisms by which HCMV regulates host signaling in CD34+ hematopoietic progenitor cells (HPCs) for the establishment and maintenance of viral latency and reactivation from latency. HCMV remains a significant cause of morbidity and mortality after solid organ and hematopoietic stem cell transplantation despite advances in diagnostics and therapeutics. HCMV latency is complex and the signaling mechanisms regulating the establishment and maintenance of HCMV latency, as well as for reactivation of HCMV, are poorly understood. We have identified a locus of viral genes in the ULb’ region of the genome that coordinates the expression of four genes, UL133, UL135, UL136, and UL138, from polycistronic transcripts. Using state-of-the art in vitro models in human CD34+ HPCs and in vivo models in humanized mice, we will define virus-host interactions modulating host signaling for the establishment of latency. Our preliminary data show that the latency determinant, UL138, interacts with host WD Repeat containing protein 48 (WDR48), which serves as a scaffold to activate ubiquitin specific protease, USP1, USP12, and USP46. WDR48-USP1 complexes regulate STAT1 and AKT signaling and we demonstrate that UL138 directs the activity of WDR48-USP1 to induce the activation of STAT1. We further show that USP1 activity it important for the establishment of a latent infection, such that when USP1 is inhibited, the virus replicates in the absence of a replication stimulus. We hypothesize that UL138 interaction directs WDR48-USP complexes to regulate innate signaling to suppress virus replication to prevent reactivation. In Aim 1, we will determine how UL138 impacts WDR48/USP complexes and function. We will map the amino acids in UL138 that are required for interaction with WDR48/USP complexes and generate recombinant viruses defective for these interactions. We will distinguish roles of the UL138-WDR48/USP1 interactions from that of UL138-EGFR interactions. Further, we have shown that UL138 sustains AKT signaling, at least in part through an interaction with EGFR, but interaction with USP12 and USP46 may provide additional avenues to the regulation of AKT. Aim 2 will determine how UL138-WDR48/USP interactions impact latency and reactivation and the role of UL138 in activating STAT1 and AKT using recombinant viruses and knockdown of host factors. This project will provide the comprehensive and mechanistic insights into the multi-faceted regulation of host signaling for the control of HCMV latency. The network of viral and host factors we have identified uniquely position us to define novel host and viral targets for antiviral strategies to control HCMV latency or reactivation.