Virus-host interactions regulating innate signaling for human cytomegalovirus latency

项目总结 我们的研究计划的目标是阐明巨细胞病毒调节宿主的分子机制。 CD34+造血祖细胞在病毒建立和维持中的信号转导 等待时间和从等待时间重新激活。人巨细胞病毒仍然是固体中毒后发病率和死亡率的重要原因 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。巨细胞病毒 潜伏期很复杂，调节巨细胞病毒建立和维持的信号机制 对于潜伏期以及HCMV重新激活的原因，人们知之甚少。我们已经确定了一个病毒基因的基因座 基因组中协调四个基因UL133、UL135、UL136和 UL138，来自多顺反子转录产物。利用最新的人CD34+造血干细胞体外模型和体内模型 在人源化小鼠的模型中，我们将定义病毒-宿主相互作用调节宿主信号 建立潜伏期。我们的初步数据显示，延迟决定因素UL138与主机相互作用 WD Repeat Containing Protein 48(WDR48)，作为激活泛素特定蛋白酶的支架， USP1、USP12和USP46。WDR48-USP1复合体调节STAT1和AKT信号转导 该UL138引导WDR48-USP1的活性以诱导STAT1的激活。我们进一步证明了USP1 活性对于建立潜伏感染很重要，这样当USP1被抑制时，病毒 在没有复制刺激的情况下进行复制。我们假设UL138相互作用指导WDR48-USP 调节先天信号的复合体，以抑制病毒复制，防止重新激活。在目标1中，我们将 确定UL138如何影响WDR48/USP复合体和功能。我们将绘制UL138中的氨基酸图谱 它们是与WDR48/USP复合体相互作用所必需的，并产生缺陷的重组病毒 这些互动。我们将区分UL138-WDR48/USP1相互作用和UL138-EGFR的作用 互动。此外，我们已经证明UL138至少部分地通过相互作用来维持AKT信号 与EGFR的相互作用，但与USP12和USP46的相互作用可能为调节AKT提供额外的途径。 AIM 2将确定UL138-WDR48/USP相互作用如何影响延迟和重新激活，以及 UL138利用重组病毒激活STAT1和AKT，并敲除宿主因子。这个项目 将提供对主机信号的多方面监管的全面和机械性的见解 控制人巨细胞病毒潜伏期。我们已经确定的病毒和宿主因素网络使我们处于独特的位置 为抗病毒策略定义新的宿主和病毒靶点，以控制HCMV潜伏期或重新激活。

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.