Role of Daxx degradation by pp71 during the Human Cytomegalovirus life cycle

描述(申请人提供)：人类巨细胞病毒是一种重要的人类病原体，它在髓系未分化细胞中建立了一个终身潜伏库，部分原因是当它进入这些细胞时，它抑制了病毒即刻早期(IE)裂解期基因的表达。虽然众所周知，病毒被膜蛋白pp71是IE基因表达的主要调节者，但这种关键的转录因子实际上是如何发挥作用的仍然是个谜。在这笔赠款的最后一个资助期，我们确定了pp71激活IE基因表达的机制。从病毒粒子被膜传递的蛋白质转位到成纤维细胞等分化细胞的核中，并降解Daxx，Daxx是一种细胞转录抑制因子和固有防御蛋白，在pp71功能之前或不存在时，会在转录上沉默感染病毒基因组。通过降解Daxx，pp71激活IE基因的表达，随后发生裂解复制。我们在上一次资助期间进一步表明，在潜伏期的建立过程中，Daxx的内在防御也抑制了IE基因的表达，在这种情况下，pp71不会使其失活。Daxx至少通过三种方式抑制细胞基因表达：阻断细胞转录因子的活性，将修饰蛋白招募到靶向启动子，以及将组蛋白变异体H3.3沉积到非复制型DNA上。我们在目标1中建议破译Daxx的每个个体活动如何有助于其在裂解性和潜伏性感染开始时沉默病毒IE基因的表达。在裂解感染的成纤维细胞中，Daxx的内在防御很快就被 Pp71，但在潜伏感染的未分化细胞中保持活性。在上一个资助期，我们 结果表明，当潜伏期确定时，Daxx并没有被灭活，因为被皮递送的pp71仍然留在细胞质中。从广义上讲，这意味着进入分化和未分化细胞的过程必须至少有一些差异。具体地说，被膜传递蛋白的亚细胞定位是不同的。在上一个资助期，我们使用分化和未分化细胞之间的异源融合来表明，分化细胞表达一种允许在未分化细胞中转运被膜的pp71的因子。在目标2中，我们建议定义HCMV感染未分化细胞并建立潜伏期的进入过程，并确定在分化细胞中发现的允许被膜递送的pp71进入细胞核的因素。

DESCRIPTION (provided by applicant): Human cytomegalovirus is a significant human pathogen that establishes a life- long latent reservoir in undifferentiated cells of the myeloid lineage in part by suppressing viral immediate early (IE) lytic phase gene expression when it enters these cells. While it was known for some time that the viral tegument protein pp71 was the master regulator of IE gene expression, how this critical transcription factor actually worked remained enigmatic. In the last funding period of this grant, we determined the mechanism through which pp71 activates IE gene expression. The protein delivered from the virion tegument translocates to the nucleus of differentiated cells such as fibroblasts and degrades Daxx, a cellular transcriptional repressor and intrinsic defense protein that, prior to or in the absence of pp71 function, transcriptionally silences infecting viral genomes. By degrading Daxx, pp71 activates IE gene expression and lytic replication ensues. We further showed during the last funding period that the Daxx intrinsic defense also represses IE gene expression during the establishment of latency, and in this context is not inactivated by pp71. Daxx represses cellular gene expression in at least three ways: blocking the activity of cellular transcription factors, recruiting modifying proteins to targeted promoters, and depositing histone variant H3.3 onto non- replicating DNA. We propose in Aim 1 to decipher how each individual activity of Daxx contributes to its ability to silence viral IE gene expression at the start of both lytic and laten infections. In lytically infected fibroblasts, the Daxx intrinsic defense is quickly inactivated by pp71, but remains active in latently infected undifferentiated cells. In the last funding period we showed that Daxx is not inactivated when latency is established because tegument-delivered pp71 remains in the cytoplasm. In broad terms, this means that the entry processes into differentiated and undifferentiated cells must have at least some differences. Specifically, the subcellular localization of tegument-delivered proteins is different. In the last funding period, w used heterologous fusions between differentiated and undifferentiated cells to show that differentiated cells express a factor that permits the nuclear trafficking of tegument delivered pp71 in undifferentiated cells. We propose in Aim 2 to define the entry process HCMV uses to infect undifferentiated cells and establish latency, and to identify the factor found in differentiated cells that permits tegument-delivered pp71 access to the nucleus.