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

描述（由申请方提供）：人巨细胞病毒是一种重要的人类病原体，其在髓系未分化细胞中建立终身潜伏储库，部分原因是当病毒进入这些细胞时抑制病毒立即早期（IE）裂解期基因表达。虽然一段时间以来人们都知道病毒被膜蛋白pp71是IE基因表达的主要调节因子，但这个关键的转录因子实际上是如何工作的仍然是个谜。在该资助的最后一个资助期内，我们确定了pp71激活IE基因表达的机制。从病毒体被膜递送的蛋白质易位到分化细胞如成纤维细胞的细胞核并降解Daxx，Daxx是一种细胞转录抑制因子和内在防御蛋白，其在pp71功能之前或不存在时，在转录上沉默感染病毒基因组。通过降解Daxx，pp71激活IE基因表达和裂解性复制增强。我们进一步表明，在最后一个资助期间，Daxx内在防御也抑制IE基因的表达，在建立潜伏期，并在这种情况下，是不是由pp71失活。Daxx以至少三种方式抑制细胞基因表达：阻断细胞转录因子的活性、将修饰蛋白募集至靶向启动子以及将组蛋白变体H3.3沉积到非复制DNA上。我们在目标1中提出，要破译Daxx的每一个单独的活动如何有助于其在裂解和潜伏感染开始时沉默病毒IE基因表达的能力。在裂解感染的成纤维细胞中，Daxx内在防御被以下因素迅速灭活： pp71，但在潜伏感染的未分化细胞中保持活性。在上一个融资期， 表明当潜伏期建立时，Daxx没有失活，因为被膜递送的pp71保留在细胞质中。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。具体而言，被膜递送蛋白的亚细胞定位是不同的。在最后一个资助期，w使用分化和未分化细胞之间的异源融合来显示分化细胞表达允许未分化细胞中被膜递送pp71的核运输的因子。我们在目标2中提出定义HCMV用于感染未分化细胞并建立潜伏期的进入过程，并鉴定在分化细胞中发现的允许被膜递送的pp71进入细胞核的因子。

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.