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

描述（由申请人提供）：人巨细胞病毒是一种重要的人类病原体，在髓系未分化细胞中建立终身潜伏库，部分原因是当病毒进入这些细胞时，抑制病毒直接早期（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基因表达的主要调控因子，但这个关键的转录因子是如何起作用的仍然是个谜。在本次资助的最后一个资助期，我们确定了pp71激活IE基因表达的机制。从病毒粒子被皮传递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，在pp71功能之前或缺失时，该蛋白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，随后进行裂解复制。在上一个资助期内，我们进一步发现Daxx内在防御在潜伏期的建立过程中也抑制IE基因的表达，并且在这种情况下不会被pp71灭活。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，募集修饰蛋白到目标启动子，以及将组蛋白变体H3.3沉积到非复制DNA上。在Aim 1中，我们建议破译Daxx的每个个体活性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。在裂解性感染的成纤维细胞中，Daxx内在防御很快被

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.