HCMV miRNA Regulation of Secretion and Formation of the Viral Assembly Compartment

 描述（由申请方提供）：原代细胞的人巨细胞病毒（HCMV）感染导致正常细胞代谢和细胞途径调节的大量改变，这些细胞途径控制细胞死亡、核酸复制、细胞膜稳态、细胞骨架完整性和蛋白质的细胞内运输。许多研究已经证明了HCMV感染后细胞基因转录的广泛失调，认为细胞表型的主要变化是在转录水平上控制的。然而，对基本和高度协调的细胞功能（如蛋白质转运和膜稳态）的粗略调节可能导致细胞环境也会限制感染性病毒的产生。我们最近的研究表明，HCMV miRNA通过靶向编码这些途径的关键组分的基因来调节感染期间的分泌/内吞途径，从而提供了HCMV对细胞功能的另一层调节。HCMV miRNA对这些途径的调节导致抑制促炎细胞因子的释放和细胞内膜的重组，以促进感染性病毒的有效产生。在本项目中，HCMV miRNAs靶向的分泌/内吞途径中的特定基因将通过PAR-CLIP方法与信息学相结合的综合方法进行鉴定，然后进行严格的验证。首先，将确定经验证的miRNA靶标在内吞和分泌途径中的运输调节中的作用，然后研究这些miRNA靶标在细胞因子运输和分泌、膜重组和感染性病毒组装中的作用。HCMV miRNA的这些细胞靶标在受感染细胞的分泌/内吞途径的调节中的作用及其对细胞因子分泌和病毒组装的表型效应的验证将通过工程化表达这些途径内靶向基因的shRNA的特定组合的重组HCMV来完成。预计这些研究将阐明这些调控RNA在细胞HCMV感染结果中的作用，并定义一种新的和新颖的调控基本细胞途径的模式，从而导致优化的细胞环境用于感染性病毒体的组装。

 DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) infection of primary cells results in massive alterations in normal cellular metabolism and regulation of cellular pathways that govern cell death, nucleic acid replication, cellular membrane homeostasis, cytoskeleton integrity, and intracellular trafficking of proteins. Numerous studies have documented the widespread dysregulation of cellular gene transcription following HCMV infection arguing that major changes in cellular phenotypes are controlled at a transcriptional level. However, crude regulation of essential and highly coordinated cellular functions such as protein transport and membrane homeostasis could result in a cellular environment that would also limit the production of infectious virus. Our recent studies have demonstrated that HCMV miRNAs regulate the secretory/endocytic pathway during infection by targeting genes encoding-key components of these pathways, thus providing another layer of HCMV regulation of cellular functions. Regulation of these pathways by HCMV miRNA results in inhibition of the release of pro-inflammatory cytokines and the reorganization of intracellular membranes to facilitate efficient production of infectious virus. In this project, the specific genes in the secretory/endocytic pathway targeted by HCMV miRNAs will be identified by a comprehensive approach combining PAR-CLIP methodology with informatics followed by rigorous validation. Initially, the role of validated miRNA targets in the regulation of trafficking in the endocytic an secretory pathways will be defined, followed by investigation of the role of these miRNA targets in cytokine trafficking and secretion, membrane reorganization, and infectious virus assembly. Validation of the role of these cellular targets of HCMV miRNAs in regulation of the secretory/endocytic pathways of the infected cells and their phenotypic effects on cytokine secretion and virus assembly will be accomplished by engineering recombinant HCMVs expressing specific combinations of shRNAs targeting genes within these pathways. It is anticipated that these studies will elucidate the role of these regulatory RNAs in the outcome of HCMV infection of the cell as well as defining a new and novel mode of regulation of essential cellular pathways leading to a cellular environment optimized for assembly of infectious virions.