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

 描述(申请人提供)：人巨细胞病毒(HCMV)感染原代细胞导致正常细胞代谢和调控细胞死亡、核酸复制、细胞膜动态平衡、细胞骨架完整性和蛋白质细胞内运输的细胞通路发生巨大变化。大量研究证明，在HCMV感染后，细胞基因转录普遍失调，认为细胞表型的主要变化是在转录水平上控制的。然而，对蛋白质运输和膜稳态等基本和高度协调的细胞功能的粗略调控可能会导致细胞环境，这也将限制传染性病毒的产生。我们最近的研究表明，在感染过程中，HCMV miRNAs通过靶向编码这些途径的关键成分的基因来调节这些途径的分泌/内吞途径，从而提供了另一层HCMV对细胞功能的调节。通过HCMV miRNA对这些途径的调控，可以抑制促炎细胞因子的释放，并重组细胞内膜，从而促进感染性病毒的高效生产。在这个项目中，HCMV miRNAs靶向的分泌/内吞途径中的特定基因将通过PAR-CLIP方法和信息学相结合的综合方法进行识别，然后进行严格的验证。首先，将确定有效的miRNA靶标在调节细胞内运输和分泌途径中的作用，然后调查这些miRNA靶标在细胞因子运输和分泌、膜重组和传染性病毒组装中的作用。验证这些HCMV miRNAs的细胞靶标在调节感染细胞的分泌/内吞途径中的作用以及它们对细胞因子分泌和病毒组装的表型影响将通过工程表达这些途径中靶向基因的特定组合的重组HCMV来完成。预计这些研究将阐明这些调控RNA在细胞中HCMV感染结局中的作用，并确定一种新的、新的调控基本细胞途径的模式，从而为感染病毒粒子的组装提供优化的细胞环境。

 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.