Control of Translation in Herpesvirus Infected Cells - Resubmission - 1

7.项目摘要/摘要 在翻译水平上对基因表达的调节使细胞和生物体能够对 生理压力和不断变化的环境。事实上，有帽的、多腺化的差异翻译 真核细胞核糖体的mRNAs在对人类健康至关重要的生物过程中起着关键作用，包括 正常的细胞生长、分化、发育、学习和记忆以及对生理应激的反应 包括病毒感染。病毒模型系统已被证明在阐述细胞翻译控制方面很有用 策略，因为它们的复制绝对依赖于宿主核糖体对病毒mRNA的翻译。这里, 我们利用疱疹病毒家族成员人类巨细胞病毒(Hcmv)来探测复杂的回路。 调节信使核糖核酸翻译。虽然对健康人无害，但巨细胞病毒是一种广泛存在的机会性病毒。 负责严重疾病的病原体免疫受损，包括器官移植 接受者和艾滋病患者。此外，先天性巨细胞病毒感染是导致出生缺陷的主要病毒原因。 在新生儿身上。我们的长期总体目标是了解巨细胞病毒感染的机制(S) 操纵细胞翻译机制来控制病毒复制。根本的一步 调节巨细胞病毒感染细胞的蛋白质合成涉及核糖体募集到m7G-caped的5‘端 病毒和宿主的mRNA。这通常涉及翻译起始因子复合体的5‘-帽识别 包含帽结合蛋白eIF4E，然后加载eIF3结合的40S核糖体。出乎意料的是，一个 另一种形式的40s核糖体装载依赖于特定eIF3亚单位eIF3d的帽识别，具有 已被描述为绕过了eIF4E的需要，并受到明显的监管。初步结果表明， 靶向eIF3d选择性抑制HCMV复制，减少多聚核糖体丰度并干扰 主要病毒晚期基因的表达和指示慢性内质网应激的宿主基因表达特征 这促进了巨细胞病毒的繁殖。根据我们的初步结果，我们假设eIF3d依赖的帽- 识别被劫持以利用病毒诱导的内质网应激和在eIF4E和eIF3d反应之间的切换 翻译可以不同地调节病毒和宿主基因在感染细胞中的表达。这一假设在以下方面得到了验证 三个具体目标旨在：i)确定巨细胞病毒感染如何影响eIF3d积累和亚细胞 分布；ii)定义eIF3d选择性地塑造基因表达图景的机制 以及iii)破译在感染巨细胞病毒的细胞中eIF3d活性是如何被调节的。该项目是 影响是因为涉及eIF3d的非规范翻译启动机制如何调节差异 病毒感染等应激反应过程中的mRNA翻译在很大程度上仍不清楚，它代表着一种 重大知识差距有望揭示干扰病毒的新生物学和治疗机会 复制或疫苗开发。此外，它还将提供对基本翻译控制的洞察 这种机制在人类疾病中很重要，比如癌症和糖尿病，这些疾病中蛋白质的产生是不受调控的。

7. PROJECT SUMMARY / ABSTRACT Regulation of gene expression at the level of translation allows cells and organisms to respond swiftly to physiological stress and changing environments. Indeed, differential translation of capped, polyadenylated mRNAs by eukaryotic ribosomes plays a critical role in biological processes vital for human health, including normal cell growth, differentiation, development, learning and memory, and responses to physiological stress including virus infection. Viral model systems have proven useful in elaborating cellular translational control strategies because their replication is absolutely reliant upon virus mRNA translation by host ribosomes. Here, we utilize a herpesvirus family member, human cytomegalovirus (HCMV), to probe the complex circuitry regulating mRNA translation. Although innocuous in healthy individuals, HCMV is a widespread, opportunistic pathogen responsible for severe disease among the immunocompromised, including organ transplant recipients and AIDS patients. In addition, congenital HCMV infection is the leading viral cause of birth defects in newborns. Our long-term overall objective is to understand the mechanism(s) through which HCMV manipulates the cellular translational machinery to control viral replication. A fundamental step regulating protein synthesis in HCMV-infected cells involves ribosome recruitment to the 5'-end of m7G-capped virus and host mRNAs. This typically involves 5'-cap-recognition by a translation initiation factor complex containing the cap-binding protein eIF4E followed by loading the eIF3-bound 40S ribosome. Unexpectedly, an alternative form of 40S ribosome loading relying upon cap-recognition by a specific eIF3 subunit, eIF3d, has been described that bypasses the need for eIF4E and is distinctly regulated. Preliminary results establish that targeting eIF3d selectively inhibited HCMV replication, reduced polyribosome abundance and interfered with expression of essential virus late genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. Based on our preliminary results, we hypothesize that eIF3d-dependent cap- recognition is hijacked to exploit virus-induced ER-stress and switching between eIF4E and eIF3d-responsive translation differentially tunes virus and host gene expression in infected cells. This hypothesis is tested in three specific aims designed to: i) determine how HCMV infection impacts eIF3d accumulation and sub-cellular distribution; ii) define the mechanism whereby eIF3d selectively shapes the gene expression landscape in HCMV-infected cells; and iii) decipher how eIF3d activity is regulated in HCMV-infected cells. The project is impactful because how non-canonical translation initiation mechanisms involving eIF3d regulate differential mRNA translation during stress responses like virus infection remains largely unknown and represents a significant knowledge gap poised to reveal new biology and therapeutic opportunities for interfering with virus replication or vaccine development. Furthermore, it will provide insight into fundamental translational control mechanisms important in human diseases, like cancer and diabetes, where protein production is dysregulated.