Nuclear rotation and cellular reorganization during Cytomegalovirus infection

项目摘要 人巨细胞病毒（HCMV）是一种β-疱疹病毒，在世界上超过60%的人中建立终身感染 人口虽然在大多数健康个体中无害，但HCMV是先天性巨噬细胞缺乏的主要感染原因。 出生缺陷，从听力或视力丧失和认知障碍，到严重的发育障碍， 小头畸形和死亡然而，公众对这一点的认识却低得惊人，这导致许多专家 将HCMV称为“沉默的全球负担”。在成人中，HCMV是再狭窄和冠状动脉粥样硬化的主要原因。 问题，与某些癌症有关，并导致免疫抑制移植的主要并发症 接受者或艾滋病患者。尽管如此，没有疫苗或治疗方法，我们仍然有一个相对 与其他病毒相比，对HCMV复制的了解有限。事实上，与其他大多数人不同， 病毒，HCMV具有跨越数天的延长的复制周期，在此期间，它形成独特的 病毒体成熟的细胞质位点，称为组装复合体（AC）。虽然最近固定成像 的方法提供了深入了解其结构，揭示它包括一个重塑的高尔基体， 通过各种宿主细胞器和囊泡，HCMV复制的详细机制的理解已经被 在很大程度上受到与AC和宿主细胞器成像相关的挑战的限制， 活细胞的周期在初步的数据支持这一建议，我们开发创新的新的多色活 细胞成像方法提供了对AC和受感染细胞行为的第一次洞察， 意想不到的发现是，AC充当新的病毒组装微管组织中心（MTOC）， 使HCMV能够旋转宿主细胞核，为细胞迁移做准备。HCMV通过以下方式实现这一点： 直接靶向和募集高度特化的微管（MT）末端结合蛋白EB 3至AC。 这用于募集MT成核和聚合的特异性EB 3相关调节剂，并允许MT成核和聚合。 AC以产生高度乙酰化的MT的专门子集。AC衍生的MT的乙酰化赋予 旋转宿主细胞核和控制细胞粘附和迁移所需的机械强度， 病毒传播。我们的数据还表明，RNA干扰（RNAi）介导的EB 3表达抑制 阻止核旋转并抑制病毒传播。独立地，我们开发了一种小豆蔻酰化肽， 它被原代细胞迅速吸收，并干扰宿主蛋白依赖EB 3募集到细胞中。 AC.这种肽，而不是对照肽，阻断HCMV诱导的细胞核旋转，并作为非- 毒性，病毒特异性抑制剂的HCMV传播。在本提案中，我们将测试几个假设和替代方案 确定AC如何通过EB 3的控制作为新型MTOC起作用的机理细节，以及 确定HCMV如何以及为什么在促进细胞迁移时诱导宿主细胞核旋转 病毒传播。实现这些目标不仅将为研究提供创新的新工具， 对活细胞中HCMV复制机制的新见解，而且还具有显着的翻译潜力。

PROJECT SUMMARY Human Cytomegalovirus (HCMV) is a β-herpesvirus that establishes life-long infection in over 60% of the world population. While innocuous in most healthy individuals, HCMV is the leading infectious cause of congenital birth defects ranging from hearing or vision loss and cognitive impairment, to severe developmental disabilities, microcephaly and death. Yet awareness of this in the general public is alarmingly low, leading many experts to refer to HCMV as the “silent global burden”. In adults, HCMV is a leading cause of restenosis and coronary problems, has been linked to some cancers, and causes major complications in immunosuppressed transplant recipients or AIDS patients. Despite this, there is no vaccine or cure, and we continue to have a relatively limited understanding of HCMV replication when compared against other viruses. Indeed, unlike most other viruses, HCMV has a protracted replication cycle spanning several days during which time it forms a unique cytoplasmic site for virion maturation, termed the Assembly Compartment (AC). While recent fixed imaging approaches have provided insights into its structure revealing that it comprises a remodeled Golgi surrounded by various host organelles and vesicles, a detailed mechanistic understanding of HCMV replication has been limited in large part by the challenges associated with imaging the AC and host organelles over extended periods in living cells. In preliminary data supporting this proposal, we develop innovative new multi-color live cell imaging approaches that provide the first insights into AC and infected cell behavior, resulting in the unexpected finding that the AC acts as a novel virus-assembled microtubule organizing center (MTOC) that enables HCMV to rotate the host cell nucleus in preparation for cell migration. HCMV accomplishes this by directly targeting and recruiting the highly specialized microtubule (MT) end-binding protein, EB3 to the AC. This serves to recruit specific EB3-associated regulators of MT nucleation and polymerization, and allows the AC to generate specialized subsets of MTs that are heavily acetylated. Acetylation of AC-derived MTs confers the mechanical strength needed to rotate host nuclei and control cell adhesion and migration, which promote virus spread. Our data also shows that RNA interference (RNAi)-mediated suppression of EB3 expression blocks nuclear rotation and suppresses virus spread. Independently, we develop a small myristoylated peptide that is rapidly taken up by primary cells and interferes with EB3-dependent recruitment of host proteins to the AC. This peptide, but not control peptides, blocks HCMV-induced rotation of the nucleus and acts as a non- toxic, virus-specific inhibitor of HCMV spread. In this proposal, we will test several hypotheses and alternatives to determine the mechanistic details of how the AC functions as a novel MTOC through control of EB3, and determine both how and why HCMV induces rotation of the host cell nucleus when promoting cell migration and virus spread. Accomplishing these Aims will not only provide innovative new tools for research alongside mechanistic new insights into HCMV replication in living cells, but also has significant translational potential.