Nuclear rotation and cellular reorganization during Cytomegalovirus infection

项目总结 人类巨细胞病毒(Hcmv)是一种β疱疹病毒，在世界上超过60%的地区造成终身感染。 人口。虽然对大多数健康人来说是无害的，但巨细胞病毒是先天性 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，公众对此的认识令人震惊地低，导致许多专家 将人类巨细胞病毒称为“无声的全球负担”。在成人中，人巨细胞病毒是导致再狭窄和冠状动脉病变的主要原因 问题，已与一些癌症有关，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，没有疫苗或治愈方法，我们继续有一个相对 与其他病毒相比，对巨细胞病毒复制的了解有限。事实上，与大多数其他公司不同 与病毒不同，HCMV有一个长达数天的复制周期，在此期间它形成了一个独特的 病毒粒子成熟的细胞质部位，称为集合室(AC)。虽然最近的固定成像 各种方法提供了对其结构的洞察，揭示了它包括一个被改造的高尔基山脉 通过不同的宿主细胞器和囊泡，人们对HCMV复制的详细机制有了详细的了解 在很大程度上受到AC和宿主细胞器过度扩展成像的挑战 活细胞中的经期。在支持这一建议的初步数据中，我们开发了创新的多色直播 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，导致 出乎意料的发现，AC作为一种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 直接靶向并招募高度特化的微管(MT)末端结合蛋白EB3到AC。 这用于招募MT成核和聚合的特定EB3相关调节因子，并允许 AC产生大量乙酰化的MTS的专门子集。AC衍生MTS的乙酰化反应 旋转宿主核和控制细胞黏附和迁移所需的机械强度，这促进了 病毒传播。我们的数据还表明，RNA干扰(RNAi)介导的EB3表达抑制 阻止核旋转，抑制病毒传播。我们独立地开发了一种小的肉豆蔻酰化的多肽 它会被原代细胞迅速吸收，并干扰依赖EB3的宿主蛋白向 交流电。这种多肽，但不是对照多肽，阻断了HCMV诱导的核旋转，并作为非 有毒的、病毒特异性的巨细胞病毒传播抑制物。在这个提案中，我们将测试几个假设和备选方案 通过控制EB3来确定AC如何作为新的MTOC发挥作用的机械细节，以及 确定当促进细胞迁移时，巨细胞病毒如何以及为什么诱导宿主细胞核旋转 以及病毒的传播。实现这些目标不仅将为研究提供创新的新工具 对活细胞中巨细胞病毒复制的机械性新见解，但也具有显著的翻译潜力。

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.