Clarifying the role of the cytoplasmic dynein motor complex in polyomavirus nuclear entry.

阐明细胞质动力蛋白运动复合物在多瘤病毒核进入中的作用。

• 批准号: 9758812
• 负责人: Chelsey Cierra Spriggs
• 金额: $ 6.16万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9758812
• 关键词: Adaptor Signaling Protein; Affinity; Antiviral Agents; Binding; Capsid; Cell Nucleus; Cell physiology; Cell surface; Cells; Complex; Cytosol; DNA Tumor Viruses; DNA Viruses; Disease; Dynein ATPase; Endoplasmic Reticulum; Event; Genetic; Genetic Transcription; Genome; Human; Immunocompromised Host; Impairment; Individual; Infection; Intracellular Transport; Lead; Life Cycle Stages; Lytic Phase; Mammalian Cell; Membrane; Merkel Cells; Microtubules; Motor; Motor Activity; Nuclear; Nuclear Pore Complex; Pathway interactions; Polyomavirus; Polyomavirus Infections; Prevention; Process; Reporting; Research Proposals; Role; Simian virus 40; Skin Carcinoma; Specificity; Structure; Testing; Viral; Viral Genome; Virion; Virus; Virus Replication; base; cell transformation; ds-DNA; dynactin; experimental study; human disease; insight; knock-down; mechanical force; metaplastic cell transformation; particle; protein complex; recruit; virus core

Project Summary/Abstract Polyomaviruses (PyVs) are small DNA tumor viruses that cause debilitating human disease, especially in immunocompromised individuals. To infect cells, these non-enveloped viruses must transport to the host nucleus where transcription and replication of the viral genome lead to lytic infection or cellular transformation. During entry, PyV sorts from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From here, the virus is disassembled in order to cross the narrow nuclear pore complex (NPC) and enter the nucleus. PyV transport from the cytosol to the nucleus is an important, yet enigmatic, step in infection. In mammalian cells, intracellular transport to the nucleus is facilitated largely by the cytoplasmic motor dynein, which moves cargo along microtubules towards the center of the cell. Using the prototypic PyV, simian virus 40 (SV40), which shares both structural and genetic organization with human PyVs as well as the same infectious life cycle, we recently reported that dynein motor activity is required for viral disassembly and nuclear arrival of the virus. The exact mechanisms by which dynein promotes this process are unknown. Processive dynein activity requires a three-protein complex composed of the dynein motor, dynactin activator, and an adaptor protein, which confers cargo specificity. Preliminary experiments reveal that in addition to dynein and dynactin, the bicaudal D2 (BICD2) cargo adaptor is also important for SV40 infection. The knockdown of BICD2 significantly impairs SV40 disassembly in the cytosol as well as its nuclear arrival. Moreover, BICD2 interacts directly with the virus and promotes its release at the NPC. In addition to activation by cargo adaptors, dynein activity can also be regulated by LIS1 with either NUDE or NDEL, co-factors that serve to anchor the motor to its microtubule track. Interestingly, we found that LIS1 and NDEL are also essential for SV40 disassembly and infection. This research proposal aims to define the role of dynein motor complex activators and regulators in the PyV entry pathway (Aim1), and whether the virus is directly capable of regulating this cellular process to complete its life cycle (Aim2). We hypothesize that SV40 recruits the dynein-dynactin-BICD2 (DDB) complex in the cytosol, which in turn transports the viral particle into the nucleus. Because the intact virus is too large to transport across the NPC, we further postulate that the coordinated action of dynein activators (BICD2) and regulators (LIS1/NDEL) produces a mechanical force that is strategically harnessed by the virus to generate a smaller core virus that can enter NPC. Upon completion of these studies, our findings will illuminate key steps in the PyV entry pathway and identify potential anti-viral targets for the prevention and treatment of PyV infection and disease.

项目总结/摘要 多瘤病毒（PyV）是小DNA肿瘤病毒，其引起使人衰弱的疾病，特别是在哺乳动物中。 免疫力低下的人。为了感染细胞，这些无包膜病毒必须运输到宿主 病毒基因组的转录和复制导致裂解性感染或细胞转化的细胞核。 在进入过程中，PyV从细胞表面分选到内质网（ER），在那里它穿透ER 膜到达胞质溶胶。从这里开始，病毒被分解，以便穿过狭窄的核孔 复合物（NPC）并进入细胞核。PyV从细胞质到细胞核的运输是一个重要的，但 神秘的，感染的步骤。在哺乳动物细胞中，细胞内转运到细胞核主要是由 细胞质运动动力蛋白，其将货物沿着微管向细胞中心移动。使用 原型PyV，猿猴病毒40（SV 40），其与人类共享结构和遗传组织 PyVs以及相同的感染生命周期，我们最近报道，动力蛋白运动活性是需要的， 病毒解体和病毒到达核。动力蛋白促进这一过程的确切机制 过程未知。进行性动力蛋白活性需要由动力蛋白组成的三蛋白复合物 马达、动力蛋白激活剂和赋予货物特异性的衔接蛋白。初步实验 揭示了除了动力蛋白和动力肌动蛋白之外，双尾D2（BICD 2）货物衔接子对于 SV 40感染。BICD 2的敲低显著损害了细胞质中的SV 40分解以及其在细胞内的表达。 核武器的到来此外，BICD 2直接与病毒相互作用，并促进其在NPC中的释放。在 除了被货物衔接子激活外，动力蛋白活性还可以被具有NUDE或NUDE的LIS 1调节。 NDEL，辅助因子，用于将马达锚在其微管轨道上。有趣的是，我们发现LIS 1和 NDEL对于SV 40的分解和感染也是必需的。本研究计划旨在界定 PyV进入途径（Aim 1）中的动力蛋白运动复合物激活剂和调节剂，以及病毒是否 直接能够调节该细胞过程以完成其生命周期（Aim 2）。我们假设SV 40 在胞质溶胶中募集动力蛋白-动力蛋白-BICD 2（DDB）复合物，该复合物又将病毒颗粒转运到 原子核因为完整的病毒太大而不能穿过NPC，我们进一步假设， 动力蛋白激活剂（BICD 2）和调节剂（LIS 1/NDEL）的协调作用产生机械力， 被病毒战略性地利用，以产生一种可以进入NPC的较小的核心病毒。完成后 通过这些研究，我们的发现将阐明PyV进入途径的关键步骤，并确定潜在的抗病毒药物 预防和治疗PyV感染和疾病的目标。