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.

项目概要/摘要 多瘤病毒 (PyV) 是一种小型 DNA 肿瘤病毒，可导致人类衰弱疾病，尤其是在 免疫功能低下的个体。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 从细胞表面分选至内质网 (ER)，并在此穿透 ER 膜到达细胞质。从这里开始，病毒被分解以穿过狭窄的核孔 复合体（NPC）并进入细胞核。 PyV 从细胞质到细胞核的转运很重要，但 神秘的感染步骤。在哺乳动物细胞中，细胞内向细胞核的转运主要是通过 细胞质运动动力蛋白，将货物沿着微管移向细胞中心。使用 原型 PyV，猿猴病毒 40 (SV40)，与人类共享结构和遗传组织 PyV 以及相同的感染生命周期，我们最近报道，动力蛋白运动活动是 病毒分解和病毒到达核。动力蛋白促进这一作用的确切机制 过程未知。动力蛋白的加工活性需要由动力蛋白组成的三蛋白复合物 马达、动力蛋白激活剂和接头蛋白，赋予货物特异性。初步实验 揭示除了动力蛋白和动力蛋白外，双尾 D2 (BICD2) 货物适配器对于 SV40 感染。 BICD2 的敲低显着损害了细胞质中的 SV40 分解及其 核到来。此外，BICD2 直接与病毒相互作用并促进其在 NPC 中的释放。在 除了通过货物适配器激活之外，动力蛋白活性也可以通过 LIS1 与 NUDE 或 NDEL，辅助因子，用于将马达锚定到其微管轨道上。有趣的是，我们发现 LIS1 和 NDEL 对于 SV40 分解和感染也至关重要。本研究提案旨在定义以下角色： PyV 进入途径 (Aim1) 中的动力蛋白运动复合体激活剂和调节剂，以及该病毒是否是 直接能够调节该细胞过程以完成其生命周期（目标2）。我们假设 SV40 在胞质溶胶中募集动力蛋白-动力蛋白-BICD2 (DDB) 复合物，该复合物反过来将病毒颗粒转运到 核。由于完整的病毒太大而无法穿过 NPC，我们进一步假设 动力蛋白激活剂 (BICD2) 和调节剂 (LIS1/NDEL) 的协调作用产生机械力， 被病毒战略性地利用来生成可以进入NPC的更小的核心病毒。完成后 在这些研究中，我们的研究结果将阐明 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。