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

项目摘要/摘要 多瘤病毒(PYV)是一种小的DNA肿瘤病毒，会导致人类虚弱的疾病，尤其是在 免疫功能受损的个体。为了感染细胞，这些未被包膜的病毒必须传输到宿主 病毒基因组的转录和复制导致裂解感染或细胞转化的核。 在进入过程中，PYV从细胞表面到内质网(ER)，在那里它穿透内质网 到达细胞质的膜。从这里，病毒被分解，以便穿过狭窄的核孔 复合体(NPC)并进入细胞核。PYV从胞浆到细胞核的运输是一个重要的，但 神秘莫测，一步一步感染。在哺乳动物细胞中，细胞内向细胞核的运输主要是通过 细胞质马达动力蛋白，将货物沿微管向细胞中心移动。使用 PYV的原型，猿猴病毒40(SV40)，它与人类具有相同的结构和遗传组织 PYV以及相同的感染性生命周期，我们最近报道了动力蛋白运动活动是 病毒的解体和核的到达。动力蛋白促进这一变化的确切机制 过程是未知的。过程中的动力蛋白活性需要由动力蛋白组成的三蛋白复合体 马达，动力蛋白激活剂，和适配器蛋白，这赋予货物特异性。初步实验 揭示了除了dynein和dynactin之外，双尾D2(BICD2)货物接头对 SV40感染。BICD2的敲除显著损害了SV40在胞浆中的拆解以及其 核子到达。此外，BICD2与病毒直接相互作用，并促进病毒在NPC中的释放。在……里面 除了被货物适配器激活外，dynein的活性也可以由Lis1来调节，可以是裸体的或者是 Ndel，用于将马达固定在其微管轨道上的辅助因素。有趣的是，我们发现LIS1和 NDel对SV40的解体和感染也是必不可少的。这项研究提案旨在定义 PYV进入途径(Aim1)中的动力蛋白马达复合体激活物和调节器，以及病毒是否 能够直接调节这一细胞过程以完成其生命周期(AIM2)。我们假设SV40 在胞浆中招募动力蛋白-动力蛋白-BICD2(DDB)复合体，后者进而将病毒颗粒输送到 原子核。由于完整的病毒太大，无法通过NPC运输，我们进一步假设 动力蛋白激活剂(BICD2)和调节剂(Lis1/ndel)的协同作用产生一种机械力， 被病毒战略性地利用来产生更小的核心病毒，可以进入鼻咽癌。在完成后 这些研究，我们的发现将阐明热带病病毒进入途径的关键步骤，并确定潜在的抗病毒药物 预防和治疗热带病病毒感染和疾病的目标。

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.