Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

多种病原体会在其侵染的宿主细胞内形成推进型肌动蛋白彗星尾（propulsive actin comet tails），以此实现感染扩散。此类病原体通过招募宿主细胞质中的肌动蛋白成核因子——Arp2/3复合物（Arp2/3 complex）与聚合调控因子来完成这一过程。由于目前对肌动蛋白彗星尾的结构组织，尤其是参与推进过程的肌动蛋白丝的空间排布信息了解有限，病原体运动的潜在机制目前仍属于推测且存在争议。本研究借助电子断层扫描技术（electron tomography），解析了驱动杆状病毒（baculovirus）运动的肌动蛋白彗星尾的三维结构——杆状病毒是迄今已知的首个劫持肌动蛋白动力装置的最小病原体。研究还发现，将病毒衣壳与纯化肌动蛋白以及最少组分的肌动蛋白调控因子混合后，可重现彗星尾的几何结构。我们证实，推进机制基于一类鱼骨状排列的肌动蛋白丝阵列：该阵列由通过分支连接点（branch junctions）相连的肌动蛋白丝亚组构成，平均每次有四根肌动蛋白丝推动病毒运动。通过能量最小化函数（energy-minimizing function），我们模拟了肌动蛋白彗星尾的结构，以及杆状病毒在体内感染细胞中所采取的运动轨迹。模拟结果排除了凝胶挤压型推进模型，并支持以下假说：肌动蛋白丝在分支成核与聚合过程中持续被拴定。鉴于单核细胞增生李斯特菌（Listeria monocytogenes）、福氏志贺菌（Shigella flexneri）、牛痘病毒（Vaccinia virus）等多种病原体与杆状病毒共享同一套核心组分工具箱，我们认为它们的肌动蛋白组织方式与推进模式遵循相同的原理。