Institut für Virologie, Philipps-Universität Marburg, D-35043 Marburg, Germany.
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14402-7. doi: 10.1073/pnas.1307681110. Epub 2013 Aug 12.
Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.
大型病毒核衣壳从复制中心运输到组装部位需要宿主细胞骨架通过细胞衔接蛋白和马达蛋白的参与。对于马尔堡病毒和埃博拉病毒等引起严重出血热的相关病毒，核衣壳的运输机制仍知之甚少。在这里，我们开发并使用活细胞成像技术，对荧光标记的病毒和宿主蛋白进行了研究，以在 4 级生物安全条件下研究马尔堡病毒感染细胞中核衣壳的运输动态和分子需求。研究表明，在病毒复制中心到出芽部位之间，核衣壳通过复杂的基于肌动蛋白的长距离运输。只有当核衣壳与质膜上的基质病毒蛋白 VP40 结合后，它们才会被招募到丝状伪足中，并与宿主肌球蛋白 10 一起向长细胞突起的尖端或侧部的出芽部位共转运。确定了三种不同的运输模式和速度：（i）在细胞质中的肌动蛋白丝上，核衣壳以约 200nm/s 的速度运输；（ii）核衣壳以约 400nm/s 的速度从一条肌动蛋白丝迁移到另一条肌动蛋白丝；（iii）VP40 相关的核衣壳在丝状伪足内以 100nm/s 的速度移动。对核衣壳的时空动力学及其与细胞骨架和马达蛋白相互作用的独特见解，可以开发出新型的抗病毒药物，干扰病毒从感染细胞中的运输和随后的释放。
