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

Electron tomography reveals the structural organization of actin comet tails generated by a baculovirus, providing an understanding of how this pathogen hijacks host machinery to propel itself between cells.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.Author Summary: Several bacteria and viruses hijack the motile machinery of cells they invade to generate networks of actin filaments (comet tails) to propel themselves from one cell to another. A proper understanding of the mechanism of propulsion has so far been hampered by a lack of information about the structure of the machinery. Using electron tomography we present here the three-dimensional structure of actin comet tails propelling a baculovirus, the smallest pathogen known to recruit the actin nano-machinery. We show that baculovirus is propelled by a fishbone-like array of actin filaments constructed from subsets linked by branch junctions, with an average of four filaments pushing the virus by their fast polymerizing ends at any one time. Using a stochastic mathematical model we have simulated comet tail organization as well as the tracks adopted by baculovirus inside cells. The simulations support a model of baculovirus propulsion in which the actin filaments are continuously tethered to the virus surface as they grow, branch, and push. Since larger pathogens like Listeria, Shigella, and Vaccinia virus generate comet tails exhibiting the same general morphology and components as those of baculovirus, the basic mechanism of their propulsion is likely a scaled up version of the one described here.