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

<div><p>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 <i>in vivo</i>. 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 <i>Listeria monocytogenes</i>, <i>Shigella flexneri</i>, 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.</p></div

Cryo-electron tomography of baculovirus actin comet tail <i>in vitro</i>.

<p>(A) Cryo-electron tomogram section (11 nm) of a comet tail formed on a purified, de-enveloped baculovirus <i>in vitro</i> in a motility cocktail containing actin, Arp2/3 complex, gelsolin, cofilin, and VASP. Insets show details of branch junctions from the squares in the overview image. (B) Model derived from tomogram, showing actin filaments in green and branch junctions in red. (C) Close-up of the model highlighting the filaments abutting the rear of the virus in yellow. See also Movie S5. Bars (A, B), 100 nm; (C) 25 nm; inset, 10 nm.</p

Mathematical simulation of comet tail tracks.

<p>(A, B) Simulations of comet tail tracks obtained by assuming that the actin filaments are tethered (A) or untethered (B) to the virus surface. (C, D) Example of a comet tail track observed by conventional negative stain electron microscopy in a CAR fibroblast. The superimposed lines, 200 nm in length, are examples of those used for quantification of angular deviations (angles marked as φ₁ and φ₂). (E) Simulated model matching the example in (D). Actin filaments are in green and branch junctions in red. (Actin filament subsets are not shown.) (F) Histograms of angular deviations of comet tail tracks in electron micrographs as in (D) compared to simulations with filaments tethered, untethered, tethered during branching, and tethered with random branching. Measured versus tethered n.s. (<i>p</i>>0.9999); measured versus tethered during branching **** (<i>p</i><0.0001); measured versus untethered **** (<i>p</i><0.0001); measured versus tethered random branching n.s. (<i>p</i> = 0.0905); tethered versus tethered during branching **** (<i>p</i><0.0001); tethered versus untethered **** (<i>p</i><0.0001); tethered versus tethered random branching **** (<i>p</i><0.0001). Data nonparametric, by Kruskal-Wallis test. Bars, (A–C) 500 nm, (D, E) 100 nm.</p

Cryo-electron tomography of a baculovirus actin comet tail <i>in vivo</i>.

<p>(A) Cryo-electron tomogram of a baculovirus comet tail in a B16 melanoma cell. Image shows 19 nm sections of the tomogram. Since the virus tail was not in one plane in the ice layer, the tomogram is shown in three images, separated by white lines, taken at different z-levels. Insets show details of branch junctions from the squares in the overview image. (B) Projection of 3D model derived from the tomogram in (A) showing the branch points as red dots and actin filaments as translucent lines. Yellow region indicates the core of the tail previously traversed by the cross-section of the virus, used for deriving (C). (C) Plot of the number of filaments transecting the core region in (B), taken as the number involved in pushing. (D) Projection from the rear of the complete comet tail model with actin filaments of the host cytoskeleton (translucent) as well as one microtubule (grey tube) superimposed. See also Movie S3. Bars (A, B), 100 nm; inset, 10 nm.</p

<i>In vitro</i> assembled baculovirus comet tails.

<p>Images of viruses and actin tails obtained in <i>in vitro</i> assays after negative staining and conventional transmission EM. (A) Budded baculovirus in the infected cell supernatant. (B) De-enveloped virus obtained after detergent treatment of the budded virus. (C–E) Actin comet tails formed on baculovirus <i>in vitro</i> in the motility cocktail after the incubation times indicated. (F, G) Effect of varying gelsolin concentration on the length of the comet tail filaments. Bars (A, B, F), 100 nm; (C, D, E, G), 500 nm.</p

Mathematical simulation of comet tail architecture.

<p>(A–C) Schematic representation of the three different models considered. (A). Tethered model. (B) Tethered during branching model. (C) Untethered model. Actin filaments are in green, branchpoints in red, and the virus is depicted in grey. Polymerizing filaments can push (1). The virus surface is continuously tethered to the barbed ends (A, 2) or to the Arp2/3 complex during branching (B, 2) or not at all (C). Tethering is modeled by a spring connection. Branches (3) are initiated by Arp2/3 complex (red) recruited to the plus ends of actin filaments at the virus surface. Elongation of filaments that can no longer push (4) continues until they are capped (5). Filaments at the rear of the tail become de-branched and depolymerize from their minus ends (6). In (B) and (C) filaments lagging behind are not tethered (7). (D) Baculovirus actin comet tail in a B16 melanoma cell observed in vitreous ice, shown in two z-sextons, 18 nm thick. (E) Model derived from tomogram in (D) showing branch points in red and actin filaments in green. The angles of filaments to the core axis are shown for three examples ξ₁–ξ₃. (F–I) Simulated comet tails for the different model scenarios: (F) tethered actin filaments; (G) filaments tethered during branching; (H) untethered filaments; (I) tethered filaments but with branching towards and away from the virus surface. Filaments in different colors belong to different subsets. (J) Histograms of angles of filaments to the core trajectory from three cryo-tomograms (<i>n</i> = 485) compared to the model simulations with tethered filaments (<i>n</i> = 49,321), tethered filaments only during branching (<i>n</i> = 58,895), untethered filaments (<i>n</i> = 51,168), and filaments with random branching (<i>n</i> = 155,556). Measured versus tethered n.s. (<i>p</i> = 0.8270); measured versus tethered during branching **** (<i>p</i><0.0001); measured versus untethered **** (<i>p</i><0.0001); Measured versus tethered random branching **** (<i>p</i><0.0001); tethered versus tethered during branching **** (<i>p</i><0.0001); tethered versus untethered **** (<i>p</i><0.0001); tethered versus tethered random branching **** (<i>p</i><0.0001). Data nonparametric, by Kruskal-Wallis test. (K) Histogram of filament lengths for the experimental data (measured) and the “tethered” simulation compared. Bars (A–C), 50 nm, (D, E) 100 nm, (F–I) 300 nm.</p

Electron tomography of negatively stained baculovirus actin comet tail <i>in vivo</i>.

<p>(A) Negatively stained comet tail in a cytoskeleton of a fish fibroblast. Image shows a 14.5 nm section of the tomogram, with the virus particle on the right (BV). Insets show details of branch junctions from the squares in the overview image. (B) Projection of 3D model derived from the tomogram in (A) showing the branch points as red dots. Actin filaments are marked as lines of different colors, with each color depicting filaments linked into a subset by branch junctions. Grey tube corresponds to a microtubule. (C) Projection of 3D model highlighting filaments subjected to polarity analysis in black and the branch points in red. Black spots mark the plus ends of the filaments. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001765#pbio.1001765.s003" target="_blank">Figure S3</a> and Movie S2. Bars (A–C), 100 nm; inset, 10 nm.</p