RSV infection induced the formation of filopodia, and ARP2 knockdown reduced the number of long RSV-induced filopodia.

<p><b>(A) RSV-induced filopodia.</b> A549 cells were transfected with siARP2 or siControl for 48 hr followed by mock infection or infection with RSV-WT (MOI = 1) for 24 hr. Cells were fixed, permeabilized, and immunostained for RSV F (<i>green</i>) and beta-tubulin (<i>cyan</i>, here a pseudocolor) by incubating with mouse mAb for F protein and rabbit mAb for beta-tubulin followed by the secondary antibodies ant-mouse AlexaFluor₄₈₈ and anti-rabbit AlexaFluor₆₄₇ respectively. The cells were further stained with rhodamine phalloidin (<i>red</i>) to detect F-actin and with the nuclear stain DAPI (<i>blue</i>). The images show all four colors (versions that individually show <i>green</i>, <i>cyan</i>, and <i>red</i> are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006062#ppat.1006062.s004" target="_blank">S4 Fig</a>). In the enlargement, examples of filopodia (<i>red</i>) are indicated with arrows, examples of what appear to be released filamentous RSV particles (<i>green</i>) are indicated with asterisks, and examples of filopodia-associated RSV filaments are indicated with arrowheads. <b>(B) ARP2 knockdown reduced RSV-induced filopodia.</b> In samples from two independent experiments similar to the experiment described for A, the number and length of filopodia on siControl- and siARP2-transfected mock-infected RSV-WT-infected cells were evaluated by automated scanning using confocal microscopy. In brief, Z-stacking for Alexafluor₄₈₈ for RSV F protein, DAPI for nuclei, rhodamine phalloidin for F-actin was performed for 50 to 100 different random fields of interest in each coverslip. The length and number of filopodia was measured on 100 cells per treatment per experiment from the surface to the tip of the filopodium. Error bar: SD.