Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

Abstract

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

A novel host factor for human respiratory syncytial virus

“…1). 7,8 Based on these results, we speculate that the increased induction of filopodia facilitated virus spread, and likely also reduced the exposure of RSV to extracellular spaces where neutralization by antibodies would be more efficient.

Figure 1.Filopodia-driven RSV cell-to-cell spread.

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“…The cells were further stained with rhodamine phalloidin (Cytoskeleton Inc.) to detect F-actin ( red ). Images were collected on a Leica TCS SP8 STED 3X system (Leica Microsystems) as described in 7 . This image illustrates that the filopodia (indicated by arrows) of the RSV-infected cell (labeled A) appear to convey RSV particles (indicated by arrow heads) to a neighboring cell (labeled B). …”

Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells

“…RSV forms pleomorphic virus particles, with a predominance of long filaments about 100 nm in diameter and up to about 10 μm in length (Bachi and Howe, 1973; Mehedi et al ., 2016). High-resolution light microscopy techniques are key to visualizing the interactions between RSV infected cells and virus particles.…”

“… Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al ., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al ., 2016).…”

“…High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al ., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging.…”