Arp2/3 complex-dependent actin remodeling is required for efficient RSV uncoating in A549 cells.

Productive human respiratory syncytial virus (RSV) cell entry requires coordinated interactions between viral proteins and host-cell factors at the plasma membrane-actin cortex interface. Branched actin networks remodel this interface, but their precise contribution to the early stages of RSV infection remains unclear. Here, we interfered with Arp2/3 complex-dependent actin filament branching by generating A549 cell lines disrupted for expression of the essential Arp2 subunit by CRISPR/Cas9. Permanent loss of Arp2 reduced the infection of the RSV long GFP reporter virus, as quantified over the first 24 h post-infection. Compromised infection efficiency in Arp2 knockout cells persisted at later time points, and also resulted in reduced syncytia formation. Arp2 re-expression restored Arp2/3 complex-dependent actin remodeling and rescued RSV infection in Arp2 KO cells. Notably, impaired RSV infection was not accompanied by obvious changes in viral host cell attachment. Moreover, photoactivated localization microscopy (PALM) studies revealed comparable receptor diffusion and clustering in cells stably expressing mEos3.2-tagged insulin-like growth factor I receptor (IGF1R). Although Arp2/3-deficient cells displayed fewer, albeit larger, macropinosomes compared with WT cells, no changes were observed for internalized RSV genome levels. In contrast, Arp2-deficient cells appeared suppressed in RSV-F mediated virus-like particle uncoating efficiency. Consequently, viral mRNA expression and the cellular type III interferon response were reduced. Together, these data reveal that Arp2/3 complex-dependent, branched actin networks contribute to the efficiency of RSV uncoating.IMPORTANCEHuman respiratory syncytial virus (RSV) is a major cause of severe respiratory disease. The infection initiates at the plasma membrane-actin cortex interface, yet the role of actin in productive RSV entry has remained unclear. Using CRISPR/Cas9 disruption of the essential Arp2/3 complex subunit Arp2 in A549 cells, we show that branched actin networks are required for efficient RSV infection. Despite actin network remodeling, photoactivated localization microscopy showed unchanged diffusion and clustering of the RSV receptor IGF1R. Although macropinocytosis was affected in Arp2-deficient cells, RSV attachment and internalization were not influenced. In contrast, a β-lactamase virus-like-particle-based assay revealed a defect in RSV-F-mediated uncoating, followed by reduced viral gene expression and a weaker type III interferon response. These findings define Arp2/3 complex-dependent branched actin networks as a host determinant of RSV-F-mediated uncoating and provide a practical approach to quantify uncoating without engineering the RSV genome.