The pneumoviruses human and bovine respiratory syncytial virus (RSV) are significant global respiratory viral pathogens responsible for causing lower respiratory tract infections in humans and cattle, respectively. The formation of progeny virions at the cell surface requires the coordinated assembly of glycoproteins trafficked to the apical surface of cells via the host secretary pathway, other viral protein complexes assembled in cytoplasm inclusion bodies and lastly viral ribonucleoproteins. Although some host proteins and pathways have been implicated in pneumovirus budding and assembly (e.g. HSP90, Rab-11 and apical recycling endosomes), the full spectrum of virus-host interactions has not been fully elucidated. Using a siRNA library targeting membrane trafficking and the Incucyte Live Cell Imaging System, we have developed and optimised a high-throughput siRNA protocol for characterising RSV egress. The siRNA library targets human proteins which are known, or predicted, to be involved in membrane trafficking or remodelling, while the Incucyte System allows near real-time imaging over an extended time-course, generating high quality data that allows the monitoring and quantification of multiple parameters such as cell viability, viral replication and syncytia formation. Using a recombinant human RSV expressing a GFP reporter we have identified a number of proteins involved in pneumovirus trafficking in infected cells. Our techniques provide a robust and sensitive mechanism for genetic screening and the identification of pneumovirus-protein interactions.

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