Respiratory syncytial virus phosphoprotein has NTPase and helicase-like activities.

Respiratory syncytial virus (RSV), a non-segmented, negative-sense RNA virus (NNSV) in the family Pneumoviridae, represents a significant global health burden causing severe lower respiratory tract infections in infants and immunocompromised adults. While RNA helicases are essential for viral replication through their RNA remodeling functions, the presence of such enzymatic activities in RSV remains unclear. Here, we reveal that the RSV phosphoprotein (P), despite lacking canonical helicase motifs, demonstrates nucleoside triphosphatase (NTPase) activity and directional 5'-to-3' RNA helix-unwinding capacity in an NTP-dependent manner. Through mutagenesis assays, we establish a functional coupling between NTP hydrolysis and helicase-like activity. Importantly, reverse genetics experiments, RSV minigenome, and antiviral-effect assays demonstrate the essentiality of RSV P's helicase-like activity for viral viability and replication. These findings identify P as an enzymatic component critical for RSV replication, providing new insights into the mechanisms of pneumovirus propagation.

Importance: RNA helicases and helicase-like viral proteins are crucial for viral RNA replication and are prime targets for antiviral development. RSV infects nearly all children by age two, causing over 30 million acute lower respiratory infections, 3.6 million hospitalizations, and 100,000 deaths annually in children under five, while also posing a significant threat to immunocompromised adults and the elderly. In this study, we demonstrate for the first time that the RSV P has NTPase activity and unwinds RNA helices in an NTP-dependent manner. Mutagenesis and reverse genetics experiments confirm that these enzymatic activities are essential for RSV viability. These findings not only redefine RSV P as a multifunctional protein but also expand our understanding of the RSV replication machinery, highlighting the potential of targeting P for antiviral therapy.