Airway protease availability governs a reversible shift between syncytial and cell-free spread.

UNLABELLED: Paramyxoviruses such as respiratory syncytial virus (RSV) and human parainfluenza virus 3 (HPIV3) spread in the airway by releasing virions and by forming syncytia. Spread phenotypes are typically attributed to the fusion protein cleavage site, yet it remains unclear whether, and if so, how, host protease context biases syncytial versus cell-free spread. We compared RSV and HPIV3-two closely related viruses with overlapping epithelial tropism but distinct F cleavage-site composition (polybasic, furin-type for RSV versus a distinct, less polybasic cleavage-site motif for HPIV3)-in respiratory cell lines and primary airway cells under defined in vitro protease conditions. In multi-cycle infections, furin-dominant, trypsin-like-low environments produced the largest syncytia, whereas trypsin-like-high environments favored smaller syncytia and more cell-free spread. Adding exogenous TMPRSS2 shifted HPIV3 toward cell-free spread with minimal impact on RSV. Using serine protease inhibitors (SERPINs) as protease-selective inhibitors, we show that inhibiting specific proteases restricts infection and remodels syncytium architecture, consistent with protease switching. Together, these results indicate that host protease availability is a reversible control point for respiratory virus dissemination. This framework has direct implications for protease-targeting therapeutics. Selective inhibition may reroute fusion activation and shift spread modality toward more syncytia-associated cytopathic effects rather than uniformly suppressing dissemination.

IMPORTANCE: Respiratory syncytial virus and human parainfluenza virus 3 are leading causes of pneumonia and bronchiolitis in infants, the elderly, and immunocompromised patients, and disease severity tracks with how efficiently these viruses spread through the airway. Spread has long been attributed to a fixed feature of the viral fusion protein. Here, we show instead that the host airway environment-specifically, the proteases that activate viral fusion proteins-acts as a reversible switch between two modes of spread: virus release and cell-to-cell fusion. Because this switch can be moved, protease-targeting drugs may not simply block infection but redirect how the virus spreads, with consequences for tissue damage. Recognizing protease availability as a controllable determinant of dissemination reframes how we think about respiratory virus pathogenesis and offers a rationale for tuning, rather than only suppressing, viral spread with host-directed therapeutics.