Respiratory syncytial virus (RSV) exploits host proteases to enhance its replication efficiency; however, the precise mechanisms remain unclear. Through high-throughput screening, we identified four matrix metalloproteinase 9 (MMP-9) inhibitors (including JNJ0966 and doxycycline hyclate) that suppress RSV infection in vitro and in vivo. Mechanistic studies revealed a proteolytic cascade wherein MMP-9 cleaves transglutaminase 2 (TGM2) at the PVP³⁷⁵↓VR site, generating an N-terminal fragment (1-375) that activates its protein disulfide isomerase (PDI) activity. This TGM2-dependent PDI activity catalyzes disulfide bond rearrangement in the RSV fusion glycoprotein (F), enabling F protein maturation, a prerequisite for membrane fusion and syncytium formation—key processes driving late-stage viral propagation. Genetic ablation of MMP-9 significantly attenuated RSV infectivity, while pharmacological inhibition reduced pulmonary viral loads and mitigated lung pathology in infected mice. Our study defines a unified MMP-9→TGM2→F axis as the core mechanism driving RSV replication and validates MMP-9 as a therapeutic target.
Key findings from the study include:

1. MMP-9 inhibitors show therapeutic efficacy against RSV in vitro/vivo.
2. MMP-9 is essential for late-stage RSV replication; MMP-9^-/- mouse model reduced infectivity.
3. MMP-9 drives pathogenesis by enhancing RSV F-mediated syncytium formation.
4. MMP-9 proteolysis of TGM2 enhances PDI activity to drive RSV-mediated syncytium formation.

DOI：10.1093/procel/pwaf063