hMPV M2-2 protein modulates host mRNA alternative splicing to favor virus replication

Human metapneumovirus (hMPV) is a widespread and prevalent respiratory pathogen, yet the mechanisms by which hMPV modulates host gene expression to facilitate replication remain incompletely characterized. Here, we systematically profiled hMPV-host protein-protein interactions by affinity purification-mass spectrometry and assessed their impact on host messenger RNA (mRNA) splicing using RNA sequencing, complemented by genetic perturbation, protein interaction assays, and fluorescence microscopy-based subcellular localization and colocalization analyses. Among the viral proteins analyzed, the regulatory protein M2-2 was found to directly interact with multiple splicing factors. Transcriptomic analysis revealed that M2-2 triggers extensive changes in alternative splicing across host genes, notably affecting genes encoding serine-threonine kinase receptor-associated protein (STRAP) and tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2), both of which exhibit distinct splicing patterns during hMPV infection. Knockdown of M2-2 using short hairpin RNA (shRNA) during hMPV infection reversed M2-2-driven changes in the splicing patterns of STRAP and TANC2, confirming its regulatory role. Functional knockout of STRAP or TANC2 significantly impaired viral replication, indicating that the M2-2-favored isoforms are required for efficient viral propagation. Collectively, our findings demonstrate a novel mechanism by which hMPV M2-2 hijacks the host splicing machinery to reprogram key factors that facilitate viral replication. This extends the functional repertoire of M2-2 and highlights the virus-spliceosome interface as a potential therapeutic target for anti-hMPV intervention.