Respiratory viral infections impose a substantial global health burden. Among these, respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in young children, immunocompromised individuals, and older adults. During the COVID-19 pandemic, co-circulation of RSV and COVID-19 led to increasing reports of coinfections, which continue to occur in endemic settings. In this study, we employed a physiologically relevant in vitro model comprising human primary respiratory epithelial cells cultured at an air–liquid interface (ALI), together with the A549-ACE2 cell line model, to investigate the impacts of sequential RSV and SARS-CoV-2 infection. This platform enabled us to examine how host responses primed by an initial RSV infection influence subsequent SARS-CoV-2 infection. Our findings demonstrate that RSV-induced host response significantly restricted SARS-CoV-2 gene expression. Notably, we identified that RSV induced phospholipid scramblase 1 (PLSCR1), an interferon-stimulated gene previously implicated in antiviral defense against SARS-CoV-2, as a novel potential mediator of this effect. Together, these mechanisms are consistent with the phenomenon of viral interference, whereby one respiratory virus transiently reduces susceptibility to another, providing a biologically plausible explanation for cross-protective effects observed in sequential infections.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants, young children, and immunocompromised individuals. Currently, FDA-approved monoclonal antibody therapies are limited to infants and young children with severe RSV disease. As a result, there is an urgent need for comprehensive studies of RSV pathogenesis to support the development of new therapeutic strategies. Exchange proteins directly activated by cAMP (EPAC) have recently emerged as key regulators in various viral infections. Our previous work identified EPAC isoform 2 (EPAC2) as a critical factor in RSV replication and host innate immune responses. However, the molecular mechanisms underlying EPAC2’s role in RSV infection remain unclear. In this study, we investigated EPAC2-mediated RSV infection by identifying EPAC2-interacting proteins. Proteomics and immunoprecipitation analyses revealed that junction plakoglobin (JUP) interacts with EPAC2 in both mock- and RSV-infected cells, with this interaction notably enhanced during RSV infection. To determine JUP’s role in RSV infection, we compared viral replication in JUP-deficient and control cells. JUP downregulation significantly reduced the production of infectious RSV particles, likely by impairing viral budding and viral gene transcription. Moreover, our findings indicate that JUP is essential for an effective cellular immune response to RSV infection. Together, these results suggest that EPAC2 and JUP may cooperatively regulate RSV replication and dissemination.