CD46 and DSG2 synergistically mediate human adenovirus type 7 infection

models, including receptor overexpression, RNA interference, competitive binding assays, antibody blockade, and surface plasmon resonance analysis, as well as differentiated human bronchial epithelial cells (HBEpiCs) and humanized mouse models, dual-receptor engagement was demonstrated to significantly enhance viral entry. Furthermore, in humanized dual-receptor transgenic mice expressing both receptors, this synergistic interaction correlated directly with increased viral replication and enhanced inflammatory pathology compared with single-receptor or wild-type controls. Mechanistic analyses further revealed that the viral fiber knob domain simultaneously can bind CD46 and DSG2 at distinct epitopes with differing affinities. This synergistic physical interaction contributes to the observed enhancement of infection efficiency. Collectively, these findings elucidate a critical aspect of HAdV-7 pathogenesis by establishing the CD46-DSG2 dual-receptor system as an important determinant of viral infectivity and virulence. These insights not only resolve long-standing controversies regarding receptor usage but also provide a structural framework for designing adenovirus vectors with optimized tissue tropism and reduced off-target effects and identify a target for broad-spectrum antivirals targeting cooperative engagement. IMPORTANCE: Human adenovirus type 7 (HAdV-7) is a clinically important pathogen associated with severe respiratory infections, yet its cellular entry mechanism has remained incompletely defined. Most existing studies have focused on individual receptor functions, leaving critical knowledge gaps unresolved. It remains unclear whether HAdV-7 relies on both CD46 and desmoglein-2 (DSG2), whether these receptors act synergistically or independently, and how their interactions influence viral infection dynamics. This study provides comprehensive evidence that HAdV-7 utilizes CD46 and DSG2 as synergistic co-receptors to mediate efficient infection, viral replication, and inflammatory pathology. This study resolves long-standing controversies regarding receptor usage in HAdV-7, elucidates a novel dual-receptor mechanism of infection, and offers a foundation for the design of novel adenovirus vectors with optimized tissue tropism.