BVDV NS5A promotes mitochondria-associated endoplasmic reticulum membrane (MAM) formation to enhance viral...

The nonstructural protein 5 A (NS5A) of bovine viral diarrhea virus (BVDV) is a multifunctional viral factor that coordinates BVDV RNA replication and assembly. Mitochondria-associated endoplasmic reticulum membrane (MAM) has emerged as a critical platform for lipid metabolism, Ca²⁺ signaling, and viral replication. However, whether BVDV NS5A promotes viral replication by targeting MAM remains unclear. In this study, we demonstrate that NS5A of BVDV promotes viral replication through increasing MAM formation. MAM is essential for efficient viral replication and is promoted by BVDV NS5A. NS5A directly interacts with the host MAM-tethering protein, vesicle-associated membrane protein-associated protein B (VAPB), thereby remodeling this organelle interface. We further mapped this interaction to a specific binding interface on NS5A centered around key residues (G246, K249, D259) and (D398, K400, T402, D405). Crucially, using a reverse genetics system, we show that mutations disrupting the NS5A-VAPB interaction abolish MAM expansion and severely impair viral replication. Conversely, re-establishing this interaction through complementation restores both MAM integrity and viral propagation. Taken together, our findings reveal a critical host reshaping mechanism in which BVDV NS5A interacts with VAPB to enlarge the MAM platform and create a pro-viral environment that is essential for efficient replication. Our work identifies the NS5A-VAPB interface as a promising target for host-directed antiviral strategies with potential applicability to related members of the Pestiviridae family.