Interactome profiling reveals the LCMV nucleoprotein co-opts DDX helicases through conserved interfaces.

The nucleocapsid protein (NP) of lymphocytic choriomeningitis virus (LCMV) serves as a multifunctional regulator of viral replication and immune evasion, yet its full repertoire of host protein interactions remains incompletely characterized. Here, we employed proximity-dependent biotinylation coupled with mass spectrometry in HEK293T cells to systematically map the global NP interactome. Functional enrichment analysis demonstrated that NP selectively recruits host RNA-binding proteins (RBPs), thereby perturbing cellular metabolic pathways and the intracellular environment. Specifically, NP associates with multiple DEAD-box RNA helicases (DDX5, DDX17, DDX36, and DDX56). Genetic validation confirmed these interactions, which are associated with dysregulation of host RNA processing, translational control, autophagy, and oxidative stress responses during viral replication. Mechanistic studies revealed that NP co-localizes with DDX helicases at potential sites of viral replication through conserved N-terminal domains, with interfacial residues (Glu/Lys/Arg) predicted to play a critical role in complex formation. All-atom molecular dynamics simulations further established that NP-DDX5 binding induces conformational allostery in the host helicase. This modulation potentially facilitates viral RNA translation and virion assembly. Collectively, these findings identify a novel mechanism in HEK293T cells whereby arenavirus NP may co-opt DEAD-box helicases to subvert host cell functions, provide a structural basis for replication complex assembly, and highlight potential therapeutic antiviral intervention.