Dissecting the Interaction Domains of SARS-CoV-2 Nucleocapsid Protein and Human RNA Helicase DDX3X and Search for Potential Inhibitors

The SARS-CoV-2 nucleocapsid protein (Np) plays multifunctional roles in the viral life cycle. By interacting with host cellular proteins, Np regulates viral RNA transcription, replication, and immune evasion. It controls genome packaging and counteracts host RNA interference mediated antiviral responses through its RNA binding activity. Previous studies revealed a physical interaction between Np and DDX3X, a human DEAD-box RNA helicase that facilitates the replication of several viruses. This interaction enhances Np affinity for double-stranded RNA and inhibits DDX3X helicase activity. Since Np-RNA binding activity promotes ribonucleoprotein complex formation, targeting this interaction is a promising antiviral strategy. We generated truncated protein variants to define interaction regions between Np and DDX3X. Using AlphaFold modelling, we identified RecA2 as the key DDX3X domain involved in Np binding. Finally, to disrupt Np-RNA complex formation, we screened a small molecule library of putative binders of Np N-terminal region and identified two candidate inhibitors for further development.