Influenza virus Non Structural protein 1 (NS1) Chaperones Nucleoprotein (NP) Oligomerization to Coordinate Ribonucleoprotein Assembly and Genome Replication

Influenza virus nucleoprotein (NP) co-transcriptionally enwraps viral genomic/antigenomic RNAs to assemble viral ribonucleoprotein complexes (RNPs), while the non-structural protein-1 (NS1) is classically recognized as an antagonist of host antiviral responses. Although NP-NS1 interaction has long been reported, the molecular basis of this protein-protein interaction and its relevance in the virus life cycle remain elusive. Here, we define the NP-NS1 interaction interface and uncover a noncanonical role of NS1 in regulating RNP assembly and viral genome replication. Using integrated biochemical, biophysical, and structural analyses, we demonstrate that the RNA-binding domain (RBD) of NS1 directly engages the NP tail-loop, conferring high affinity for monomeric NP. Functional assays reveal that a cytoplasm-restricted NS1 variant acts as an NP-specific chaperone by stabilizing monomeric NP through sequestration of its intrinsically flexible tail-loop and by scaffolding NP-NP interactions. During influenza virus infection, phosphorylation at the homotypic interface restricts NP in a replication-competent monomeric state. Cytoplasmic NS1 recruits these phosphorylated monomeric NPs into assembling RNPs, thereby facilitating viral genome replication and virus propagation. Collectively, our findings establish NS1 as a selective cytoplasmic chaperone of NP and reveal a novel regulatory axis governing influenza virus RNP biogenesis and replication.