INTRODUCTION
Influenza virus poses a long-standing public health issue worldwide, causing both recurring seasonal flu and occasional pandemics in humans and other animals. Current vaccines and antivirals predominantly target the envelope proteins, which are prone to mutations, leading to decreased efficacy as well as drug resistance over time. By contrast, the ribonucleoprotein (RNP) complexes inside the virus are more evolutionarily conserved, which makes them promising targets for broad-spectrum antiviral development. Each RNP complex consists of a viral genomic RNA segment bound with multiple nucleoprotein (NP) subunits and a polymerase complex, which carries out transcription and replication of the viral genome in the host cell. Despite their critical functional roles, a high-resolution structural understanding of RNP assembly and its working mechanism has remained elusive.
RATIONALE
Although the structures of individual influenza NP and polymerase have been characterized, the complete RNP complex has been refractory to high-resolution structural analysis owing to its inherent flexibility. Previous cryo–electron microscopy (cryo-EM) studies of influenza RNP were limited to nanometer resolution, and the polymerase complex within the RNP could not be visualized. To address this gap, we combined cryo-EM single-particle analysis (SPA) and cryo–electron tomography (cryo-ET) to investigate both reconstituted and native RNPs in distinct functional states. Specifically, we reconstituted the shortest RNP segment—the nonstructural (NS) segment—of influenza D virus to reduce flexibility for detailed structural analysis of the NP-RNA packaging, and we used cryo-ET to capture the polymerase functioning on individual RNPs in three dimensions for subtomogram averaging. The overall picture of the RNP in the act could highlight important molecular interactions for RNP function and guide the focus of influenza inhibitor design.
RESULTS
Cryo-EM SPA of the reconstituted influenza D NS RNP revealed a right-handed, antiparallel double helix of NP polymers, with the RNA encapsidated in the minor groove and adjacent NP subunits linked by a conserved tail loop. Subtomogram averaging of native RNP purified from influenza A virus particles confirmed these features and further identified multiple different conformations indicative of dynamic interstrand motion within the double helix. The polymerase complex, visualized in preinitiation and elongation states, consistently associates with the RNP exterior, which supports a helical strand sliding model in which the polymerase processes along the RNA template for RNA synthesis while maintaining the overall double-helical architecture of RNP. This mechanism is enabled by the flexible tail loop connecting neighboring NPs, which renders the double-helical RNP highly dynamic and prone to sliding motion with a low energy barrier. Virtual screening against the tail loop binding interface of NP identified lead compounds that effectively inhibited influenza virus replication in cell-based assays.
CONCLUSION
By integrating high-resolution cryo-EM and cryo-ET approaches, we delineate the structural basis of influenza RNP assembly and reveal how the polymerase-driven RNA synthesis proceeds without disrupting the helical framework—potentially enhancing processivity and/or evading host immune detection. These findings offer insights for the design of next-generation, broad-spectrum anti-influenza therapeutics targeting the conserved RNP components.