NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals. Synopsis: The cryo-EM structure of the influenza A virus Pol-NS2 complex shows a highly ordered barrel-like hexamer. This study explains how the NS2-FluAPol interaction inhibits viral transcription and promotes genome replication. The structure of the full-length NS2 as part of the FluPol-NS2 complex is resolved at ~3.0 Å. NS2 forms a pseudo two-fold domain-swapped dimer. The binding of NS2 to FluAPol impairs FluAPol transcriptase activity by hampering FluAPol-Pol II CTD interactions. NS2 stalls FluAPol in the replicase conformation. The cryo-EM structure of the influenza A virus Pol-NS2 complex shows a highly ordered barrel-like hexamer. This study explains how the NS2-FluAPol interaction inhibits viral transcription and promotes genome replication. [ABSTRACT FROM AUTHOR]