Host ANP32A mediates the assembly of the influenza virus replicase

Aquatic birds represent a vast reservoir from which novel pandemic influenza A viruses can emerge(1). Influenza viruses contain a negative-sense segmented RNA genome which is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein (vRNP) complexes(2,3). RNA polymerases of avian influenza A viruses (FluPol(A)) replicate viral RNA poorly in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity(4). Interestingly, host adaptive mutations, particularly a glutamic acid to lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit (PB2(627)), allow avian FluPol(A) to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here, we report cryo-EM structures of influenza C virus polymerase (FluPol(C)) in complex with human and chicken ANP32A. In both structures, two FluPol(C) molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain (LRR) of ANP32A. The C-terminal low complexity acidic region (LCAR) of ANP32A inserts between the two juxtaposed PB2(627) domains of the asymmetric FluPolA dimer, providing insight into the mechanism behind the PB2E(627K) adaptive mutation in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a vRNP.