Influenza A virus RNA polymerase structures provide insights into viral genome replication

Influenza A viruses (IAV) are responsible for seasonal epidemics, and pandemics can arise from novel zoonotic influenza A viruses transmitting to humans(1,2). IAV contain a segmented negative sense RNA genome that is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the PB1, PB2, and PA subunits(3–5). Although the high-resolution crystal structure of bat IAV polymerase (FluPol(A)) has been reported(6), there are no complete structures available for human and avian FluPol(A). Furthermore, the molecular mechanisms of viral RNA (vRNA) replication, which proceeds through a complementary RNA (cRNA) replicative intermediate and requires polymerase oligomerisation(7–10), remain largely unknown. Here we report 3.0 – 4.3 Å resolution structures of polymerases from human A/NT/60/1968 (H3N2) and avian A/duck/Fujian/01/2002 (H5N1) IAVs, obtained by crystallography and cryo-electron microscopy (cryo-EM), in the presence or absence of cRNA or vRNA template. In solution, FluPol(A) forms dimers of heterotrimers through the PA C-terminal domain and the PB1 thumb and PB2 N1 subdomains. A cryo-EM structure of a monomeric FluPol(A), bound to cRNA template, reveals a binding site for the 3′ cRNA at the dimer interface. Using a combination of cell-based and in vitro assays we show that the FluPol(A) dimer interface is required for initiation of vRNA synthesis during viral genome replication. Furthermore, we show that a nanobody, a single-domain antibody, which interferes with FluPol(A) dimerisation, inhibits vRNA synthesis and consequently virus replication in infected cells. Our study provides the first high-resolution structures of medically relevant FluPol(A) and offers novel insights into the replication mechanisms of the viral RNA genome. Furthermore, it identifies novel sites of FluPol(A) that could be targeted for antiviral drug development.