Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site

Abstract

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site¹ and reportedly harbours endonuclease activity², whereas PB2 is responsible for cap binding^2,3,4. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease⁵ and protease activities⁶ as well as viral RNA/complementary RNA promoter binding⁷. Here we report the 2.2 ångström (Å) crystal structure of the N-terminal 197 residues of PA, termed PA_N, from an avian influenza H5N1 virus. The PA_N structure has an α/β architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX_N(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA_N provide further evidence that PA_N holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA_N holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA_N is an important target for the design of new anti-influenza therapeutics.