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 site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 ångström (A) 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 alpha/beta 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.

Bunyavirus N: eIF4F surrogate and cap-guardian

“…PB2 is a cap-binding peptide mediating association with the caps of pre-mRNAs. [45][46][47] Recent data indicates that the endonuclease activity for pre-mRNA scission and generation of the capped oligomer resides with PA. 48,49 PB1 has intrinsic polymerase activity and is responsible for both mRNA elongation and correct association with the viral RNA template. 50 In contrast to influenza virus, bunya-and arenavirus replication is exclusively cytoplasmic and caps are thus derived from mRNA rather than pre-mRNA.…”

The influenza virus RNA synthesis machine

“…95 Although it has been reported that alternative binary complexes of the polymerase subunits might be responsible for transcription or replication, 120,121 a number of other reports indicate that the polymerase heterotrimer is the functional unit in both processes. 8,48,49,63,66,122 Altogether, the genetic approaches indicated above enabled the proposal of a new model for influenza RNA replication, which is depicted in the diagram presented in Figure 5. Activation of vRNA synthesis would result from the interaction of a soluble polymerase complex (Non-resident polymerase-NRP1-) with the cRNP (Fig.…”

“…2). In addition to the subunit interfaces mentioned above, the atomic structures of domains of the PA and PB2 subunits have been determined 47,55,56,[63][64][65][66][67] but no information on the structure of the polymerase active site in the PB1 subunit is yet available. The data reported allowed the identification of the polymerase cap-binding site in PB2 and established that the endonuclease responsible for cap-snatching resides at the N-terminal region of PA, in agreement with previous genetic contacts detected in the cryo-EM structure of the mini-RNP.…”

“…47,68 The fold of the endonuclease domain and its biochemical properties are similar to those of type II restriction endonucleases. 63,66 In addition of the endonuclease active site, protease activities have been associated to the PA subunit of the polymerase [69][70][71] and a correlation has been reported between such an activity and the capacity of the polymerase to replicate. 72,73 The mechanism by which the polymerase subunits interact, the site where the trimeric complex is formed and its transport to the nucleus have been analysed in detail.…”

Comparative aspects of infectious salmon anemia virus, an orthomyxovirus of fish, to influenza viruses

“…ISAV, as with the infl uenza viruses, has an U residue at the 3'-end of the vRNA but this U residue is lost in the cap-stealing process, and the penultimate C residue at the 3'-end of the vRNA then becomes the fi rst nucleotide transcribed by the ISAV polymerase, which is similar to how infl uenza virus vRNA is transcribed [26,27]. The capstealing endonuclease activity of infl uenza virus lies in the PA subunit of the polymerase [28,29], and this subunit could thus be an attractive target for anti-infl uenza drugs. The capstealing endonuclease activity of ISAV has not been mapped in detail to any particular polymerase subunit.…”