The structural basis for cap binding by influenza virus polymerase subunit PB2

Abstract

Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

“…To clarify which function of the polymerase is impaired in avian strains, we performed a PB2 complementation assay in human cultured cells using the polymerase reconstitution system. In this assay, we co-expressed a modified AvianPr PB2 subunit (PB2-E627K-Tdef) that harbours both the adaptive mutation E627K as well as mutations in the cap-binding region that diminish the cap-binding activity 18 and therefore leads to inefficient transcription of mRNA from vRNA. If AvianPr is only defective for RNA replication in human cultured cells, the avian precursor polymerase should be able to complement the transcription defect of PB2-E627K-Tdef, whereas no complementation should occur if the AvianPr polymerase also has a defect in transcription (Fig.…”

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.…”

“…However, they all appear to have undergone convergent evolution to derive similar capbinding pockets. 45,46 In each of these three peptides the 5' cap is sandwiched between two aromatic amino acids (W, Y or F) and adjacent supplementary acidic residues (D or E) further contribute to peptide-cap stability. As might be expected, there are multiple aromatic amino acids within N that might fold into a cap-binding pocket in a manner parallel to that of CBP20, VP39 and eIF4E.…”

Nouvelle stratégie pour cibler la réplication du virus de la grippe

“…Bien que sujet à discussion, ce mécanisme de vol de la coiffe a finalement fait l'objet d'un consensus [1]. La sous-unité PB2 lierait de manière spécifique, par un site de fixation que notre laboratoire a réussi à caractériser [2], la coiffe des pré-ARNm cellulaires. L'ARNm serait ensuite clivé 10-13 nucléotides en aval de la coiffe par une activité endonucléase [1], qui fut un temps associée à la sous-unité PB2 [3] comportant un motif PD-(D/E)XK.…”

“…Nos données concernant le domaine PANter, combinées aux données déterminées précédemment par notre laboratoire pour le domaine de liaison de la coiffe de la sousunité PB2 [2], nous permettent de proposer un nouveau schéma pour le mécanisme du vol de la coiffe dans lequel la sous-unité PA est largement impliquée (Figure 2). La sousunité PB2 lierait spécifiquement, via son domaine de fixation, la coiffe des pré-ARNm cellulaires (Figure 2A).…”