Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites

Abstract

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP.

Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

“…PB2 interacts with PB1, PA, and NP (49)(50)(51)(52). To examine whether PB2-S1 also binds to these viral proteins, we performed a coimmunoprecipitation assay.…”

Stabilization of Influenza Virus Replication Intermediates Is Dependent on the RNA-Binding but Not the Homo-Oligomerization Activity of the Viral Nucleoprotein

“…The rest of the vRNA interacts with multiple copies of NP, each molecule covering approximately 24 nucleotides (reviewed in reference 17). NP binds ssRNA with high affinity but little or no sequence specificity (2, 9, 22) and has been shown to homo-oligomerize and interact with the PB1 and PB2 subunits of the viral RdRp (4,8,14,15,23). Although cryoelectron microscopy reconstitution images of a mini-RNP containing nine NP molecules have been obtained (6), a detailed high-resolution structure of the vRNP is still lacking.…”

The influenza virus RNA synthesis machine

“…Interactions between the NP and polymerase subunits PB1 and PB2 have been identified biochemically, 39,40 but so far it is not possible to correlate such information with the polymerase-NP viruses to the mammalian hosts has revealed that host-dependent specific interactions of the virus polymerase and RNP with cellular factors are at the basis of an efficient replication in humans of influenza viruses transferred from the avian reservoir. [11][12][13][14][15] Here we summarise new structural, biochemical and genetic evidences that have set the stage for a more profound understanding of influenza virus transcription and replication, and discuss alternative models to describe the mechanisms of these processes (reviewed in ref.…”

“…Other virus factors, as for instance NS1 protein, may also be involved in virus genome amplification, as temperature-sensitive mutations in this is needed to encapsidate the cRNA product of replication, as both RNA-binding and NP oligomerisation are needed to support RNP replication, 34,87 but, in addition, it has been proposed that direct interaction of free NP with the polymerase could be involved in favouring de novo initiation over transcription. 39,40,88 Alternatively, it has been proposed that an interaction of newly synthesized NP with the RNP template could alter the relative ability for de novo versus primed initiation. 30,89,90 However, excess expression of NP did not result in an increase of RNA replication versus mRNA synthesis in a recombinant replicon system.…”