Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5.
The genome of influenza A virus (IAV) comprises eight RNA segments (vRNA) which are transcribed and replicated by the heterotrimeric IAV RNA-dependent RNA-polymerase (RdRp). RdRp consists of three subunits (PA, PB1 and PB2) and binds both the highly conserved 3'- and 5'-ends of the vRNA segment. The IAV RdRp is an important antiviral target, but its structural mechanism has remained largely elusive to date. By applying a polyprotein strategy, we produced RdRp complexes and define a minimal human IAV RdRp core complex. We show that PA-PB1 forms a stable heterodimeric submodule that can strongly interact with 5'-vRNA. In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain. Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding. Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Christopher
C
Swale
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.
Alexandre
A
Monod
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Laura
L
Tengo
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Alice
A
Labaronne
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|EMBL Grenoble, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.
Frédéric
F
Garzoni
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Jean-Marie
JM
Bourhis
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|EMBL Grenoble, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.
Stephen
S
Cusack
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Guy
G
Schoehn
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|EMBL Grenoble, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|The School of Biochemistry, University of Bristol, Clifton BS8 1TD, United Kingdom.
Imre
I
Berger
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Rob W H
RW
Ruigrok
Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|CNRS, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.|Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
Thibaut
T
Crépin
D015967:Gene Expression Regulation, Viral; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism*; D007251:Influenza, Human / Q000378:metabolism* / Q000821:virology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism*
C107940:IPO5 protein, human; D021122:Protein Subunits; D012367:RNA, Viral; D014764:Viral Proteins; D028961:beta Karyopherins; D012324:RNA-Dependent RNA Polymerase