Illustrations
Figure 1
A. Overview of the different polymerase structures. The overall shape of the polymerase complexes in four resolved structures are shown with a fixed position of the central core. The PB1 subunit is colored beige, PB2 blue, PA green. The pdb are as follows: bat FluA bound to 5’+3’vRNA pdb 4WSB [16] ; human FluB bound to 5’ + 3’vRNA pdb4WSA [15] ; human FluB bound to 5’cRNA pdb 5EPI [17] , human apo-FluC pdb5d9 [14] . B. Shematics of the PA /P3, PB1 and PB2 domain organization. Numbering is given for human bat FluA, human FluB and human FluC as indicated. Hatched domains are those constituting the central core polymerase.
Figure 1
Figure 2
The catalytic core of influenza polymerase. A polymerase fold (pdb 4WRT) displaying the unique resolved structure of the priming loop. Example of the polymerase fold of the flu structure is given (pbd 4WRT) which is the only containing a resolved structure of the priming loop. A. Ribbon diagram of the PB1 subunit, with the modules typical of an RNA dependent RNA polymerase fold indicated. The conserved functional motifs of RNA polymerase lying in the palm domain are colored in purple, the priming loop in red and the PB1 bipartite NLS in rust. B. Diagram representing the polymerase fold of fluA PB1 flanked by the PA-C on one side and by the PB2-N on the other side of the thumb. PB1, PB2-N and PA-C are colored beige, blue and green respectively the NLS of PB1 is represented with rust spheres.
Figure 2
Figure 3
RNA promoter binding to the polymerase. All images are generated from the FluB structure pdb 4WRT [15] which is the only crystal containing full-length 5’ and 3’ vRNA strands used as surrogate of viral promoter. A. Representation of the 5’ and 3’ vRNA promoter, highlighting the base pairing of the distal region and configuration of single-strand extremities. B. Surface representation of the polymerase promoter-binding interface, showing the RNA-protein binding interface with the 3’ vRNA extremity. The 5’ and 3’ ends of vRNA strands are indicated. C. Surface representation of the vRNA 3’end at the entry of the putative template canal modeled from the superimposition with the primer-template structure of the Norwalk virus polymerase [15] . The 3’ end of the 3’ vRNA strand is indicated. D. Surface representation of the polymerase promoter-binding interface highlighting the binding 5’vRNA hook in a pocket formed by PB1 and PA. The 5’ end of the 5’ vRNA strand is indicated.
Figure 3
Figure 4
Cartoon representation of PB2 folding in promoter-bound (shown for FluB pdb 4WSA) and in the 5’cRNA bound (FluB pdb 5EPI) polymerase configurations. The step-wise representation of the PB2 structure starts with the rigid axis constituted by the cap-627 linker (purple) and mid domain (pink), then with the cap (light blue), 627 (cyan) and NLS (violet) subdomains, last in full PB2 protein (PB2-N in sky blue). N- and C-terminus extremities of the corresponding polypeptides are indicated.
Figure 4
Figure 5
The transcription-competent polymerases. A. Structures of 5’ + 3’ vRNA bound polymerases bat FluA (pdb 4WSB) and human FluB (pdb 4WSA), shown with the PB2-C and PA-ENDO domains in ribbon diagram and the rest of the polymerase in a surface view. A cap bound to the PB2 cap-binding domain is shown in red, and the catalytic site of PA endonuclease domain is colored purple. Color code for PB1, PB2 and PA subunits are as in figure 1 . B. Surface view of the human-fluB polymerase conformation (pdb 4WSA) highlighting the orientation of a cap bound to the PB2 cap binding site facing the catalytic center. The view position is across the PB2 cap binding domain as indicated in the panel a. C. Surface representation of the transcription-competent polymerase (from pdb 4WSA) highlighting the position of template exit indicated by the location of the obstructing PB2 helical lid (colored brown), and of the putative mRNA exit tunnel.
Figure 5
Figure 6
The different arrangements of the PB2 subunit in polymerase configurations. A. Representation of the structures of the FluB polymerase when bound to the 5’ + 3’ vRNA viral promoter (pdb 4WSA) or to the 5’cRNA only (pdb 5EPI), and of the apo-FluC polymerase (pdb 5d9). The polymerase core is represented in grey surface, the PB2-C is colored in a ribbon representation for cap domain (light blue), cap-627 linker (purple) and mid domain (pink). PB2-627 (cyan) and NLS (dark blue) domains are in surface representation. The PA-ENDO is shown in ribbon and colored green. Polymerase structures are represented with similar orientation of the polymerase core to highlight the rearrangements of the PB2-C domains in the polymerase complex. B. Surface representation of PB2-CAP bound to a cap represented in red spheres in the 5’cRNA polymerase, highlighting the obstruction of the cap-binding site through apposition to the PB2 cap-627 linker.
Figure 6
Figure 7
Polymerase dimer formation A. Dimer observed for FluA H5N1 subcomplex 1 (pdb3j9). Top: Ribbon representation of the dimer, PB1 is colored beige, PA is colored green, PB2-αhelices (attributed to aa 86 to 130) are shown in cyan (monomer 1) or blue (monomer 2). The PA α-helices involved in the dimer interface are shown in light pink (monomer 1) and dark pink (monomer2), PB2 helices involved in dimer interface are shown in cyan (monomer 1) and dark blue (monomer 2). Amino acids of PB1 proposed to be involved in tetramer formation are in colored yellow. Bottom: the surface of the dimer interface highlights the contacts between each monomer. B. Structural elements proposed for dimer interface are shown in surface representation colored as in one monomer of fluA H5N1 subcomplex 1, in the 5’cRNA bound form of fluB polymerase (pdb 5 EPI) and in the apo-fluC polymerase (pdb5d9). They are shown with the rest of the polymerase in ribbon (left) and in surface (right) representation to highlight both the contiguity and the accessibility of the intervening α-helices in the dimer formation.
Figure 7
Figure 8
Interaction with NP. A. Interaction interface with NP. The PB1 β ribbon interacting with NP is colored beige, 5’ v or cRNA in yellow, 3’vRNA in orange, PB2 N-terminal α helices involved in NP binding colored blue, the PB1-C-terminal α helices involved in the PB2-N/PB1-C interaction interface observed in isolated domains colored beige. The position of the PB1 NLS in the PB1β ribbon is labeled red for the FluB 5’ + 3’vRNA-bound and 5’c-RNA bound polymerases, but not in the FluC apo polymerase structure where it is not conserved. Both PB1 β ribbon and RNA strands are shown in ribbon relative to the rest of the polymerase in white surface. Images are built from pdb 4WSA for the FluB promoter-bound polymerase, pdb 5EPI for 5’cRNA FluB bound polymerase, pdb 5j9 for apo-FluC polymerase. B. Location of the interaction interfaces with NP in the promoter-bound FluB (left, pdb 4WSA), the 5’cRNA-bound fluB (right, pdb 5EPI) and the apo-FluC (pdb5d9) polymerases relative to the putative exit tunnels for products (dark blue arrow for mRNA product, red arrow for replicate product) and to the putative position of template exit, indicated by the location of the obstructing PB2 helical lid (colored brown). Note that the putative template exit is indicated on the face of the polymerase where the template is expected to emerge. The PB2-627K domain colored is cyan, residues involved in interaction with NP are in red. The PB2 627K, PB1C/PB2N helices bundle and PB1 β ribbon are in ribbon representation.
Figure 8
Figure 9
Adaptative Residues in the polymerase A. Adaptative residues of influenza polymerase. Residues are shown at the surface of the transcription-competent (pdb 5WSA, left) and 5’cRNA-bound (pdb 5EPI, right) forms of the FluB polymerase. Adaptative residues are colored red in PB2, purple in PA and brown in PB1, positions are given for the PA and PB1 subunits. B. Adaptative residues of the PB2 subunit colored in red are shown in a surface representation of the 5’cRNA-bound FluB polymerase, with transparency to highlight residues lying along the interaction interface between PB2 and PB1 or PA (indicated by arrows). C. Adaptative resides in the 627/NLS domains of PB2 are colored distinctly, their accessibility is shown in the transcription competent (left), and 5’-cRNA bound (right) forms of the fluB polymerase.
Figure 9
Figure 10
Polymerase interactions with host factors. A. Interaction with importin α. Top (Left) Ribbon representation of the NLS domain of 5’cRNA-bound FluB polymerase (pdb 5EPI) colored dark blue, with the rest of the polymerase shown in a surface representation. The residues involved in interaction with importin α are colored red. (right) surface representation of the PB2 NLS in the same polymerase configuration. Bottom-the NLS domain in the promoter-bound FluB conformation (pdb 4WSA), only partly resolved, is shown in ribbon representation, colored dark blue. The extreme C-terminus peptide containing the NLS lacks electron density reflecting, a flexible non-folded state and is represented with a dashed line. B. Interaction with hCLE. The interaction interface of PA with the human factor hCLE is shown in surface representation and colored in red in the FluB polymerase associated to 5’ + 3’ vRNA promoter (pdb 4WSA). Residues PB2 504 and PA 550 involved in RNA polII degradation are represented in purple spheres.
Figure 10
Figure 11
Potential distinctive binding interfaces. Surface representation of the FluB polymerase bound 5’ + 3’ vRNA (pdb4WSA, right) or to 5’cRNA bound (pdb 5EPI right). Surfaces specifically exposed in the 5′ + 3’vRNA polymerase are colored brown (PB2) and purple (PA), surfaces more exposed in the 5’cRNA-bound form are colored salmon (PB2) and pink (PA). The PB2 residue 627K is colored yellow.
Figure 11
Auteurs
1 Unité de génétique moléculaire des virus à ARN, CNRS, UMR 3569, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France
2 Université Paris Diderot, Sorbonne Paris Cité, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris cedex 15, France
Influenza viruses are segmented negative-sense RNA viruses whose RNA dependant RNA polymerase (RdRp) multiple activities are central for the viral life cycle. The RdRp is composed of three subunits, PB1, PB2 and PA. It binds to the extremities of each vRNA segments encapsidated with multiple copies of the Nucleoprotein (NP), altogether constituting the viral ribonucleoproteins (vRNPs). The RdRp performs both vRNA transcription and replication in the context of vRNP in the nuclei of infected cells. The temporal regulation of RdRp-associated activities is essential for the successful completion of the virus life cycle, but its understanding has been limited by the lack of structural information about the polymerase complex. The atomic-resolution of polymerase complexes from influenza virus type A, type B and type C came out in the past two years. We compile here the data provided by the near-concomitant resolution of several influenza polymerase crystal structures. We will highlight how structural information can contribute to our understanding of the interactions between the RdRp and viral or host factors.