Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms - PubMed (original) (raw)
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Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms
Stéphane Boivin et al. J Biol Chem. 2010.
Abstract
The heterotrimeric RNA-dependent RNA polymerase of influenza viruses catalyzes RNA replication and transcription activities in infected cell nuclei. The nucleotide polymerization activity is common to both replication and transcription processes, with an additional cap-snatching function being employed during transcription to steal short 5'-capped RNA primers from host mRNAs. Cap-binding, endonuclease, and polymerase activities have long been studied biochemically, but structural studies on the polymerase and its subunits have been hindered by difficulties in producing sufficient quantities of material. Recently, because of heightened effort and advances in expression and crystallization technologies, a series of high resolution structures of individual domains have been determined. These shed light on intrinsic activities of the polymerase, including cap snatching, subunit association, and nucleocytoplasmic transport, and open up the possibility of structure-guided development of new polymerase inhibitors. Furthermore, the activity of influenza polymerase is highly host- and cell type-specific, being dependent on the identity of a few key amino acid positions in the different subunits, especially in the C-terminal region of PB2. New structures demonstrate the surface exposure of these residues, consistent with ideas that they might modulate interactions with host-specific factors that enhance or restrict activity. Recent proteomic and genome-wide interactome and RNA interference screens have suggested the identities of some of these potential regulators of polymerase function.
Figures
FIGURE 1.
Cap-snatching transcription mechanism of influenza polymerase. The PA-PB1-PB2 complex is localized in the nucleus of the infected cell. During transcription, the PB2 subunit binds the 5′,7-methylguanosine cap of a host pre-mRNA molecule (red), which is subsequently cleaved 10–15 nucleotides downstream by the PA endonuclease. The resulting short capped RNA primer is used to initiate polymerization by the RNA-dependent RNA polymerase of the PB1 subunit using 5′- and 3′-bound vRNA (green) as template, resulting in capped, polyadenylated, chimeric mRNA molecules (red and blue) that are exported to the cytoplasm for translation into viral proteins.
FIGURE 2.
Features of the polymerase subunits. Linear representations of the three polymerase subunits are annotated to show the structurally characterized domains (large boxes). Residues implicated in host adaptation (67) are shown in black. X-ray crystal structures of interaction regions of PA-PB1 (33, 34) and PB1-PB2 (48) are presented with helix colors as in the linear representations.
FIGURE 3.
X-ray structures of the cap-snatching domains. a, the PA endonuclease domain (37) exhibits a fold similar to members of the PD-(D/E)_X_K family of nucleases. The key active-site residues and two manganese ions (pink) are shown. b, the PB2 cap-binding domain complexed with m7GTP (55) shows a novel fold but binds the positively charged base through a sandwich of aromatic residues in a manner similar to other cap-binding proteins. The binding site is shown with ligand-contacting residues.
FIGURE 4.
Residues at the C terminus of PB2 implicated in host adaptation. a, the PB2 627-NLS domain comprises two independently folded domains, the 627 domain (dark gray) and the NLS domain (light gray), interacting via a polar interface (57). Residues implicated in host adaptation (67) are shown in yellow. b, the crystal structures show electrostatic surface potentials of the human 627 domain with Lys-627 (left) and a point mutant with the avian-like Glu-627 residue (right). Formation of a large basic surface occurs upon host adaptation that is mediated almost universally by an E627K mutation.
References
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