Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit

Abstract

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

Keywords: DSF; PB2; biophysics; cap-binding domain; crystal structure; influenza virus; isothermal titration calorimetry (ITC); ligand-binding protein; mRNA cap analog.

FIGURE 1.

FluB PB2^cap exhibits elevated structural flexibility in solution. A, size-exclusion chromatography elution profiles of FluA (red) and FluB (blue) PB2^cap analyzed by HiLoad 16/60 S200. The elution positions of Bio-Rad molecular mass markers were also indicated in gray. Inset: SDS-PAGE gel of FluA and FluB PB2^cap. mAU, milliabsorbance units. B and C, DSF implicates direct binding of m⁷GTP and GTP to FluA and FluB PB2^cap. The interactions of m⁷GTP and GTP with Flu PB2^cap were analyzed by thermal shift assay. Purified FluA PB2^cap and FluB PB2^cap were tested with different concentrations of m⁷GTP or GTP. Mean values out of three independent experiments are depicted for the melting temperature (A) and thermal shift (B) graphs.

FIGURE 2.

Crystal structures of FluB PB2^cap. A–C, ribbon diagram of the FluB PB2^cap in unliganded form (green) overlaid with FluA PB2^cap (blue, A), m⁷GTP-bound form (B), and GTP-bound form (C). D–F, close-up view of the FluB PB2 active site in unliganded form (D), m⁷GTP-bound form (E), and GTP-bound form (F). Active residues and ligands are shown in ball-and stick mode with green carbon atoms; hydrogen bonds involving ligands are represented as dashed lines. The 1.9 Å 2|F_o| − |F_c| omit electron density is contoured at 2.5σ (blue mesh).

FIGURE 3.

Comparison of binding modes of various cap analogs in FluA and FluB PB2^cap. A–C, comparison of the conformations of methylated cap analogs in FluA PB2^cap (A), FluB PB2^cap (B), and FluB PB2^cap Q325F (C). D and E, comparison of the conformation of GTP in FluB PB2^cap (D) and GDP in FluB PB2^cap Q325F (E). Active residues and ligands are shown in ball-and stick mode; FluA, FluB, and FluB Gln-325 are colored in green, blue, and salmon, respectively. Hydrogen bonds involving ligands are represented as dashed lines.

FIGURE 4.

Characterization of mRNA cap analogs binding to FluA and FluB PB2^cap by ITC. A, ITC measurement for the interactions between FluA PB2^cap and m⁷GTP, FluA PB2^cap and GTP, FluB PB2^cap and m⁷GTP, and FluB PB2^cap and GTP (from left to right). B, table summary of ligand-binding isotherms derived from ITC.