Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit

Abstract

Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain) exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design experiments to elucidate the effects of mutations on polymerase-host factor interactions.

Figure 1. Structure and sequence alignment of C-terminal domains of influenza polymerase PB2 subunit.

(A) Ribbon diagram of the 627-domain showing secondary structure elements and the position of human specific lysine 627. Helices are in red and beta-strands in yellow as defined by DSSP . The conformation of the C-terminal tail (residues 676–693) is determined by crystal contacts. The structure shown is of the SeMet labelled protein. (B) Ribbon diagram of the 627-NLS-double domain showing the position of lysine 627. The 627-domain is in red and yellow, the core NLS-domain in cyan and blue and the truncated nuclear localization peptide in purple. The flexible inter-domain linker is in green. Figure 1A and Figure 1B were drawn with MOLSCRIPT and rendered with RASTER3D . (C) Sequence alignment of C-terminal regions of PB2 from influenza A and B viruses with superimposed secondary structure. The coloured bar under the alignment indicates the 627-domain (red), linker (green), core NLS-domain (cyan) and the bipartite NLS (purple). The seven host specific residues identified in this region are indicated with a blue square in the coloured bar. Alignment figure produced with ESPript .

Figure 2. Effect of the K627E mutation on the electrostatic surface of the 627-domain.

The electrostatic surface potentials were calculated from the crystal structures of (A) the Lys627 human determinant-containing domain, and (B) the Glu627 avian-like variant using DelPhi and displayed using PyMol . The potential scale ranges from -4 kT/e (red) to 4 kT/e (blue). The maps reveal that the K627E substitution disrupts a prominent basic surface patch which also includes residues Lys586, Arg589, Arg597, Arg630 and Arg646.

Figure 3. Identification of host species determinant sites.

Surface representations highlight the position of the sites on the (A) 627-domain and (B) 627-NLS-domain in two different orientations. In (B) the 627-domain and NLS domain are respectively in grey and dark grey. Major host specificity determinant residues are shown in green and with those for HxN2 subtype in blue. Residues 714 and 701 (yellow) were identified as host specificity determinants in a laboratory model of avian to mouse transmission .