The NS1 protein of influenza virus is an important virulence factor and has been demonstrated to interact with a wide variety of viral and cellular biomolecules (Hale, Randall et al., 2008 ). In particular, crystallographic structures have been obtained of the N-terminal RNA-binding domain (RBD) in complex with dsRNA (Cheng et al., 2009 ) and of the C-terminal effector domain (ED) in complex with the F2F3 portion of the cellular processing and specificity factor CPSF30 (Das et al., 2008 ) and with the iSH2 domain of the PI 3-­kinase regulatory subunit p85β (Hale, Kerry et al., 2010 ). Furthermore, both domains form homodimers in vitro (Bornholdt & Prasad, 2006 ; Chien et al., 1997 ; Hale, Barclay et al., 2008 ; Xia et al., 2009 ). While the conformation of the RBD dimer appears to be conserved, two forms of the ED dimer have been proposed: the strand–strand dimer and the helix–helix dimer (Hale, Barclay et al., 2008 ; Xia et al., 2009 ; Bornholdt & Prasad, 2006 ). The helix–helix dimer is present in all wild-type NS1 ED structures (Kerry et al., 2011 ) and in the structure of full-length NS1 (Bornholdt & Prasad, 2008 ). In contrast, the canonical strand–strand dimer has only been observed in crystallographic contacts in a few NS1 ED structures obtained using NS1 from the A/Puerto Rico/8/34 (PR8) strain. The structure of the full-length NS1 protein from an H5N1 strain has also been reported to exhibit this interface, although in a somewhat distorted form, leading to claims that the strand–strand dimer is involved in NS1 oligomerization (Bornholdt & Prasad, 2008 ). The introduction of a W187A mutation into the ED induces a monomeric phenotype (Hale, Barclay et al., 2008 ; Kerry et al., 2011 ; Xia & Robertus, 2010 ) and, intriguingly, two crystal structures of the PR8 NS1 ED containing this mutation also exhibit this strand–strand interface.

The construction of the pRSFDuet plasmid expressing a His₆-tagged version of PR8 NS1 ED (W187A) (residues 73–230) has been described previously (Kerry et al., 2011 ). The plasmid was transformed into Escherichia coli Rosetta (DE3) expression strain (Novagen) for protein expression. The transformed E. coli cells were inoculated into Luria–Bertani (LB) medium with 50 µg ml⁻¹ kanamycin at 310 K. 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce protein expression when the optical density at 600 nm (OD₆₀₀) of the culture reached 0.6. Cell culture continued for 16 h at 295 K before harvesting by centrifugation at 7000g for 20 min at 277 K. The harvested pellet was resuspended in 20 ml phosphate-buffered saline supplemented with 1 mM MgCl₂, 20 µg ml⁻¹ DNase (Sigma), 200 µg ml⁻¹ lysozyme (Sigma) and protease-inhibitor cocktail tablets (two tablets per 20 ml; Roche Diagnostics) and incubated for 2 h at 295 K, after which the crude cell extract was centrifuged at 20 000g for 15 min at 277 K. The supernatant was then supplemented by the addition of 5 ml 2 M NaCl and 0.5 ml 250 mM imidazole before loading onto a 15 ml nickel column (GE Healthcare). Bound protein was eluted using 500 mM imidazole and then dialysed for 16 h at 295 K against 200 mM NaCl, 50 mM Tris pH 7.4, 1 mM dithiothreitol, during which time cleavage of the His₆ tag was achieved by addition of 200 U tobacco etch virus protease (Invitrogen). Uncleaved protein was removed from the dialysed fraction by loading it onto a 15 ml nickel column. The flowthrough fraction was then pooled for gel filtration using a 120 ml HiLoad 16/60 Sephadex 75 column (GE Healthcare).

The novel crystal form of PR8 NS1 ED (W187A) shows the α/β fold common to all structures of NS1 ED (Bornholdt & Prasad, 2006 ; Kerry et al., 2011 ). In contrast to the majority of NS1 structures, only one molecule of PR8 NS1 ED (W187A) was present within the asymmetric unit, although a strand–strand interface homologous to those observed for previous structures obtained using this con­struct was observed between symmetry-related monomers (Fig. 1 a). As expected from its monomeric form, the helix–helix dimer present within all wild-type structures of NS1 was not observed in the crystal lattice (Fig. 1 b). This is in agreement with previous structures obtained using this construct, which also lack the helix–helix dimer interface (Kerry et al., 2011 ). Therefore, since the PR8 NS1 ED W187A mutant is monomeric in vitro (Kerry et al., 2011 ), it appears highly likely that the helix–helix dimer is the predominant interface for ED homodimerization. However, it is intriguing to observe that the strand–strand dimer is conserved among all three PR8 NS1 ED (W187A) structures that have now been solved. Additionally, this interface is also employed in two of the four wild-type PR8 NS1 ED structures [PDB entries 2gx9 (Bornholdt & Prasad, 2006 ) and 3o9u (Kerry et al., 2011 )]. This is a remarkable coincidence, especially considering that the crystals leading to these five structures belonged to different space groups (P3₂21, P2₁2₁2₁, C222₁, P4₃22 and P6₄) and that conservation between mutant and wild-type structures is not observed for any other NS1 ED interface. Comparison of the strand–strand dimers indicates two distinct orientations of the monomers relative to one another (Supplementary Fig. 1¹). Although one orientation predominates, the AF and BD dimers of 3o9u adopt a slightly twisted arrangement, indicating that there may be some flexibility in the contacts formed at this interface. The partial con­servation of the strand–strand packing interface indicates that while it may not allow ED dimerization, extension of the β-sheet is a possible method of interaction with cellular and viral binding partners. Interestingly, this interface is not observed in other structures of the NS1 ED, even when mutations preventing helix–helix dimerization are introduced [e.g. W187Y (PDB entry 3kwi) and W187A (PDB entry 3kwg); Xia & Robertus, 2010 ]. Therefore, it could be concluded that any functional properties of this interface may vary between influenza virus strains. Such strain-specificity has been observed for some functions of NS1, most notably interaction with CPSF30, which is associated with NS1s from H3N2 and H2N2 subtypes but not all isolates of the H1N1 subtype (Kuo et al., 2010 ; Hale, Steel et al., 2010 ). However, while the other NS1 ED solved from an H1N1 subtype (A/California/07/09; PDB entry 3m5r; Center for Structural Genomics of Infectious Diseases, unpublished work) does not exhibit a strand–strand dimer, it does show antiparallel strand–strand interactions via an alternative arrangement (Supplementary Fig. 2¹). In structures of NS1 from PR8 the strand–strand dimer is formed by residues 88–91 of the two monomers interacting with one another to form a contiguous β-sheet; however, in the case of 3m5r residues 80–85 of chain A are sandwiched between residues 87–92 of chain G and residues 86–89 of chain B. This variant on the β-sheet augmentation theme suggests that such interactions may not be restricted by the sequences and arrangements observed in the strand–strand packing interface.