X-ray structure of NS 1 from a highly pathogenic H 5 N 1 influenza virus

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains— a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker—is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-Å-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection. NS1 of influenza virus is a multifunctional protein associated with a number of regulatory functions during a viral infection, including conferring resistance to antiviral interferon (IFN) induction. Although the mechanisms for how NS1 antagonizes IFN response remains to be fully understood, both the RNA binding domain (RBD) and the effector domain (ED) have been implicated in this activity. Our goal was to determine the X-ray structure of the fulllength NS1 protein to provide a more complete structural framework for understanding the functional aspects of NS1, including the effects of various mutations in H5N1 strains. In contrast to RBD and ED, which were individually amenable for crystallographic analysis, full-length NS1 readily aggregates and precipitates at the concentrations needed for crystallographic studies. We determined that two mutations, R38A and K41A, completely abrogated aggregation and allowed full-length NS1 to be sufficiently concentrated for crystallographic studies. The H5N1 NS1 structure was determined using molecular replacement techniques with previously reported structures of the RBD and ED as search models (see Methods and Supplementary Table 1). In the full-length H5N1 NS1 monomer structure, the RBD and ED are formed by residues 1–72 and residues 84–220, respectively (Fig. 1). The density corresponding to the five residues (75–79) in the linker region is not well defined, indicating the flexible nature of the linker. The electron density is relatively weak for residues 67–74, and these residues exhibit higher than average temperature factors (,70 Å). The proposed connectivity between RBD and ED (Fig. 1),