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. 2014 Feb 21;343(6173):881-5.
doi: 10.1126/science.1247749. Epub 2014 Feb 6.

Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system

David L Akey  1 , W Clay Brown, Somnath Dutta, Jamie Konwerski, Joyce Jose, Thomas J Jurkiw, James DelProposto, Craig M Ogata, Georgios Skiniotis, Richard J Kuhn, Janet L Smith
Affiliations

Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system

David L Akey et al. Science. .

Abstract

Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis, and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus nonstructural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system and are a basis for elucidating the molecular mechanism of NS1 function.

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Figures

Figure 1
Figure 1
NS1 dimer structure. (A) NS1 dimer with one subunit in gray and the other colored by domain (blue β-roll, yellow wing with orange connector sub-domain, red central β-ladder). Disulfides are shown as yellow spheres and N-linked glycosylation sites as sticks with black C. A 20-residue disordered region is indicated with dotted lines. (B) Topology diagram for NS1 monomer, colored in blue, yellow, orange and red as in A. Glycosylation sites are indicated with green hexagons and disulfides with yellow circles. (C) Perpendicular views of NS1 from the edge (left) and the end (right) of the β-ladder. The β-roll (blue) and β-connector subdomain (orange) of the wing form a protrusion on one face of the β-ladder with the spaghetti loop (pink) and glycosylation sites on the other face. The wing domain is omitted from the left image for clarity.
Figure 2
Figure 2
Hydrophobic protrusion for membrane interaction. (A) NS1 electrostatic surface potential at pH 6.5 colored from electropositive in blue (+5 kT) to electronegative in red (-5 kT) with bound detergent (sticks with green C) and glycosylation sites (sticks with black C), viewed on the left as in Fig. 1A with the β-roll circled and facing the reader, and on the right as in Fig. 1C (right panel). (B) Effect of WNV NS1 on liposome structure. The negative-stain EM images show how NS1 treatment remodels liposomes (10% cholesterol, 90% phosphatidyl choline) into nano-particles, leaving almost no free NS1 when mixed in a 585:1 ratio of lipid:NS1 hexamer. (Control images in Fig. S5).
Figure 3
Figure 3
NS1 hexamer association. (A) NS1 hexamers: the symmetric hexamers in DEN2 NS1 (left) and WNV NS1 form 2 (center) and the splayed hexamer in WNV NS1 crystal form 1 (right). Molecular surfaces are green and white for two dimers. The third, front-most dimer has a red β-ladder, blue β-roll and yellow wings. The β-roll is entirely inside the hexamer. (B) Association of hydrophobic protrusions at the center of the NS1 hexamer: The electrostatic surface potential illustrates the hydrophobicity of the surfaces. Conserved side chains that form this surface are shown as sticks with green C. Fragments of the detergent molecules (Triton X-100) bound to the hydrophobic surface are shown as sticks with gray C. For clarity, the splayed WNV NS1 form 1 viewed from the open end. (C) Comparison of WNV NS1 hexamers in solution with the symmetric NS1 hexamer in crystals. Two-dimensional class averages (top row) are very similar to reprojections calculated from the WNV NS1 hexamer in crystal form 2 (bottom row).
Figure 4
Figure 4
NS1 and the immune system (A) Linear epitopes to NS1 mapped on the structure. The molecular surface of hexameric NS1 is colored from white to red based on the frequency of 108 NS1-mapped epitopes (http://www.iedb.org). The disordered loop at the periphery of the wing domain, modeled as poly-alanine and circled, is a frequent epitope. (B) Similarity of the NS1 wing α/β subdomain to the RIG-I family of innate immune proteins. An SF2 helicase domain of RIG-I (blue, 3TBK(25)) is superimposed on the WNV NS1 wing domain (yellow).
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