Structural basis for dsRNA recognition by NS1 protein of influenza A virus

Abstract

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7Å. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel α-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Dimer Interface of the Effector Domain of Non-structural Protein 1 from Influenza A Virus

“…9, left inset), as established in recent structural studies of NS1A RBD-dsRNA complexes (18,19). In fact, in the previous model, the orientation of the RBD with respect to the bound dsRNA is not consistent with the orientation of dsRNA relative to the conserved basic surface of the RBD defined by structures of NS1A RBD-dsRNA complexes (18,19). Moreover, in our model, the CPSF30-binding pocket is buried because NS1A oligomerization is mediated by this same interface.…”

“…In particular, based on the nature of the conserved dsRNA-binding surface of the RBD, we propose that the RBD is on the inside of the oligomer with its conserved tracks of basic residues determining the orientation of its interaction with dsRNA (Fig. 9, left inset), as established in recent structural studies of NS1A RBD-dsRNA complexes (18,19). In fact, in the previous model, the orientation of the RBD with respect to the bound dsRNA is not consistent with the orientation of dsRNA relative to the conserved basic surface of the RBD defined by structures of NS1A RBD-dsRNA complexes (18,19).…”

“…Domains corresponding to full-length NS1A dimers (mediated by RBD-RBD interactions) are shown in similar colors; ED subunits are labeled. The model was made using the coordinates from the x-ray crystal structures of PR8 NS1A RBD-dsRNA complex (PDB ID 2ZKO (19)), preserving the known dsRNA binding-topology observed in this complex, and Ud NS1A ED (PDB ID 3EE9 (22)), featuring a helix-helix dimer interface, by overlaying multiple copies of the RBD-dsRNA complex coordinates onto canonical dsRNA and manually docking the ED coordinates onto separate RBDs. In this schematic model, the protein wraps around the dsRNA via its RBD, forming a tube with the dsRNA in the center.…”

“…Left inset: view of the dsRNA-binding epitope of NS1A RBD. The relative orientation of the dsRNA parallel to the axes of helices 2 and 2Ј is determined by highly conserved tracks of basic residues (18) and other polar dsRNA-binding residues (19) lining the dsRNA-binding face, shown in dark and light blue, respectively. Right inset: view of the intermolecular ED-ED interface (22).…”

“…The isolated RBD domains from both NS1A (16,17) and NS1B (the NS1 protein of influenza B virus) (18) form unique sixhelical head-to-tail symmetric homodimers featuring a conserved dsRNA-binding epitope (18), which binds the major groove of A-form dsRNA (19). The isolated effector domain of NS1A also forms a homodimer, with each monomer subunit adopting a novel ␣-helix ␤-crescent fold (20 -22).…”