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, 76 (24), 13028-33

Role of the Hemagglutinin-Neuraminidase Protein in the Mechanism of Paramyxovirus-Cell Membrane Fusion

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Role of the Hemagglutinin-Neuraminidase Protein in the Mechanism of Paramyxovirus-Cell Membrane Fusion

Toru Takimoto et al. J Virol.

Abstract

Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth beta sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.

Figures

FIG. 1.
FIG. 1.
3D structure of NDV HN protein and locations of mutated residues. The mutations we characterized in the first (A) and second (B) series of experiments are shown in the structure of HN-Neu5Ac2en complex. The side view in panel A shows the locations of mutations that affected fusion promotion activity in the first series of analysis. The structures were generated using WebLab ViewerPro 3.5 (Molecular Simulations Inc.).
FIG. 2.
FIG. 2.
The fusion promotion domain is located at the large hydrophobic surface of the HN protein. (A) The surface area of HN involved in the dimer interface of the HN-Neu5Ac2en complex is yellow. The area involved in the dimer interface of HN alone is blue. The dimer interface area involved in both forms of crystals is green. (B) GRASP software was used to calculate the electrostatic potential of the surface of HN. Areas of positive potential (blue) and areas of negative potential (red) are shown. (C) Residues that affected the fusion promotion activity of HN are green, and those that did not affect it are purple. All of the structures in panels A to C are shown in the same orientation.
FIG. 3.
FIG. 3.
Receptor binding-induced conformational change of HN protein. A space-filling model of a monomer of HN-Neu5Ac2en depicts the protein colored according to the root mean square deviation of the α-carbon atoms between the two crystal forms of HN. The red regions are those regions that move more than 0.5 Å and show that the primary conformational change is restricted to a small region connecting the sialic acid-binding/hydrolysis site to the large hydrophobic surface, which is to the left of the image.
FIG. 4.
FIG. 4.
Models of the role of HN protein in membrane fusion. (A) In the first model, HN protein forms a dimer through its hydrophobic site and physically interacts with the fusion (F) protein trimer to form an HN-F complex. HN binding to the receptor triggers a conformational change near the hydrophobic site, which results in the structural change of dimer formation of HN and the dissociation of the HN-F complex. The receptor-binding/NA pocket is shown as a gray spot. (B) In the second model, two HN molecules align to form a dimer. HN protein interacts with the F protein at its hydrophobic surface and prevents the exposure of the fusion peptide to the environment. Receptor binding induces a structural change near the hydrophobic surface of HN, which causes the dissociation of the HN-F complex and releases the fusion peptide to initiate membrane fusion. Yellow spots in the F trimer indicate the locations of radial channels that may sequester the fusion peptide. The hydrophobic surface of the HN is shown in red. (C) In both models shown above, HN protein prevents the conformational change of the fusion peptide (blue arrow) of the F protein until the virus reaches the target cell membrane. Receptor binding and the subsequent conformational change of HN result in the dissociation of the HN-F interaction and the activation of the F protein. The F protein then inserts the fusion peptide into the target membrane and merges the two membranes by forming a coiled-coil structure between the two heptad repeat regions near the fusion peptide and TM domains.

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