Biological Significance of the Second Receptor Binding Site of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein

Abstract

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-␣(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

“…The NDV HN protein forms tetramers on the virion, and similar to the PIV5 HN, the isolated head NA domains exist primarily as monomers (4)(5)(6)(16)(17)(18). The HN stalk provides a significant driving force for oligomerization, but the TM domains are also thought to play a role (19).…”

Addition of N-Glycans in the Stalk of the Newcastle Disease Virus HN Protein Blocks Its Interaction with the F Protein and Prevents Fusion

“…On the side of the helix at which N-glycan addition decreases HAd, the N-glycan may be disrupting HNЈs second sialic acid binding site formed by the dimeric interface, thereby decreasing HNЈs receptor binding avidity. This idea is based on the demonstrated role of the second sialic acid binding site in attachment (3). N-glycan addition to the other side of the HR2 helix increases receptor binding avidity and may do so by stabilizing the receptor-binding structure of HN, resulting in a more efficient interaction with receptors.…”

Residues in the Stalk Domain of the Hendra Virus G Glycoprotein Modulate Conformational Changes Associated with Receptor Binding

“…For the HN glycoproteins both receptor binding activity and the neuraminidase active site are localized to the globular head domain, as with human parainfluenza virus 3 (hPIV-3) and NDV HN (10,16,30,52). In addition to receptor binding and specificity, the attachment glycoproteins also possess the fusion promotion activity and specificity for the F glycoprotein.…”