Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

Abstract

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

“…Recently solved atomic structures of PIV5 HN and NDV HN show distinct arrangements of the globular head domains (19,23,41), with the globular head dimers of HN either attaining a "down" position, where one monomer of each dimer interacts with the stalk 4HB (23), or an "up" position, where this interaction is absent (19). A third hybrid arrangement of PIV5 HN globular heads "2 heads up, 2 heads down" was also observed recently.…”

“…Recently obtained atomic structures of HN stalk domains from NDV HN (23) and PIV5 HN (29) showed the stalks to be four-helix bundles (4HB), possessing a C-terminal linear coiled-coil region with an 11-mer hydrophobic central core repeat, adjacent to an N-terminal left-handed supercoiled region with 7-mer repeats. A large body of data suggests that F interacts with the attachment protein through the HN, H, or G stalk domains (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41); however, the exact nature of the interaction and nature of the F activation process are not clear.…”

Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies

“…promoting region ("four-heads-down" conformation) (12); (v) different crystal structures of PIV5 HN tetramers have revealed the heads can adopt a four-heads-up conformation or a hybrid two-heads-up/two-heads-down conformation (8,30); (vi) there is an energetic requirement for the movement of the heads, likely indicating that receptor binding is the trigger (41).…”

“…Recently obtained atomic structures of HN stalk domains from NDV HN (12) and PIV5 HN (18) showed the stalks to be four-helix bundles (4HB). A large body of data suggests that F interacts with the attachment protein through the stalk domains (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

“…4). Together with biochemical and structural data of paramyxovirus attachment proteins (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), a general model of paramyxovirus fusion has been proposed: upon activation by the attachment protein, metastable prefusion F undergoes a series of large-scale, ATP-independent conformational changes, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. The energy released during F refolding is believed to facilitate membrane fusion to create a pore between the virus and host cell through which the viral ribonucleoprotein complex can enter the target cell.…”