A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade

The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F''s heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t_1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t_1/2, 1.9 to 7.5 min) or second (t_1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.Nipah (NiV) and Hendra (HeV) viruses are emerging members of the new Paramyxoviridae genus Henipavirus (12, 19). The Paramyxoviridae family comprises important viral pathogens, such as measles, mumps, human parainfluenza, respiratory syncytial, and Newcastle disease viruses and the henipaviruses (HNV), and NiV is its deadliest known member (4, 5). NiV has a broad host range and causes respiratory and neurological symptoms that often lead to encephalitis and a mortality rate of up to 75% in humans (21, 47). It can also spread efficiently and cause morbidity in economically important livestock (21). NiV is a biosafety level 4 (BSL4) pathogen and is considered a select agent with bio- and agro-terrorism potential. Both animal-to-human and human-to-human transmissions have been documented (4, 5), underscoring the need for research and treatment development. Since microvascular endothelial cell-cell fusion (syncytium formation) is a pathognomonic hallmark of NiV infection (50), understanding virus-cell and cell-cell membrane fusion should assist in the development of therapeutics to target this aspect of NiV pathobiology.Paramyxovirus membrane fusion requires the coordinated action of the attachment (G, HN, or H) and fusion (F) glycoproteins, and numerous canonical structural and functional features of G/HN/H and F proteins are conserved among paramyxoviruses (20, 23, 46, 48). G/HN/H proteins have a receptor-binding globular domain formed by a six-bladed beta-propeller connected to its transmembrane anchor via a flexible stalk domain (10, 51). For NiV and HeV, both ephrinB2 (B2) and ephrinB3 (B3) can be used as cell receptors (8, 33, 34), although B2 appears to be the higher-affinity receptor (34). B2 or B3 receptors bind to and activate G, which in turn triggers a conformation cascade in F that leads to membrane fusion (1). HNV F proteins are trimeric class I fusion proteins with structural/functional features common to their class (23, 52). HNV F proteins are synthesized as precursors that are cleaved and hence activated into a metastable conformation, poised for enabling membrane fusion. Cleavage generates a new N terminus that contains a hydrophobic fusion peptide (48). For NiV and HeV, the precursor (F₀) reaches the plasma membrane uncleaved, but endocytosis exposes F₀ to cathepsin L in the endosomes, cleaving F₀ to generate mature disulfide-linked F₁ and F₂ subunits that are trafficked back to the cell surface (14, 31). The structures of the retroviral Moloney murine leukemia virus p15E, lentiviral human immunodeficiency virus type 1 (HIV-1) gp41, Ebola virus GP2, influenza virus HA, and paramyxovirus SV5 and NiV-F fusion proteins all share similar trimeric coiled-coil core structures (6, 11, 17, 27, 53) and, in general, similar membrane fusion mechanisms (22, 23, 48).Receptor binding to paramyxoviral G/HN/H triggers a conformational cascade in F, leading to membrane fusion (Fig. ​(Fig.1).1). Although the determinants for F triggering on G/HN/H have not been defined clearly, evidence suggests that the stalk domain (7, 13, 24, 28, 29) and, at least for NiV, a region at the base of the globular domain of G (1) are involved in F triggering. Additionally, recent evidence indicates an interaction between the stalk region of the measles virus H protein and the globular domain of the cognate F protein (35). Once triggered, F progresses through a prehairpin intermediate (PHI) (Fig. 1A and B). In the PHI conformation, the fusion peptide is harpooned into the host cell membrane, and the N- and C-terminal heptad repeat domains (HR1 and HR2, respectively) are exposed. The HR domains then coalesce into the postfusion six-helix-bundle (6HB) hairpin conformation. In the 6HB, the transmembrane and fusion peptide domains are juxtaposed, bringing viral and target cell membranes together and driving membrane fusion (Fig. ​(Fig.1C)1C) (30, 48). Much evidence suggests that 6HB formation is coincident with membrane merger and that synthetic HR1 and HR2 peptides only bind to and inhibit fusion intermediates (e.g., PHI) prior to 6HB formation (9, 30, 37, 43, 48). Additionally, HR1 peptides can inhibit an earlier fusion intermediate than that inhibited by HR2 peptides (43), and HR2 peptides are invariably more potent inhibitors of fusion than HR1 peptides. HR2 peptides trap the PHI by binding to the radial interstices formed by the trimeric HR1 core, inhibiting 6HB formation and membrane fusion (22, 23, 48). Altogether, there is much evidence to support the fusion cascade shown in Fig. ​Fig.11 and the use of HR2 peptides to physically capture fusion intermediates (9, 30, 43, 48).Open in a separate windowFIG. 1.Nipah virus fusion cascade. The schematic shows the NiV fusion cascade broken down into three major stages. (A) EphrinB2 or ephrinB3 binding to NiV-G triggers the metastable NiV-F protein through allosteric mechanisms that are still being elucidated. (B) After F is triggered, it forms the PHI, in which a fusion peptide is harpooned into the host cell membrane. The PHI can be captured by peptides that mimic the NiV-F HR1 (orange-striped cylinder) or HR2 (green-striped cylinder) region and bind the F HR2 or HR1 region, respectively. (C) The HR1 and HR2 regions in the PHI coalesce to form the 6HB conformation, bringing the viral and cell membranes together and facilitating virus-host membrane fusion and viral entry. The viral membrane can be replaced by a cell membrane expressing the F and G glycoproteins in cell-cell fusion, resulting in syncytium formation. We term the transitions from A to B and from B to C phases I and II, respectively, of the fusion cascade. (D) Schematic representation of the F-triggering assay, showing its four main steps: (1) receptor binding at 4°C, (2) biotinylated HR2 peptide addition and induction of F triggering at 37°C, (3) fixation at 4°C with paraformaldehyde, and (4) signal amplification at 4°C. In the “time-of-addition” and “time-of-stopping” experiments, step 2 was modified as indicated in the text. The HR2 peptide (green hatched column) is shown with its N-terminal biotin modification (red star). Blue stars, streptavidin-APC; black, three-pronged symbols, activator; blue symbols with red octagons, enhancer.We previously developed a fluorescence-activated cell sorting (FACS)-based NiV-F-triggering assay by measuring the amount of HR2 peptide binding to F/G-expressing cells triggered by cell surface ephrinB2 (1). In this study, we further optimized our assay for robust quantification of HR2 peptide binding and used this assay to monitor the differential degree of F triggering induced by B2 or B3. In addition, through “time-of-addition” and “time-of-stopping” experiments (described below), we show that this HR2 binding assay can measure the half-lives of various fusion intermediates, i.e., the transition times from the prefusion (PF) state to PHI and from PHI to 6HB. Using a panel of hyper- and hypofusogenic mutants, we show that hyper- and hypofusogenicity can each be manifested through distinct effects on the half-lives of these fusion intermediates and/or the absolute amounts of F triggering. Thus, we elucidated the impacts of different mutations on individual steps of the fusion cascade. Since HR2 peptides can generally capture the PHI of class I fusion proteins, our assays should help efforts to understand fusion processes mediated by other class I fusion proteins.