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

Abstract

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 ϳ2-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.

Cysteines in the Stalk of the Nipah Virus G Glycoprotein Are Located in a Distinct Subdomain Critical for Fusion Activation

“…F triggering involves the formation and exposure of heptad repeat region 1 (HR1), which is coincident with fusion peptide insertion into the target cell membrane. The HR1 and HR2 in this prehairpin intermediate then fold together to form the 6-helix bundle that drives membrane fusion (2). To test our hypothesis, we used a previously established assay for F triggering that relies on the binding of labeled HR2 peptides to the trimeric HR1 core, which is exposed during prehairpin intermediate formation (1,2).…”

“…The HR1 and HR2 in this prehairpin intermediate then fold together to form the 6-helix bundle that drives membrane fusion (2). To test our hypothesis, we used a previously established assay for F triggering that relies on the binding of labeled HR2 peptides to the trimeric HR1 core, which is exposed during prehairpin intermediate formation (1,2). WT or cysteine mutant G proteins were coexpressed with NiV-F in receptor-deficient CHO cells.…”

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…To investigate whether the chimeric pro- teins G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 are defective at initial F triggering or at this later stage in the fusion process, we determined whether the defect is before or after insertion of the fusion protein into the target cell. Insertion of F's fusion peptide into the target cell indicates that the prehairpin intermediate has formed (18,28,(41)(42)(43); at this stage, F mediates attachment to the target cells, and disengagement of the receptor binding protein does not lead to release from the target cell (13,18,28,40,41,44). To determine whether the G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 chimeric proteins activate NiV F up to the stage of the prehairpin intermediate with fusion peptide inserted but then fail to complete the fusion process, we modified the assay described in Fig.…”

Circulating Clinical Strains of Human Parainfluenza Virus Reveal Viral Entry Requirements for In Vivo Infection

“…Human parainfluenza viruses possess a paired receptor-binding protein (HN) and fusion protein (F) that in concert mediate fusion between the viral and host cell membranes. Inhibitory peptides interact with specific intermediate stages of F during fusion activation and inhibit only once F has begun its structural rearrangement and has extended, before refolding into the ultimate 6HB (16,17,(32)(33)(34). The period of availability of this extended state-the peptide's window of time for inhibition-depends on factors such as the fusion promotion phenotype of the HN partner as well as properties intrinsic to F.…”