A novel receptor-induced activation site in the Nipah virus attachment glycoprotein (G) involved in triggering the fusion glycoprotein (F).
Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (beta6S4/beta1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.
Aguilar
Hector C
HC
Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA. haguilar@ucla.edu
Ataman
Zeynep Akyol
ZA
Aspericueta
Vanessa
V
Fang
Angela Q
AQ
Stroud
Matthew
M
Negrete
Oscar A
OA
Kammerer
Richard A
RA
Lee
Benhur
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D036388:Ephrin-B2 / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D009154:Mutation; D045405:Nipah Virus / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization / Q000187:drug effects
D000911:Antibodies, Monoclonal; D036388:Ephrin-B2; D000939:Epitopes; C510755:F protein, Nipah virus; D014759:Viral Envelope Proteins