Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change.
Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.
Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA.
Matthew
M
Brecher
Kathryn L
KL
Schornberg
Sue E
SE
Delos
Marnie L
ML
Fusco
Erica Ollmann
EO
Saphire
Judith M
JM
White
D056668:Cathepsin L / Q000378:metabolism*; D002460:Cell Line; D029043:Ebolavirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology; D019142:Hemorrhagic Fever, Ebola / Q000201:enzymology* / Q000821:virology; D006801:Humans; D008561:Membrane Fusion; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D056668:Cathepsin L