Cathepsin Cleavage Potentiates the Ebola Virus Glycoprotein To Undergo a Subsequent Fusion-Relevant Conformational Change

Abstract

Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptorbinding 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 ϳ10°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.T he filovirus Ebola virus (EBOV) is an extremely lethal pathogen for which there are currently no approved vaccines or antiviral agents (18). Entry of the virus into cells is mediated by GP, the sole glycoprotein species present on the virion surface (32). GP is a class I fusion protein (22,53) that mediates binding to the cell surface and fusion with an intracellular membrane. Similar to many other class I fusion proteins (e.g., influenza virus hemagglutinin), native GP is a trimer of heterodimers composed of a receptor binding subunit (GP1) and a fusion subunit (GP2) (31). The two subunits arise by cleavage of a precursor polypeptide as GP transits the Golgi compartment and remain associated through a disulfide bond and noncovalent interactions. GP1 is a large subunit (apparent molecular mass of 130 kDa) that includes a glycan cap containing 4 N-linked carbohydrate addition sites and a heavily O-glycosylated mucin-like domain. The mucin-like domain is dispensable for infection of cells in culture; pseudovirions and virus-like particles bearing genetically engineered GP with a deletion of the mucin-like domain (GP⌬) are fully competent for entry and infection (17,29,46). The two carbohydraterich domains of GP1 sit atop a head domain, which contains a receptor-binding region (RBR) (5,17,29,35,39). In turn, the head domain of GP1 sits atop a base that interacts extensively with the fusion subunit (GP2), which houses the fusion loop (26).The endosomal proteases cathepsin B (Cat B) and cathepsin L (Cat L) are requir...

Post-exposure treatments for Ebola and Marburg virus infections

“…filoviruses to host cells has been associated with several attachment factors [80][81][82][83][84][85] , but entry and deposition of replication machinery for both Ebolaviruses and Marburgviruses have been directly linked to intravesicular cleavage of GP by host proteases, such as cathepsins 86 , and subsequent fusion of viral GP with the host protein Niemann-Pick C1 (NPC1) 87,88 .…”

Ebolavirus Entry Requires a Compact Hydrophobic Fist at the Tip of the Fusion Loop

“…This example shows that the ability to fuse membranes is not simply an effect of the hydrophobicity of individual residues, but some mutations can have long-range effects in these larger FLs with tertiary structure. Mutation of F535 to an arginine was previously shown to significantly reduce GP-mediated virus entry as well as binding of the GP ectodomain to liposomes (16,21). Collectively, our findings indicate that the scaffold formed by L529, F535, and I544 is strictly required to form the fist with the consolidated hydrophobic surface at the tip of the FL and that this specific structure is crucial for a sufficiently deep membrane insertion of the FL, as well as its ability to promote membrane fusion and virus entry.…”

“…The current work on Ebov GP2 not only represents a major step forward toward a thorough understanding of the structural features of the Ebov FL under fusion-competent conditions but hopefully will also stimulate similar future work on the FLs of other viruses. Such information combined with structural knowledge of the fusion proteins in their prefusion states and pathways to postfusion states, as is emerging for Ebov GP (4,7,11,12,16), may facilitate the development of therapeutics aimed against the fusion machinery.…”

“…Viral fusion ultimately occurs in endosomes, where GP1 is cleaved by cathepsins B and L to an ϳ19-kDa species (11,12), which engages Niemann-Pick C1, a late endosomal protein essential for Ebov entry (13)(14)(15). A final unknown trigger causes conformational changes in GP (16,17) that expose the fusion loop (FL) in GP2. The Ebov FL is clamped by a disulfide bond and has a hydrophobic region at its tip.…”

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

“…These steps include binding to the host cell surface, internalizing into endosomes through macropinocytosis (21), trafficking through the endocytic pathway, and cleaving the GP 1 subunit by endosomal cathepsins (15,22,23). Later steps of viral entry include binding of cleaved (19 kD) GP to the endolysosomal membrane Niemann-Pick C1 (NPC1) protein (24)(25)(26), triggering of fusion (15,(27)(28)(29), and finally fusing of viral and endolysosomal membranes, which releases the nucleocapsid into the cytoplasm to begin replication.…”