Proteolytic disassembly of the ebolavirus glycoprotein in viral entry

Abstract

Ebolaviruses are enveloped, negative-sense, single-stranded RNA viruses that can cause a deadly hemorrhagic fever with case fatality rates as high as 90%. Like other enveloped viruses, ebolavirus enters cells by fusing its viral envelope with a cell membrane. Membrane fusion is mediated by ebolavirus GP, a viral Class I fusion protein structurally similar to influenza HA and retrovirus Env. Previous work has shown that the host endosomal cysteine proteases cathepsin L (CatL) and cathepsin B (CatB) play accessory and essential roles, respectively, in cleaving ebolavirus Zaire (ZEBOV) GP to generate distinct GP intermediates during entry. Although extensive proteolytic remodeling is required for entry, its structural consequences and purpose in the fusion mechanism are poorly understood.;To investigate the role of proteolysis in ZEBOV entry, we employed a forward genetic strategy. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing ZEBOV GP (rVSV-GP) as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We selected mutations at six amino acid positions in GP that independently confer complete CatB independence during cell entry. Structural modeling of these mutations suggests that they disrupt the interface between the two subunits of GP, thereby reducing pre-fusion GP stability. Consistent with this prediction, biochemical studies show that certain mutations dramatically enhance the susceptibility of the GP to terminal proteolysis. Pseudorevertants selected for resistance to rapid proteolytic inactivation are partially CatB-dependent, indicating that decreased GP stability may be important for bypassing the CatB requirement. A pre-fusion conformation-specific epitope was found to denature at a lower temperature in cleaved GP than in uncleaved GP, providing further evidence that proteolysis destabilizes GP. Among factors altering the epitope's thermal stability, GP cleavage status is most significant, while reducing equivalents are slightly less so and acid pH, which destabilizes many other viral envelope proteins, has no independent effect. Altogether, these results demonstrate that the cleavages mediated by CatL and CatB progressively destabilize the pre-fusion conformation of GP, facilitating the conformational rearrangements that occur during the membrane fusion reaction.