Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein Trimers

Ebola virus causes severe hemorrhagic fever, often leading to death in humans. The trimeric fusion glycoprotein (GP) is the sole target for neutralizing antibodies and is the major focus of vaccine development. Soluble GP ectodomains are unstable and mostly monomeric when not fused to a heterologous trimerization domain. Here, we report structure-based designs of Ebola and Marburg GP trimers based on a stabilizing mutation in the hinge loop in refolding region 1 and substitution of a partially buried charge at the interface of the GP1 and GP2 subunits. The combined substitutions (T577P and K588F) substantially increased trimer expression for Ebola GP proteins. We determined the crystal structure of stabilized GP from the Makona Zaire ebolavirus strain without a trimerization domain or complexed ligand. The structure reveals that the stabilized GP adopts the same trimeric prefusion conformation, provides insight into triggering of GP conformational changes, and should inform future filovirus vaccine development. [Display omitted] •Filovirus GP expression increases by stabilizing mutations in hinge loop and base helix•Charged lysine in base helix and GP1 N terminus are trapped in metastable conformation•Crystal structure of stabilized Makona Δmucin GP confirms successful stabilization•These findings may be useful for understanding fusion mechanisms and vaccine design Rutten et al. describe structure-based stabilization of soluble filovirus GP trimers to obtain high yields of trimers. The crystal structure of a stabilized Makona Δmucin GP shows how the introduced substitutions stabilize the trimer and that the N terminus of GP1 plays a crucial role in refolding.