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Protein Sci. 2015 Apr;24(4):446-63. doi: 10.1002/pro.2578. Epub 2014 Oct 31.

Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets.

Author information

1
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, 84112-5650.

Abstract

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.

KEYWORDS:

N-trimer; designed coiled coil; ebolavirus; ebolavirus GP2; filovirus entry; mirror-image phage display; phage display; prehairpin intermediate

PMID:
25287718
PMCID:
PMC4380977
DOI:
10.1002/pro.2578
[Indexed for MEDLINE]
Free PMC Article

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