Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosomes

Emily Happy Miller,Joseph S Harrison,Sheli R Radoshitzky,Chelsea D Higgins,Xiaoli Chi,Lian Dong,Jens H Kuhn,Sina Bavari,Jonathan R Lai,Kartik Chandran

doi:10.1074/jbc.m110.207084

Abstract

Ebola virus (EboV) and Marburg virus (MarV) (filoviruses) are the causative agents of severe hemorrhagic fever. Infection begins with uptake of particles into cellular endosomes, where the viral envelope glycoprotein (GP) catalyzes fusion between the viral and host cell membranes. This fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike that ultimately result in formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Infection by other viruses employing similar viral entry mechanisms (such as HIV-1 and severe acute respiratory syndrome coronavirus) can be inhibited with synthetic peptides corresponding to the native CHR sequence (“C-peptides”). However, previously reported EboV C-peptides have shown weak or insignificant antiviral activity. To determine whether the activity of a C-peptide could be improved by increasing its intracellular concentration, we prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, which is known to accumulate in endosomes. We found that this peptide specifically inhibited viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. We determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Mechanistic studies suggested that the peptide acts by blocking a membrane fusion intermediate.

Highlights

Ebola virus (EboV) and Marburg virus (MarV) are the causative agents of severe hemorrhagic fever
To determine whether the activity of a C-peptide could be improved by increasing its intracellular concentration, we prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 the localization sequence (Tat), which is known to accumulate in endosomes
Watanabe et al [36] synthesized and tested a peptide corresponding to GP2 residues 610 – 633 (Fig. 1) and found that large amounts of this peptide were required for modest inhibition of infection by VSV particles bearing VSV-GP. (ϳ1700 ␮M was needed for ϳ70% inhibition.) Netter et al [37] observed no inhibition of EboV entry with a peptide corresponding to GP2 residues 610 – 634 at peptide concentrations up to ϳ70 ␮M, but they found that C-peptides derived from the structurally related ASLV Env glycoprotein had potent activity against ASLV entry

Summary

The abbreviations used are

EboV, Ebola virus; MarV, Marburg virus; GP, glycoprotein; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; ASLV, avian sarcoma/leukosis virus; rVSV, recombinant vesicular stomatitis virus; eGFP, enhanced green fluorescent protein; mRFP, monomeric red fluorescent protein; m.o.i., multiplicity of infection. Endosomal Targeting of an Ebola Virus C-peptide triple-stranded coiled coil, projecting the N-terminal fusion peptides into the endosomal membrane This configuration results in a putative “extended intermediate” conformation in which the entire GP2 ectodomain bridges the viral and host membranes. The three CHRs fold back and pack into grooves along the NHR core trimer, creating a stable post-fusion “six-helix bundle” configuration. These rearrangements juxtapose the viral and host lipid bilayers, catalyzing their mixing and coalescence, and lead to delivery of the viral nucleocapsid into the cytoplasm. C-peptides inhibit membrane fusion and inhibit viral infection by competing with the endogenous CHR for binding to the NHR core trimer of the extended intermediate, thereby arresting the transition to the six-helix bundle. Mechanistic experiments indicate that the endosome-targeted C-peptide acts on an intermediate in the filovirus entry pathway

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION