Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events

Abstract

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.

FIG. 1.

Alignment of EBOV and MARV GP1 proteins. A ClustalW alignment of the amino-terminal 300 amino acids of four strains of EBOV and one strain of MARV identified conserved regions. The residues highlighted in black are identical, and those in gray are similar. Fifteen regions that are numbered are found to be identical in all EBOV strains. The regions similar between all EBOV strains and MARV are boxed. ZEBOV, Zaire EBOV; CIEBOV, Cote D'Ivoire EBOV; SEBOV, Sudan EBOV; REBOV, Reston EBOV.

FIG. 2.

Ability of FIV virions pseudotyped with EbolaΔO GP1 mutant glycoproteins to compete with wild-type EbolaΔO-GP1-pseudotyped virus for binding to permissive cells. Mutant and wild-type (wt) EbolaΔO-pseudotyped FIV particles containing a luciferase reporter gene were normalized for RT activity, and equivalent numbers of particles were incubated with SNB-19 cells. The cells were subsequently challenged with EbolaΔO GP-pseudotyped FIV β-gal (multiplicity of infection, 0.004). β-gal expression was determined 4 days after transduction. The data are represented as percentages of the wild-type competition. Data represent the averages and standard errors from three experiments performed in duplicate. Significant differences between the ability of wild-type and mutant GPs to compete for binding was determined by Student's t test. *, P < 0.05; **, P < 0.001.

FIG. 3.

Cathepsin L and B proteolysis of mutant EBOV GP1s. The mutant particles were normalized for GP1 content (A) and then cleaved with catL (B) or catB (C). The digested particles were separated by SDS-PAGE and immunoblotted for GP1. The blot is a representative blot from three experiments. Overexposure of the gel was required to see the catB cleavage product for G143A and E305A/E306A. Overexposure of lanes containing P146A/C147A, F153A/H154A, F159A, F160A, and R164/L165A did not result in detectable cleavage products.

FIG. 4.

R164A/L165A constitutes an immunodominant epitope of the 18- to 22-kDa cleavage product. Cleavage of wild-type EBOV and R164A/L165A by catL was examined over time (A). Silver-stained SDS-PAGE of catL cleavage products (B). Bands corresponding to uncut GP1, FIV capsid protein (CA), and the catL cleavage products are identified, and molecular size markers are shown.

FIG. 5.

Summary of mutant phenotypes. Mutants were classified after examining GP1 incorporation, transduction efficiency, binding competition, and cathepsin cleavage assays. Residues in black capital letters transduced cells at wild-type levels (65% to 150% of wild type), those that did not incorporate GP1 well are in boldface italics; residues that are involved with receptor binding are highlighted in black; residues that are involved in GP1 conformation are double underlined; residues that are involved in a postbinding, cathepsin cleavage-independent step are highlighted in gray; and residues that transduced more than 150% of the wild type are underlined once.