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.Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fevers that have high mortality rates. The viruses infect a variety of cell types, including alveolar macrophages, dendritic cells, and endothelial cells (32). The cellular receptor(s) has yet to be firmly established, but several cellular proteins have been implicated in filoviral entry. Folate receptor-␣ was initially reported to be a receptor for the filoviruses (4), but it has since been shown that cells that do not express the folate receptor are permissive for entry (25,26). Lectin molecules, including DC-SIGN, mannose binding lectin (MBL), and human macrophage galactose-and N-acetylgalactosamine-specific C-type lectin (hMGL), have been shown to enhance EBOV entry, but they are not essential (1,3,11,14,17,24,28). Integrin ␤1 has been postulated to facilitate entry (29), and, most recently, members of the Tyro3 protein kinase family of proteins (23) have been demonstrated to play to role in mediating filovirus uptake.EBOV and MARV are both members of the Filoviridae family and have been classified as separate genera due to a lack of serological cross-reactivity and genetic differences. Consistent with the taxonomic separation of the viruses, MARV and EBOV outbreaks have distinct geographical distri...

The Primed Ebolavirus Glycoprotein (19-Kilodalton GP 1,2 ): Sequence and Residues Critical for Host Cell Binding

“…Previous work has shown that many residues throughout GP 1 are important for EBOV GP 1,2 -mediated entry into host cells (3,15,17), but to date there have been no direct binding studies with mutant GP 1,2 proteins. Since the smallest RBR we analyzed (RBR-7; GP 1 residues 90 to 149) bound specifically to permissive cells, we engineered Ala substitutions (into RBR-1-Fc) at each residue between residues 90 and 149 that had been suggested to be important for receptor binding (based on infectivity studies) and that did not compromise GP 1,2 incorporation into pseudovirions: these were K95, K114/K115, K140, and G143 (see Fig.…”

“…Since the smallest RBR we analyzed (RBR-7; GP 1 residues 90 to 149) bound specifically to permissive cells, we engineered Ala substitutions (into RBR-1-Fc) at each residue between residues 90 and 149 that had been suggested to be important for receptor binding (based on infectivity studies) and that did not compromise GP 1,2 incorporation into pseudovirions: these were K95, K114/K115, K140, and G143 (see Fig. S2 in the supplemental material) (3,15,17). We also created three mutants with composite mutations, which we denoted 3mer (K114A/K115A/K140A), 4mer (K95A/K114A/K115A/K140A), and 5mer (K95A/K114A/ K115A/K140A/G143A).…”

“…Binding of WT and mutant RBR-1-Fc proteins. Ala substitutions were made individually and in combination within RBR-1-Fc at K95, K114 and K115, K140, and G143 (3,15,17). 3mer, K114A/ K115A/K140A mutant; 4mer, K95A/K114A/K115A/K140A mutant; 5mer, K95A/K114A/K115A/K140A/G143A mutant.…”

“…Moreover, 19-kDa GP 1 clearly contains receptor binding activity, since virus binding and infection are actually enhanced after the mucin-like domain is removed (GP 1,2 ⌬) (11,21). Additionally, 19-kDa GP 1 most likely includes residues from the amino-terminal portion of GP 1 , since mutations within the first 150 residues (residues 33 to 183) impair virus infection (3,13,15,17) and since a recombinant protein including residues 54 to 201 binds specifically to permissive cells and inhibits EBOV GP 1,2 -mediated infection (13). Cys 53 should also be present, since 19-kDa GP 1 remains attached to GP 2 by a disulfide bond (4; data not shown).…”

Rho GTPases Modulate Entry of Ebola Virus and Vesicular Stomatitis Virus Pseudotyped Vectors

“…Plasmids expressing adeno-associated virus 2 encoding luciferase (AAV2-luciferase) and AAV2-nuclear green fluorescent protein (GFP) were supplied within the laboratory. All plasmids used to generate feline immunodeficiency virus (FIV) and VSV core pseudovirions have been previously described (3,42). All plasmids used to generate HIV and murine leukemia virus (MuLV) core pseudovirions are commercially available from Invitrogen and Clontech, respectively.…”