Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

Abstract

Human CD4 (cluster of differentiation 4) is well known as the primary receptor for human immunodeficiency virus (HIV) entry into the cells. The virus binds to CD4 molecules to induce a conformational change in the viral glycoprotein (GP) gp120, which exposes the co-receptor binding site for coreceptors CCR5 or CXCR4. The co-receptor binding then leads to membrane fusion for viral entry. Since the CD4 molecule has a high affinity for gp120, soluble CD4 (sCD4) and CD4-mimetic small molecules (CD4mcs) have been extensively studied as potential inhibitors for HIV infection. Surprisingly, we have found that human sCD4 and some CD4mcs are able to inhibit Ebola virus (EBOV) infection. Evidence is provided that the compounds block viral entry by targeting the GP binding site for the endosomal receptor Niemann-Pick C1 (NPC1). This finding reveals virus-receptor binding similarities between two remote viruses (HIV and EBOV) and suggests new possibilities for EBOV entry inhibitors.

Keywords: Biological sciences; Immunology; Microbiology; Natural sciences; Virology.

Graphical abstract

Figure 1

Inhibition assay of sCD4 and CD4-derived protein molecules against pseudotyped and infectious Ebola viruses (A) sCD4 (2D), soluble first two-domain CD4; sCD4 (4D), soluble four-domain CD4; CD4-Ig, first two-domain CD4 linked with human Fc region (Ig domain); CD4-IgG2, first two-domain CD4 with human Fc region (Ig domain); EBOV, Zaire Ebola virus; BDBV, Bundibugyo ebolavirus. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments. (B) sCD4 (4D) inhibits infectious Ebola virus infection. This experiment was conducted in BSL-4 containment in triplicates, and the error bars represent standard deviations.

Figure 2

Inhibition assay of CD4-mimetic small molecules against pseudotyped Ebola viruses (A) NBD-556 and analogs inhibiting EBOV infection. (B) Newly designed and synthesized compounds with phenyl ring substituted molecules in the region-I inhibiting EBOV. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Figure 3

Specificity assay of CD4-IgG-2 and JRC-II-191 against pseudo-typed HIV, EBOV, VSV, and A-MLV CD4-mimetic molecules CD4-IgG2 and JRC-II-191 were evaluated against four different viruses: HIV (YU2), EBOV, VSV (vesicular stomatitis virus), and A-MLV (amphitropic murine leukemia virus). All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Figure 4

Binding affinity and kinetics assay of sCD4 (2D) and NBD-556 binding to EBOV receptor binding domain by biolayer interferometry Two-domain CD4 (sCD4-2D) (A) and NBD-556 (B) at two pH conditions, pH7.4 and pH6.1. Open circle in (B) was not included in the fit of the data. (See the STAR Methods BLI section for details).

Figure 5

Binding competition assay of sCD4 and CD4-mimetic compounds with receptor NPC1 (A) sCD4, NBD-556, JRC-II-191, and DY-III-228 competing with NPC1 receptor. (B) Dose response (0, 10, 20, and 40 μM) of NBD-556 in binding competition with NPC1 receptor. (C) Comparison of wild-type (EGPDCM-WT) and mutant (EGPDCM-mut) (WF/AA, 86W/A, and 88F/A) receptor binding domain (RBD) of EBOV binding to the NPC1 receptor. The mutant (WF/AA) surface expression level was confirmed to be comparable to the wild type (see Figure S7). Representative of 3 experiments.

Figure 6

Molecular docking analysis of sCD4 and CD4mcs (A) Molecular docking analysis of sCD4 and CD4mcs binding to the EBOV-GP. sCD4 docking using HDOCK program. The docking energy is −150.41 kcal/mol. CD4mcs docking using AutoDock program. (a) sCD4-RBD ribbon model; (b) sCD4-RBD surface binding model; (c) sCD4-RBD interactions: hydrogen bond: GP T83:OG1-CD4 L44:N (green) and salt bridge: GP K84:NZ-CD4 D56:OD2 (brown); (d) NBD-556 docking ribbon model (−6.477 kcal/mol); (e) NBD-556 docking surface model; and (f), superimposed of NBD-556 (yellow), JRC-II-191 (magenta), and DY-III-228 (cyan). (B) Comparisons of two receptor binding sites of CD4bs and NPC1-bs. (a) CD4bs^, of HIV gp120 (based on PDB 1G9N, YU2 strain). (b) NPC1bs^, of EBOV-GP (based on PDB 5F1B, Zaire EBOV). Showing the accommodation of CD4-mimetic compound NBD-556 in the red dash circle. Hydrophobic surface in gray; CD4bs of HIV gp120 in magenta dash circle; NPC1bs of EBOV-GP in red dash circle.