Cell entry by a novel European filovirus requires host endosomal cysteine proteases and Niemann-Pick C1

Abstract

Lloviu virus (LLOV), a phylogenetically divergent filovirus, is the proposed etiologic agent of die-offs of Schreibers's long-fingered bats (Miniopterus schreibersii) in western Europe. Studies of LLOV remain limited because the infectious agent has not yet been isolated. Here, we generated a recombinant vesicular stomatitis virus expressing the LLOV spike glycoprotein (GP) and used it to show that LLOV GP resembles other filovirus GP proteins in structure and function. LLOV GP must be cleaved by endosomal cysteine proteases during entry, but is much more protease-sensitive than EBOV GP. The EBOV/MARV receptor, Niemann-Pick C1 (NPC1), is also required for LLOV entry, and its second luminal domain is recognized with high affinity by a cleaved form of LLOV GP, suggesting that receptor binding would not impose a barrier to LLOV infection of humans and non-human primates. The use of NPC1 as an intracellular entry receptor may be a universal property of filoviruses.

Keywords: Cuevavirus; Ebola; Endosomal cysteine proteases; Filoviridae; Filovirus; Lloviu virus; NPC1; Niemann–Pick C1; Viral entry; Viral glycoprotein; Viral membrane fusion; Viral receptor.

Fig. 1. LLOV GP is incorporated into VSV particles and filovirus-like particles, and is competent to mediate cell entry

(A) Vero cells were exposed to recombinant VSVs (rVSVs) expressing eGFP, and VSV G or different filovirus glycoproteins. Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=4) are shown. (B) Incorporation of LLOV GP into rVSVs was determined by SDS-PAGE and western blotting with an LLOV GP1-specific antiserum. (C) Vero E6 cells generating filovirus-like particles (VLPs) consisting of EBOV VP40 and LLOV GP were visualized by transmission electron microscopy. Red arrows, filamentous VLPs released from cells. Blue arrows, spike-like projections from the surface of a VLP, likely corresponding to LLOV glycoprotein spikes.

Fig. 2. The LLOV GP precursor is cleaved by furin into GP1 and GP2 subunits, and is extensively N-glycosylated

(A) rVSV-LLOV GP and rVSV-EBOV GPΔMuc produced in WT CHO cells or the FD11 CHO cell line lacking furin (Furin⁰) were subjected to reducing SDS-PAGE, and their GP1 subunits were visualized by western blotting with peptide-specific antisera. (B) rVSV-LLOV GP was incubated with protein N–glycosidase F (PNGase F) to remove N–linked glycans, and GP1 was visualized by western blotting.

Fig. 3. LLOV GP-mediated entry requires endosomal acid pH

Vero cells were pre-treated with ammonium chloride (20 mM), and then exposed to rVSVs bearing VSV G or filovirus glycoproteins. Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=4) are shown. *, p<0.05. ***, p<0.001.

Fig. 4. Cysteine cathepsin activity is required for LLOV GP-dependent entry, but cathepsin B (CatB) is dispensable

(A–B) Vero cells were pre-treated with 1% DMSO (vehicle control), the broad-spectrum cysteine cathepsin inhibitor E-64 (300 μM) (A), or the CatB-selective inhibitor CA074 (B) for 4 h at 37°C, and then exposed to rVSVs bearing EBOV or LLOV GP. Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=4) are shown. *, p<0.05. **, p<0.01. ***, p<0.001. ns, not significant.

Fig. 5. LLOV GP1 is rapidly cleaved to a primed ≈17 kDa species (GP1_CL) in vitro

rVSVs bearing LLOV GP or EBOV GP were exposed to thermolysin (0, 6.25, 12.5, 25, 50, 100, 200 μg/ml) for 1 h at 37°C. Enzyme digestion was stopped with phosphoramidon (0.5 mM) and GP1 was were visualized by western blotting with peptide-specific antisera that detect an N–terminal region of GP1 (residues 83–97).

Fig. 6. Cleaved LLOV GP cannot overcome the entry requirement for endosomal cysteine cathepsins

Vero cells were pre-treated with 1% DMSO (control) or E-64 (300 μM) for 4 h at 37°C, and then exposed to rVSVs bearing uncleaved (GP) or thermolysin-cleaved GP proteins (GP_CL). Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=2) from a representative experiment are shown. *, p<0.05. ***, p<0.001.

Fig. 7. Niemann-Pick C1 (NPC1) is an essential entry receptor for LLOV

(A) Primary NPC1-mutant human fibroblasts from Niemann-Pick type C disease patients (NPC1^mut), NPC1^mut fibroblasts engineered to express WT human NPC1 (NPC1^mut+huNPC1), and fibroblasts from control individuals were exposed to rVSVs bearing LLOV or EBOV GP. Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=4) are shown. (B) NPC1-deficient Chinese hamster ovary (CHO) cells and NPC1⁰ cells engineered to express human NPC1 (NPC1⁰+huNPC1) were exposed to rVSVs bearing LLOV or EBOV GP. Infected cells were visualized by fluorescence microscopy at 12–16 h post-infection. Averages ± SD (n=4) are shown. (A–B) Asterisks indicate measurements below the limit of detection. (C) Cleaved LLOV GP recognizes the second luminal domain (C) of NPC1. Biotin-labeled rVSVs bearing uncleaved or cleaved filovirus glycoproteins were captured onto streptavidin-coated plates, and binding of purified FLAG-tagged NPC1 domain C was detected by ELISA with an anti-FLAG antibody conjugated to horseradish peroxidase. **, p<0.01. ***, p<0.001. ****, p<0.0001. ns, not significant. <, measurement below the threshold of detection.

Fig. 8. Secreted LLOV glycoprotein, Δ peptide, inhibits LLOV GP-mediated entry

(A) Recombinant LLOV and SUDV Δ-peptides fused to the Fc sequence of an immunoglobulin G were expressed and purified by protein A affinity chromatography, and visualized by SDS-PAGE and Coomassie Blue staining. (B) Vero cells were treated with LLOV or SUDV Δ-Fc in increasing concentrations in the presence of eGFP-expressing Moloney murine leukemia virus particles bearing MARV GP. Cells were detached at 48 h post transduction, and the percentage of eGFP-positive cells was measured by flow cytometry. Averages ± SD (n=3) are shown. **, p<0.01.