Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses

Abstract

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

Keywords: T-cell immunoglobulin mucin domain proteins; apoptotic mimicry; phosphatidylethanolamine; phosphatidylserine receptors; virus entry.

Fig. 1.

PE is a ligand for TIM1. (Upper) A total of 1 μg per well of phospholipids was dried out on ELISA plates and incubated with hTIM1-, hTIM3-, or hTIM4-mIg. (Lower) Same as upper panels except that increasing amounts of phospholipids and 1 nM hTIM-mIg proteins were used. Data, expressed as arbitrary units of absorbance at OD₆₅₀, are shown as mean ± SD of duplicates and are representative of at least three independent experiments.

Fig. 2.

Duramycin binds PE in viral membranes. (A) Duramycin specifically binds PE. (Left) A total of 1 μg per well of phospholipids was dried out on ELISA plates and incubated with increasing concentrations of biotin-Duramycin. (Right) Same as left panel except that increasing amounts of lipids and 100 nM of biotin-Duramycin were used. (B) Duramycin binds enveloped viruses. ELISA plates coated with antibodies directed against DENV2, EBOV, WNV, or AdV5 were incubated with these viruses or VLPs, followed by biotin-Duramycin (Left). Proper capture of AdV5 was verified by sandwich ELISA (Right). (A and B) Data, expressed as arbitrary units of absorbance at OD₆₅₀, are shown as mean ± SD of duplicates and are representative of three independent experiments. (C) PLC digestion eliminates Duramycin binding to virions. ELISA plates coated with anti-DENV2 antibody were incubated with DENV2, followed by PLC and biotin-Duramycin. A sandwich ELISA specific for DENV2 was performed to verify that PLC digestion did not cause the virus to detach. Duramycin and anti-DENV2 antibody binding to PLC-treated DENV2 were normalized to those of untreated DENV2. The average ± SD of five experiments is shown (***P < 0.0001).

Fig. 3.

PE contributes to hTIM1-mediated viral entry of EBOV, DENV2, and WNV. (A–C) DENV2, IAV (H1N1), and AdV5 (A); or EBOV and VSV VLPs (B); or WNV VLPs and LASV PVs (C) were preincubated with biotin-Duramycin and used to infect hTIM1-293T cells. Parental 293T cells were also infected to show TIM1 use of these viruses. Infection levels are normalized to that of hTIM1-293T cells infected in the absence of Duramycin. The average ± SD of three independent duplicated experiments is shown (*P < 0.01, **P < 0.001, and ***P < 0.0001). Representative experiments without normalization are shown in Fig. S4.

Fig. 4.

Contribution of PE in PS receptor-mediated viral entry is physiological. (A, Left) Vero cells were stained for TIM1 expression. Rabbit IgG was used as a control. (Right) WNV VLPs and LASV PVs were preincubated with biotin-Duramycin and used to infect cells. Infection levels are normalized to the level without Duramycin treatment. The average ± SD of three duplicated experiments is shown (***P < 0.0001). (B) Selected fluorographs (Left) and phase-contrast micrographs (Right) from an experiment shown in A. (C) Same as A except that A549 cells and DENV2 were used. IAV (H1N1) was used as a control. The average ± SD of three duplicated experiments is shown (***P < 0.0001). (D) EBOV VLPs were either preincubated or not with biotin-Duramycin and used to infect naïve peritoneal macrophages. VP40-Bla VLPs without GP (No GP VLP) were used as a negative control. Infection levels were normalized to those macrophages infected with untreated EBOV VLPs. (Left) Infection levels of one experiment are shown as histograms. (Right) Average of relative infection ± SD of two different batches of EBOV VLPs tested in the same preparation of macrophages.

Fig. 5.

PE is exposed on apoptotic cells and promotes TIM1-mediated phagocytosis. (A) Duramycin binds apoptotic cells. DMSO- (live) or actinomycin D-treated (apoptotic) Jurkat cells were stained with nothing, biotin-annexin A5, or biotin-Duramycin, followed by FITC-avidin. Cells were loaded with SYTOX Red dye for 10 min before analysis to distinguish necrotic cells from apoptotic cells. (B) Phagocytosis of apoptotic Jurkat cells by hTIM1-293T cells. pHrodo green-loaded, apoptotic Jurkat cells were cocultured with hTIM1-293T cells, and analyzed by flow cytometry to detect engulfed Jurkat cells. (C) Duramycin inhibits hTIM1-mediated phagocytosis. Similar experiments as in B except that pHrodo green-loaded apoptotic Jurkat cells were incubated with biotin-Duramycin before coculture with 293T, hLSIGN-, or hTIM1-293T cells. (Left) Leakage of pHrodo dye was assessed before phagocytosis assay. Results were normalized to pHrodo fluorescence in untreated Jurkat cells and expressed as relative pHrodo signal. (Right) Jurkat cells were cocultured with 293T, hLSIGN-, or hTIM1-293T cells at a 1.5:1 ratio of Jurkat cells to phagocytes. Results were normalized to the phagocytosis in hTIM1-293T cells without Duramycin treatment, and expressed as relative phagocytosis. The average ± SD of four duplicated experiments is shown (***P < 0.0001).