Exosome mimicry by a HAVCR1–NPC1 pathway of endosomal fusion mediates hepatitis A virus infection

Abstract

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR–Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1–NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.

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Fig. 1: Exosomes require HAVCR1 for cargo delivery.
Fig. 2: Cargo delivery of free HAV RNA from the lumen of exo-HAV RNA into the cytoplasm.
Fig. 3: NPC1 is required for exosome cargo delivery.
Fig. 4: Exosome cell entry by CME and cargo delivery via the HAVCR1–NPC1 pathway.

Data availability

The authors declare that the data supporting the findings of this study are available from the corresponding author upon reasonable request. Numerical and statistical source data that underlie the graphs in figures, extended data and supplemental data are provided with the paper.

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Acknowledgements

This work was supported by funding from the US Food and Drug Administration Intramural Program to G.K. and a Medical Countermeasures Initiative to G.K. This project was supported in part by appointments to the Research Fellowship Program at the Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the FDA (to M.I.C., A.A. and H.L.).

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Authors

Contributions

M.I.C. and G.K. were responsible for the overall design of the study. M.I.C. carried out most of the experiments, and A.A. and H.L. performed the confocal microscopy studies. G.K. performed the studies with NPC1 mutants, block of infectivity by liposomes and characterization of exosomes by western blot analysis and flow cytometry. M.I.C., A.A. and G.K. analysed the data. G.K. wrote the manuscript and M.I.C helped with the editing. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Gerardo Kaplan.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Fusion of R18-labeled liposomes is temperature dependent and requires phosphatidylserine and cholesterol.

a, Huh7 cells were treated with PS:PC:Chl-R18 liposomes for 30 min at 37 °C, 15 °C, or 4 °C to test for liposome fusion (red). Nuclei were stained with DAPI (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken using a 40x oil objective. b, Quantitative analysis of the fusion of R18-labeled liposomes in endosomes from (a). The R18 fluorescence intensity (red) of 20 cells from (a) was measured using ImagJ software and the experiment was repeated 3 times (n = 60). c, Huh7 cells were treated with PS:PC:Chl-R18, PS:PC-R18, PC:Chl-R18, or PC-R18 during 30 min at 37 °C. Nuclei were stained with DAPI (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken with a 40x oil objective. d, Quantitative analysis of the fusion of R18-labeled liposomes in endosomes from (c). R18 fluorescence intensity (red) of 20 cells from (c) was measured using ImagJ software, and the experiment was repeated 3 times (n=60). Scale bars in (a) and (c) represent 50 mm. In (b) and (d), box and whiskers plots were done using the Tukey method. Box limits are upper and lower quartiles, center lines are the medians, whiskers are 1.5x interquartile range, and points represent outliers. P values were determined by two-sided Mann-Whitney test. Source data

Extended Data Fig. 2 Intracellular fusion of R18-labeled liposomes is pH-dependent.

a, Liposome fusion (red) in Huh7 cells treated with 2 µM Monensin, 30 µM Chloroquine diphosphate,or 5 mM Amonium chloride (NH4Cl) prior to the addition of PS:PC:Chl-R18 liposomes. Nuclei were stained with DAPI (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken using a 40x oil objective. Scale bars represent 50 µm. b, Quantitative analysis of the fusion of R18-labeled liposomes in endosomes from (a). The R18 fluorescence intensity (red) of 20 cells from (a) was measured using ImagJ software and the experiment was repeated 3 times (n=60). Box and whiskers plot was done using the Tukey method. Box limits, upper and lower quartiles; Center line, median; whiskers, 1.5x interquartile range; points, outliers; P values between untreated and treated cells were determined by two-sided Mann-Whitney test. c, Lack of cytotoxicity of the compounds was confirmed by flow cytometry using a caspase-3 apoptosis assay based on the irreversible binding of cell-permeable inhibitor DEVD-fmk conjugated to sulfo-rhodamine (Red-DEVD-fmk) to activated caspase-3. Stained cells were analyzed with a FACSCanto II (BD Biosciences) using FlowJo v8.5 software. Apoptotic/dead Huh7 control cells were prepared by heat treatment at 55 °C for 7 min. d, Liposome fusion with intracellular membranes. Huh7 cells were treated with PS:PC:Chl-R18 liposomes (R18 liposome fusion events, red) for 15 min at 37 °C, the plasma membrane was stained with CellMask (green) for 15 min at 37 °C, and nuclei were stained with DRAQ5 (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken with 40x oil objective. Scale Bars represent 25 µm. Liposome fusion events detected by R18 fluorescence (red) that did not co-localize with plasma membrane stained with Cell Mask (green). Results are representative of 3 independent experiments. Source data

Extended Data Fig. 3 Characterization of exosomes purified from HAV-infected cells.

a, Presence of PS at the surface of exosomes from HAV-infected cells. Purified exo-HAV/Bsd treated with (+) or without (-) biotinylated ANX5 (ANX5 –Biotin) and anti-biotin mAbs was bound to magnetic beads, washed extensively, and extracted HAV RNA was quantitated by RT-qPCR. Data are mean ± sem, n=3. P values between complete and incomplete immunomagnetic sandwiches were analyzed by one-way ANOVA with Dunnett’s post-test. b, Purification by isopycnic ultracentrifugation of supernatants of HAV.8Y- Bsd-infected Huh7 cells. Gradient collected in 25 fractions from the top. H AV RNA in fractions 5–24 was quantified by RT-qPCR showing peaks of exo-HAV (fractions 11–13, 1.08–1.10 g cm-3) and vpHAV (fraction 21–23, 1.23–1.33 g cm-3). Data are mean ± sd of RT-qPCR duplicates. c, Enrichment of exosomal markers HSP70, FLOT-1, and TSG101 but not Golgi marker GOLGA1 in exo-HAV assessed by Western blot analysis of gradient fractions 5–25 from (b). Fractions aligned with (b). Cell extracts from uninfected Huh7 cells were used as controls. Migration of molecular weight markers is shown in kDa. d, Flow cytometry analysis of cell surface expression of CD9, CD63, and CD81 on Huh7 cells using PE-labeled anti-human CD9, CD63, or CD81 mAs compared to a PE-labeled isotype control. Gating strategy in left panel. e, Flow cytometry analysis of gradient fractions 2–24 from (b) absorbed to latex-beads and stained with mAbs as in (d). f, Content of CD63 and CD81 on exo-HAV (fraction 11 from b) compared to control vpHAV (fraction 22 from b) by flow cytometry as described in (e). Gating strategy in left panel. g, Sizing of exo-HAV (fractions 11–13 from (b) by DLS analysis in a Zetasizer nano ZS90 instrument performed in 3 runs (red, blue, and green lines) of 10 measurements each. Data are representative of 3 independent experiments. Source data

Extended Data Fig. 4 HAVCR1 is an exo-HAV cellular receptor.

a, Specific infectivity of purified exo-HAV and vpHAV from Fig. 1c in Huh7 cells was determined as the ratio between HAV RNA genome equivalents (GE) assessed by RT-qPCR and infectious particles assessed by ARTA. Data are mean ± sem, n=5 from 5 different titrations. b, Expression of HAVCR1 but not HAVCR1 N94A restored exo-HAV cell entry in Huh7 HAVCR1 KO cells. HAVCR1, HAVCR1 N94A, or vector transfectants were infected with exo-HAV or vpHAV from Fig. 1c, and tested for Bsd-resistant CFU. Data are mean ± sem, n=4 from two independent experiments with biological duplicates. c, Flow cytometry analysis of the cell surface expression of HAVCR1 (red dots) or HAVCR1DIgV/ANX5 (blue dots) compared to vector-transfected cells (grey dots) on Huh7 HAVCR1 KO cell transfectants stained with PE-labeled anti-HAVCR1 mucin mAb1750. Gates contain cells expressing the HAVCR1 mucin 1750 epitope. Data representative of 4 independent experiments. d, Huh7 HAVCR1 KO cell transfectants expressing HAVCR1 or HAVCR1DIgV/ANX5 bind apoptotic cells. Monolayers of vector, HAVCR1, or HAVCR1DIgV/ANX5 cell transfectants or control Huh7 parental cells were incubated with CMFDA-labeled apoptotic Jurkat cells and washed extensively. Phase contrast (DIC) and green fluorescence (CMFDA) micrographs were taken with an Axiovert 200 fluorescence microscope using a 20x objective. Scale bar represents 50 mm. Data representative of 3 independent experiments. e, Expression of an annexin V fusion protein at the cell surface restores infectivity of exo-HAV. Huh7 HAVCR1 KO cell transfectants from (c) were infected with purified exo-HAV from Fig. 1c using a m.o.i of 0.1–0.5 for 48 h at 37oC. Total RNA was extracted from the cells and HAV RNA was quantitated by HAV RT-qPCR. Data are mean±sem, n=4 from 2 independent experiments with 2 biological replicates. P values were determined by two sided Mann-Whitney test. Source data

Extended Data Fig. 5 Knock out of NPC1 in Huh7 cells blocks EBOV cell entry and endosomal liposome fusion.

a, Growth of rVSV-EBOVgp-GFP in Huh7 parental and NPC1 KO cells. Huh7 parental and NPC1 KO cells were infected with rVSV-EBOVgp-GFP for 24 h. At different times p.i., GFP fluorescence was assessed using a fluorescence plate reader. Data are mean ± sem, n=4 from 4 independent experiments. P values between Huh7 parental and N PC1 KO cells were determined by two-sided Mann-Whitney test. b, Quan titative analysis of the fusion of R18-labeled liposomes in endosomes. Huh7 parental, HAVCR1 KO and NPC1 KO cells were treated with PS:PC:Chl-R18 liposomes 10 min at 15 °C and incubated or not for 30 min at 37 °C to test for liposome fusion (red). Nuclei were stained with DAPI (blue). Cells were analyzed using a LSM 700 confocal microscope (Carl Zeiss), and micrographs were taken using a 40x oil objective. Scale bar represents 50 mm. c, Quantitative analysis of the fusion of R18-labeled liposomes in endosomes from (b). The R18 fluorescence intensity (red) of 25 cells was measured using ImagJ software, and the experiment was repeated 3 times (n=75). Box and whiskers plots were done using the Tukey method. Box limits, upper and lower quartiles; Center line, median; whiskers, 1.5x interquartile range; points, outliers; P values between mean fluorescence intensity (MFI) of Huh7 HAVCR1 KO or Huh7 NPC1 KO cells compared to Huh7 parental cells were determined by two-sided Mann-Whitney test. Source data

Extended Data Fig. 6 Interaction of NPC1 with HAVCR1 in Huh7 NPC1 KO cell transfectants.

a, Schematic representation of bimolecular fluorescence complementation (BiFC) assay based on the complementation of the monomeric Kusabira green (mKG) protein57 168 amino acid (aa) N-terminus fragment (mKG(N)) fused to the C-terminus of NPC1 (NPC1-mKG(N)) and 51 aa C-terminus fragment (mKG(C)) fused to the C-terminus of HAVCR1 (HAVCR1-mKG(C)). The interaction of NPC1 and HAVCR1 results in BiFC of mKG9 that emits peak fluorescence at 507 nm upon excitation at 492 nm. b, Huh7 NPC1 KO cells transfected with vector, plasmid coding for NPC1-mKG(N) wild type or NPC1 mutants L175A/L176A (double mutation in NT D cholesterol binding pocket prevents function of NPC130), P202A/F203A (double mutation in NTD in the rim of the cholesterol binding pocket prevents function of NPC130), F503W (mutation in domain C increases binding of GP31), F503Y (mutation in domain C reduces infectivity of rVSV-ZEBOV GP by 2 logs31), L656F (mutation in sterol-sensing domain increases cholesterol binding, Millard 200533), G660R (Mutation transmembrane helix 3 prevents function of NPC130,32, or P691S (mutation in transmembrane domain results in defect in cholesterol uptake and trafficking33) and co-transfected or not with a plasmid coding for HAVCR1-mKG(C) for 48 h to determine HAVCR1-NPC1 interaction (green). Nuclei were stained with Hoechst 33342 (blue). Cells were analyzed in an Axiovert 200 fluorescence microscope, and micrographs were taken using a 40x objective. Scale bars represent 50 mm. Results are representative of 3 independent experiments.

Extended Data Fig. 7 Effect of NPC1 mutations and cell entry inhibitors in exo-HAV infection.

a, Huh7 NPC1 KO cells were transfected with cDNA of NPC1 WT, NPC1 mutants L173A/L176A, P202A/F203A, F503W, F503Y, L656F, G660R, or P691S, or vector and infected at 48 h p.t. with purified exo-HAV from Fig. 1c. Total RNA was extracted 4 days p.i. and analyzed by HAV RT-qPCR. Data are mean ± sem, n=3 from 3 independent experiments. P values between NPC1 WT and NPC1 mutants or vector were determined by one-way ANOVA with Dunnett’s post-test. b, Cell transfectants as in (a) were infected with rVSV-EBOVgp-GFP for 24 h, and virus in the supernatant was titrated by a fluorescence endpoint dilution assay in 96-well plates containing Huh7 monolayers. Viral titers were determined at 72 h p.i. using the ID50 program. Data are log10 TCID50/ml ± s.d., n=3. P values were determined as in (a). c, Viability of Huh7 cells treated with cell entry inhibitors for 72 h by dual-fluorescence Acridine Orange / Propidium Iodide assay. Data are mean ± sem, n=4 from two independent experiments with biological duplicates. d, Huh7 cells were treated with inhibitors for 1 h, infected with rVSV-EBOVgp-GFP, and GFP fluorescence was quantified at 16–20 h p.i. in a fluorescence plate reader. Data are mean ± sem, n=4 from two independent experiments with biological duplicates. e, Huh7 cells were pretreated with inhibitors for 1 h, infected with purified vpHAV from Fig. 1c. HAV replication was quantitated at 72 h p.i. by RT-qPCR. Data are mean ± sem, from left to right n=5, 5, 5, 3 3, and 3 biological replicates. P values between DMSO and inhibitors were determined as in (a). In (c) and (d), P values between DMSO and inhibitors were determined by two-sided Mann-Whitney test. Source data

Extended Data Fig. 8 Chlorpromazine and Dynasore inhibit clathrin-mediated endocytosis.

a, Huh7 cells were treated with inhibitors of endocytic pathways (5 µg/ml CPZ, 80 µM Dynasore, 10 µM EGA, 40 µM EIPA, 10 µM LY294002) or DMSO prior to the addition of tetramethylrhodamine conjugate-transferrin (Tritc-Transferrin), a marker of clathrin-mediated endocytosis. Tritc-Transferrin (red fluorescence) uptake in the presence of inhibitors was analyzed using an LSM 700 confocal fluorescence microscope. Nuclei were stained with DAPI (blue fluorescence). Micrographs were taken with a 40x oil objective. Scale bars represent 50µm. b, Quantitative analysis of the endocytosis of Tritc-Transferrin from (a). The Tritc-Transferrin fluorescence intensity (red) of 16 cells was measured using ImagJ software, and the experiment was repeated 3 times (n=48). Box and whiskers plot was done using the Tukey method. Box limits, upper and lower quartiles; Center line, median; whiskers, 1.5x interquartile range; points, outliers; P values between DMSO and inhibitors were determined by two-sided Mann-Whitney test. Source data

Extended Data Fig. 9 Dynasore but not chlorpromazine inhibit caveolae-mediated endocytosis.

a, Huh7 cells were treated with BODIPY FL C5-Lactoceramide complexed to BSA (LAC-cer), a marker of caveolae-mediated endocytosis, and treated with inhibitors of endocytosis (5 µg/ml CPZ, 80 µM Dynasore, 10 µM EGA, 40 µM EIPA, 10 µM LY294002) or DMSO as control. LAC-cer (green) uptake was analyzed using a LSM 700 confocal microscope. Nuclei were stained with DRAQ5 (blue). Micrographs were taken with a 40x oil objective. Scale bars represent 50µm. b, Quantitative analysis of the endocytosis of LAC-cer from (a). The LAC-cer fluorescence intensity (green) of 20 cells was measured using ImagJ software, and the experiment was repeated 3 times (n=60). Box and whiskers plot was done using the Tukey method. Box limits, upper and lower quartiles; Center line, median; whiskers, 1.5x interquartile range; points, outliers; P values between DMSO and inhibitors were determined by two-sided Mann-Whitney test. Source data

Extended Data Fig. 10 Effect of endocytosis inhibitors in different cellular compartments.

Huh7 cells in 8-wells chamber slides were infected with the CellLight Bacman 2.0 reagents fused to GFP (green) or RFP (red) at a multiplicity of infection of 30 particles per cell, incubated at 37oC for 10–12 h, treated with 80 mM Dyn asore hydrate, 5mg/ml Chlorpromazine hydrochloride solution, 10 mM EGA, or a similar volume of DMSO vehicle as negative control, and incubated for additional 12–14 h at 37oC. Nuclei were stained with DRAQ5 (blue), cells were fixed with 4% PFA, coverslips mounted with ProLong Gold antifade reagent, and slides analyzed in a LSM 700 confocal microscope. Micrographs were taken using a 63X oil objective. Cells were infected with CellLight Bacmam 2.0 driving the expression of markers of: a, Mitochondria (leader sequence of E1 alpha pyruvate dehydrogenase fused to RFP); b, Lysosomes (Lamp1 fused to RFP); c, Golgi (human Golgi-resident enzyme N-acetylgalactosaminyltransferase 2 fused to RFP); d, Peroxisomes (peroxisomal C-terminal targeting sequence fused to GFP); e, Early endosomes (EE, Rab5a fused to GFP); or f, Late Endosomes (LE, Rab 7a fused to GFP). Scale Bars represent 25µm. Results are representative of 3 independent experiments.

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Costafreda, M.I., Abbasi, A., Lu, H. et al. Exosome mimicry by a HAVCR1–NPC1 pathway of endosomal fusion mediates hepatitis A virus infection. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0740-y

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