Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus

Nature Microbiology volume 5, pages 1069–1078 (2020)Cite this article

Subjects

Abstract

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes2,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection2. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides—preferentially disialogangliosides—function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1+ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Genome-wide CRISPR screen identifies gangliosides as essential host factors for nHAV and eHAV virus entry.
Fig. 2: Gangliosides are essential for nHAV and eHAV entry in Huh-7.5 cells.
Fig. 3: Gangliosides bind to the HAV capsid and mediate a post-endocytosis step in viral entry.
Fig. 4: Airyscan microscopy images of UGCG-KO1.3 and sgCtrl cells infected with gradient-purified 18f nHAV.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are included in the paper and the accompanying Supplementary Information. MAGeCK output files with results from the two CRISPR screens are included in the Source Data files for Fig. 1c and Extended Data Fig. 1c.

References

  1. Zell, R. Picornaviridae—the ever-growing virus family. Arch. Virol 163, 299–317 (2018).

    CAS  PubMed  Google Scholar 

  2. Feng, Z. et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496, 367–371 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Hirai-Yuki, A. et al. MAVS-dependent host species range and pathogenicity of human hepatitis A virus. Science 353, 1541–1545 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Hirai-Yuki, A., Hensley, L., Whitmire, J. K. & Lemon, S. M. Biliary secretion of quasi-enveloped human hepatitis A virus. mBio 7, e01998-16 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. Wang, X. et al. Hepatitis A virus and the origins of picornaviruses. Nature 517, 85–88 (2015).

    CAS  PubMed  Google Scholar 

  6. Rivera-Serrano, E. E., Gonzalez-Lopez, O., Das, A. & Lemon, S. M. Cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses. eLife 8, e43983 (2019).

    PubMed  PubMed Central  Google Scholar 

  7. Kaplan, G. et al. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus. EMBO J. 15, 4282–4296 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Das, A., et al. TIM1 (HAVCR1) is not essential for cellular entry of either quasi-enveloped or naked hepatitis A virions. mBio 8, e00969-17 (2017).

  9. Das, A., Maury, W. & Lemon, S. M. TIM1 (HAVCR1): an essential ‘receptor’ or an ‘accessory attachment factor’ for hepatitis A virus? J. Virol. 93, e01793-18 (2019).

  10. Lemon, S. M. et al. Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination. J. Virol. 65, 2056–2065 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Tomicic, M. T., Thust, R. & Kaina, B. Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation. Oncogene 21, 2141–2153 (2002).

    CAS  PubMed  Google Scholar 

  12. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Groux-Degroote, S., Guerardel, Y. & Delannoy, P. Gangliosides: structures, biosynthesis, analysis, and roles in cancer. ChemBioChem 18, 1146–1154 (2017).

    CAS  PubMed  Google Scholar 

  14. van der Spoel, A. C., Mott, R. & Platt, F. M. Differential sensitivity of mouse strains to an N-alkylated imino sugar: glycosphingolipid metabolism and acrosome formation. Pharmacogenomics 9, 717–731 (2008).

    PubMed  Google Scholar 

  15. Barrientos, R. C. & Zhang, Q. Isobaric labeling of intact gangliosides toward multiplexed LC-MS/MS-based quantitative analysis. Anal. Chem. 90, 2578–2586 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Schwarzmann, G., Hoffmann-Bleihauer, P., Schubert, J., Sandhoff, K. & Marsh, D. Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study. Biochemistry 22, 5041–5048 (1983).

    CAS  PubMed  Google Scholar 

  17. Eckels, K. H., Summers, P. L. & Dubois, D. R. Hepatitis A virus hemagglutination and a test for hemagglutination inhibition antibodies. J. Clin. Microbiol. 27, 1375–1376 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Sanchez, G. et al. Capsid region involved in hepatitis A virus binding to glycophorin A of the erythrocyte membrane. J. Virol. 78, 9807–9813 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Mobius, W., Herzog, V., Sandhoff, K. & Schwarzmann, G. Intracellular distribution of a biotin-labeled ganglioside, GM1, by immunoelectron microscopy after endocytosis in fibroblasts. J. Histochem. Cytochem. 47, 1005–1014 (1999).

    CAS  PubMed  Google Scholar 

  20. Daniotti, J. L. & Iglesias-Bartolome, R. Metabolic pathways and intracellular trafficking of gangliosides. IUBMB Life 63, 513–520 (2011).

    CAS  PubMed  Google Scholar 

  21. Wang, X. et al. Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site. Proc. Natl Acad. Sci. USA 114, 770–775 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Baggen, J. et al. Enterovirus D68 receptor requirements unveiled by haploid genetics. Proc. Natl Acad. Sci. USA 113, 1399–1404 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Zocher, G. et al. A sialic acid binding site in a human picornavirus. PLoS Pathog. 10, e1004401 (2014).

    PubMed  PubMed Central  Google Scholar 

  24. Kim, D. S. et al. Porcine sapelovirus uses alpha2,3-linked sialic acid on GD1a ganglioside as a receptor. J. Virol. 90, 4067–4077 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Cohen, L., Benichou, D. & Martin, A. Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis. J. Virol. 76, 7495–7505 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Probst, C., Jecht, M. & Gauss-Muller, V. Intrinsic signals for the assembly of hepatitis A virus particles. Role of structural proteins VP4 and 2A. J. Biol. Chem. 274, 4527–4531 (1999).

    CAS  PubMed  Google Scholar 

  27. Zhou, D. et al. Unexpected mode of engagement between enterovirus 71 and its receptor SCARB2. Nat. Microbiol. 4, 414–419 (2019).

    CAS  PubMed  Google Scholar 

  28. Qian, M., Cai, D., Verhey, K. J. & Tsai, B. A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection. PLoS Pathog. 5, e1000465 (2009).

    PubMed  PubMed Central  Google Scholar 

  29. Ravindran, M. S., Bagchi, P., Cunningham, C. N. & Tsai, B. Opportunistic intruders: how viruses orchestrate ER functions to infect cells. Nat. Rev. Microbiol. 14, 407–420 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Dupzyk, A. & Tsai, B. Bag2 is a component of a cytosolic extraction machinery that promotes membrane penetration of a nonenveloped virus. J. Virol. 92, e00607–18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Ewers, H. et al. GM1 structure determines SV40-induced membrane invagination and infection. Nat. Cell Biol. 12, 11–18 (2010).

    CAS  PubMed  Google Scholar 

  32. Ewers, H. & Helenius, A. Lipid-mediated endocytosis. Cold Spring Harb. Perspect. Biol. 3, a004721 (2011).

    PubMed  PubMed Central  Google Scholar 

  33. Spriggs, C. C., Harwood, M. C. & Tsai, B. How non-enveloped viruses hijack host machineries to cause infection. Adv. Virus Res. 104, 97–122 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Pinto, R. M., et al. Hepatitis A virus codon usage: implications for translation kinetics and capsid folding. Cold Spring Harb Perspect. Med. 8, a031781 (2018).

  35. Zhang, H. C. et al. An infectious cDNA clone of a cytopathic hepatitis A virus: genomic regions associated with rapid replication and cytopathic effect. Virology 212, 686–697 (1995).

    CAS  PubMed  Google Scholar 

  36. Yi, M., Bodola, F. & Lemon, S. M. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 304, 197–210 (2002).

    CAS  PubMed  Google Scholar 

  37. Lee, W. M., Monroe, S. S. & Rueckert, R. R. Role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome. J. Virol. 67, 2110–2122 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Wakita, T. et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11, 791–796 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Blight, K. J., McKeating, J. A., Marcotrigiano, J. & Rice, C. M. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J. Virol. 77, 3181–3190 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. MacGregor, A. et al. Monoclonal antibodies against hepatitis A virus. J. Clin. Microbiol. 18, 1237–1243 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR–Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. Szklarczyk, D. et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 (2019).

    CAS  PubMed  Google Scholar 

  45. Stapleton, J. T. et al. Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles. J. Virol. 67, 1080–1085 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Gonzalez-Lopez, O. et al. Redundant late domain functions of tandem VP2 YPX3L motifs in nonlytic cellular egress of quasi-enveloped hepatitis A virus. J. Virol. 92, 1308–1318 (2018).

    Google Scholar 

  47. Svennerholm, L. & Fredman, P. A procedure for quantitative isolation of brain gangliosides. Biochim. Biophys. Acta 617, 97–109 (1980).

    CAS  PubMed  Google Scholar 

  48. Zavesca Scientific Discussion (European Medicines Evaluation Agency, 2005); https://www.ema.europa.eu/en/documents/scientific-discussion/zavesca-epar-scientific-discussion_en.pdf

Download references

Acknowledgements

We thank K. Jacobson for helpful discussions. This research was supported in part by grants from the US National Institutes of Health (R01-AI103083, R01-AI131685 and R01-AI150095 (to S.M.L.), R01-DK123499 (to Q.Z.), R01-GM134531 (to M.K.) and T32-GM007092 (to R.M.)).

Author information

Author notes

  1. Anshuman Das & Aravind Asokan

    Present address: Departments of Surgery and Molecular Genetics and Microbiology, Duke University, Durham, NC, USA

  2. Rodell Barrientos

    Present address: US Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Walter Reed Army Institute of Research, Silver Spring, MD, USA

  3. Victoria Madigan

    Present address: Broad Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

  4. Ewelina Kaluzna

    Present address: Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Anshuman Das, Tomoyuki Shiota, Kevin L. McKnight, Lu Sun, You Li, Ewelina Kaluzna & Stanley M. Lemon

  2. Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA

    Rodell Barrientos & Qibin Zhang

  3. UNCG Center for Translational Biomedical Research, North Carolina Research Campus, Kannapolis, NC, USA

    Rodell Barrientos, Zhucui Li & Qibin Zhang

  4. Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Victoria Madigan, Ichiro Misumi, Aravind Asokan & Jason K. Whitmire

  5. Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Rita M. Meganck

  6. Department of Cell Biology & Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Maryna Kapustina

  7. Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Stanley M. Lemon

  8. Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Stanley M. Lemon

Authors
  1. Anshuman Das
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rodell Barrientos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomoyuki Shiota
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victoria Madigan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ichiro Misumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kevin L. McKnight
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lu Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhucui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rita M. Meganck
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. You Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ewelina Kaluzna
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Aravind Asokan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jason K. Whitmire
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Maryna Kapustina
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Qibin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Stanley M. Lemon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.D. and S.M.L. conceived the study and wrote the manuscript. A.D. established the HAV cell death screen. A.D., T.S., K.L.M., I.M., E.K. and L.S. performed the subsequent validation experiments. V.M. and R.M.M. performed PCR and sequencing of sgRNA integrants. Y.L. performed the bioinformatics analysis of sgRNA sequencing data. R.B., Z.L. and Q.Z. performed and analysed MS data. M.K. acquired confocal microscopy images and carried out image analyses. A.A. and J.K.W. provided research materials and technical expertise. S.M.L. supervised all aspects of the study. All authors commented on the manuscript.

Corresponding author

Correspondence to Stanley M. Lemon.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genome-wide CRISPR screen for essential hepatovirus host factors.

a, Frequency distribution of the number of reads mapping to individual sgRNA integrants in high-throughput sequencing of control (GCV-treated only) versus selected (18f-Tat virus-infected and GCV-treated) HeLa-tkGFP cells transduced with lentiviruses expressing the Brunello sgRNA library in the first of two independent screens (screen #1). b, Number of reads (log2) mapping to individual sgRNAs in control versus selected cells in screen #1. The total number of individual sgRNAs to which reads mapped is shown at the bottom. c, Scatterplot showing correlation of the positive selection scores accorded genes (n = 19,111) on the basis of sgRNA enrichment in two independent screens. Red symbols indicate genes related to NeuNAc or ganglioside synthesis. Spearman’s rank correlation coefficient r = 0.267, p < 0.0001. d, STRING analysis of functional associations of proteins encoded by genes comprising the top 39 hits identified in the combined analysis of the two independent screens. Common associations are annotated. Protein-protein interaction enrichment p-value = 3.11 × 10-15. e, Normalized counts of reads mapping to sgRNAs targeting selected genes involved in ganglioside synthesis (see main manuscript Fig. 1c) in control versus selected cell populations. Ctr, control; Sel, selected.

Source data

Extended Data Fig. 2 Miglustat inhibition of gradient-purified naked and quasi-enveloped hepatovirus infection.

a, (top) Schematic showing the organization of the HM175/18f-NLuc virus genome, and (bottom) distribution of viral RNA associated with naked and quasi-enveloped eHAV 18f-NLuc virions from supernatant fluids of infected cells in fractions of an isopycnic iodixanol gradient. b, Miglustat inhibition of naked 18f-NLuc virus replication. Data shown are mean NLuc expression in Huh-7.5 cells pre-treated with miglustat for 72 hrs prior to infection, and lysed and assayed for NLuc activity 18 hrs after infection. n = 3 technical replicates. The estimated 50% inhibitory concentration (IC50) was 44.5 µM (95% CI = 29.8-66.0) using a four-parameter, variable slope nonlinear regression model (R = 0.900). LU, light units. c, NLuc activity expressed by Huh-7.5 cells 18 hrs after infection with naked (nHAV) or quasi-enveloped (eHAV) virus. Cells were treated with the indicated concentration of miglustat beginning 72 hrs prior to infection. Data shown are means of 3 technical replicates. d, ATP assay for viability of Huh-7.5 cells treated with indicated concentrations of miglustat for 72 hrs (CellTiter-Glo, Promega). Data shown are means of 4 technical replicates.

Source data

Extended Data Fig. 3 Characterization of clonal UGCG-KO cell lines generated with two different sgRNAs.

a, Schematic showing exons (boxed segments, translated region deeply shaded) and introns (horizontal lines) in the UGCG gene. Chromatograms obtained by Sanger sequencing of the indicated regions of UGCG in the Huh-7.5 sgCtrl and two UGCG-knockout clones, KO1.3 (exon 1) and KO2.1 (exon 2). The locations of indels disrupting the open reading frame and resulting in truncated protein expression are highlighted in red. The AUG codon in the first exon at which translation originates is highlighted in green. b, Detection of GM1 ganglioside on the surface of sgCtrl, UGCG-KO1.3 and UGCG-KO2.1 cells by flow cytometry using Alexa-488 fluorophore conjugated cholera toxin (CtB). Isotype indicates parallel staining with an Alexa-488 conjugated anti-mouse secondary antibody. Absence of detectable GM1 ganglioside on the surface confirmed both clones are functional knockouts of ganglioside synthesis. Data representative of two independent experiments. c, Laser-scanning confocal images of HAV antigen (K24F2 mAb, green) in sgCtrl and UGCG-KO1.3 cells 5 days after inoculation with naked (nHAV) and quasi-enveloped (eHAV) HM175/18f virus confirm a lack of replication in the knockout cells. Images shown are representative of 3 random fields in one experiment. Scale bar = 10 μm. d, Mean human rhinovirus B14 (RV-B14) RNA abundance measured by real-time RT-PCR in sgCtrl and UGCG knockout cells at the indicated times post infection. n = 2 technical replicates. e, Mean hepatitis C virus (HCV) RNA abundance measured by real-time RT-PCR (normalized to actin mRNA) in sgCtrl and UGCG knockout cells at 16 and 64 hrs after infection with JFH1 virus. n = 2 technical replicates.

Source data

Extended Data Fig. 4 Mass spectrometry identification of gangliosides present in Huh-7.5 cells.

a, Extracted ion chromatograms (EIC) of GM2 ganglioside species showing their elution order. b, Representative ESI-MS spectrum of gangliosides showing the accurate mass of [M + 2 H]2+ precursor ion of GM2 d18:1/16:0. c, Fragmentation of precursor ion (shown in b) at NCE = 26 in QExactive HF. The inset shows TMT6 reporter ion region and the respective channel assignments. Identification of each ganglioside species is based on the presence of diagnostic B1, O”, and Y0’ ions. Only 14 gangliosides in Huh-7.5 cells passed these criteria (shown in d). d, Mean relative intensities ±s.e.m. of gangliosides detected by ESI-MS in 3 independent samples of Huh-7.5 cells treated with 50 or 200 µM miglustat, each with two technical replicate analyses (4 for control samples). e, Mean relative abundance ±s.e.m. of multiple GM2 ganglioside species with varying lengths of fatty acyl tail detected by ESI-MS in 3 independent samples of Huh-7.5 cells treated with miglustat at the indicated concentration. Each sample was subjected to 2 replicate ESI-MS analyses (4 for control samples). Note that the maximum concentration of miglustat used in these experiments was over 20-fold the clinical plasma Cmax of Zavesca® (miglustat) which is approximately 9 µM48.

Source data

Extended Data Fig. 5 Ganglioside supplementation of UGCG-KO cells.

a, Mean fold-change in viral RNA between 6 and 24 hrs in sgCtrl cells or UGCG-KO cells supplemented with 50 µM GD3 or vehicle (DMSO) for 24 hrs prior to infection with nHAV (left) or eHAV (right). Results are normalized to DMSO-treated cells at 6 hrs. n = 2 technical replicates. b, Mean NLuc activities in UGCG-KO2.1 cells supplemented with gangliosides (12.5-50μM) for 24 hrs prior to 18f/NLuc nHAV infection. sgCtrl cells were infected without supplementation. Cells were harvested for NLuc assay at 6 hpi. n = 3 technical replicates. c, Mean NLuc activity 18 hrs after nHAV infection of UGCG-KO1.3 cells supplemented with GM3, GD3 or GM2. n = 3 technical replicates. d, Mean relative NLuc activities ±s.d. in UGCG-KO2.1 cells supplemented with GM3 or GD3 (20-50 µM) 18 hrs after infection with nHAV (n = 5 independent experiments) or eHAV (n = 3 independent experiments). NLuc activities were normalized to GM3 supplementation (100 light units) in each experiment. Statistical comparison was by one-way ANOVA with Holm-Sidak’s multiple comparison test. e, Mean NLuc activity in Huh-7.5 cells 6 hrs after infection with nHAV pre-incubated with GM3, GM2 or GM1. n = 6 technical replicates. f, Immunoblot of ST8SIA1 (GD3 synthase, arrow) in lysates of polyclonal ST8SIA1-KO cells generated with different sgRNAs and sgCtrl cells. Actin included as a loading control. Data are from a single experiment. g, Mean NLuc activities in ST8SIA1-KO cells 6 and 24 hrs after 18f-NLuc nHAV infection. n = 4 technical replicates from one of 2 experiments with similar results. h, (left) Binding of eHAV to UGCG-KO1 vs. sgCtrl cells at 4 °C for 1 hr. Bars are means of n = 3 technical replicates. (right) Uptake of eHAV by UGCG-KO1 vs. sgCtrl cells following 2 hrs adsorption, removal of the inoculum, and 4 hrs additional incubation at 37 °C. Bars are means of n = 2 technical replicates i, Thermostability of 18f-NLuc nHAV following overnight incubation with 50 μM GD3 or DMSO at pH 5.5 and 4 °C, followed by additional incubation at the temperature indicated for 15 min prior to inoculation onto Huh-7.5 cells. Light units obtained with NLuc were normalized to light units from virus incubated for 15 min at 37 °C; lines reflect means of n = 2 technical replicates. All results shown as technical replicates were confirmed in independent experiments.

Source data

Extended Data Fig. 6 Purity of commercial ganglioside standards.

a, Compositional analysis of the GM3 standard using RPLC-ESI-HRMS. Extracted ion chromatograms (plotted with 10 ppm tolerance within the expected m/z value) of individual ganglioside molecular species are shown for the GM3 standard on the left, with the results of monitoring for potential ganglioside impurities to the right.The absence of GD3 and GD1a species in the GM3 standard was confirmed by monitoring for each species with no corresponding peaks observed. b, Similar analysis of the GD3 standard. A trace amount of GM3 ( < 1.2%) was detected in the GD3 standard, along with a greater quantity of GD1a as an impurity (~7%). c, GD1a standard. A trace amount of GD3 was detected as an impurity in GD1a (~0.2%), whereas the absence of GM3 species in the GD1a standard was confirmed by monitoring for the species with no corresponding peaks observed. NL: normalized level, that is peak intensity. Impurities were calculated from the ratio of summed intensity, assuming similar ionization efficiency between different ganglioside molecular species. Data from one of 2 replicate runs generating similar results for each commercial standard.

Extended Data Fig. 7 Endolysosomal location of HAV capsids in UGCG-KO1.3 cells.

a, Airyscan microscopic image of 18f-infected UGCG-KO1.3 cells labelled with antibodies to Rab-7a (green), HAV (JC, red) and LAMP1 (magenta). Virus was adsorbed to cells for 2 hrs at 37 °C, which were then washed with PBS and reincubated at 37 °C for 4 hrs prior to fixation. Scale bar = 10 µm. b, Expanded view of the area bordered by the yellow rectangle in panel (a) demonstrating that most HAV capsids are localized within endolysosomes staining for Rab-7a+ and/or LAMP1+. The arrows indicate capsids present in late endosomes staining only for Rab-7a. Scale bar = 3 µm. c, Single and dual fluorescence signals present in the image shown in panel (b). Scale bar = 3 µm. Images are representative of findings in two independent experiments with similar results.

Extended Data Fig. 8 Super-resolution stimulated emission depletion (STED) microscopy imaging of HAV-infected UGCG-KO1.3 cells.

a, Comparative (left) conventional confocal microscopic image versus (right) STED image of the same field of cells. 18f virus was allowed to adsorb to cells for 2 hrs at 37° then washed off and the cells incubated for an additional 4 hrs before being fixed and labelled with antibodies to LAMP1 (confocal: grey; STED: magenta) and HAV: K34C8 mAb (confocal: green; STED: yellow) and JC polyclonal human convalescent antibody (confocal, red; STED, cyan). Large confocal puncta with high intensity fluorescence are resolved into multiple individual particles by STED. b, Overlays of confocal and STED signals for (left) LAMP1, (center) K34C8, and (right) JC from the images shown in panel (a). c, STED microscopy demonstrates that most virus particles inside endolysosomes are in close proximity to the luminal membrane surface. STED images shown are representative of 7 randomly selected cells in one experiment and confirm findings made by conventional confocal microscopy. All scale bars = 1 µm.

Extended Data Fig. 9 Intracellular distribution of GM3 following supplementation of UGCG-KO1.3 cells.

a, Cells were incubated with GM3 conjugated at C11 to dipyrrometheneboron difluoride undecanoic acid (GM3-TopFluor, green) for 15 hr at 37 °C, then fixed and labelled with anti-LAMP1 (magenta) and anti-Rab-7a (red). Airyscan images from left to right show four optical sections along the Z-axis and demonstrate GM3-TopFluor within cells. At the far right, non-supplemented cells stained with anti-LAMP1 show no green fluorescence. b, Enlarged images of the area bounded by the yellow rectangle in panel (a) showing TopFluor within Rab-7a+ late endosomes and LAMP1 + endolysosomes. Some endolysosomes are labelled with antibodies to both LAMP1 and Rab-7a. c, 3D reconstruction of GM3-TopFluor and DAPI (nucleus) fluorescence signals in the images shown in panel (a). d, UGCG-KO1.3 cells were incubated with GM3-TopFluor (green) for 13 hr, then infected with nHAV virus for 2 hr, washed and re-incubated for 4 hrs before being fixed and labelled with anti-HAV antibody (K34C8, red). Optical sections recorded along the Z-axis demonstrate relative positions of HAV and GM3-TopFluor. e, 3D reconstruction of the image shown in panel (d) (see also Supplementary Movie 2). f, (left) Imaris 3D reconstruction of GD3 fluorescence signals in uninfected UGCG-KO1.3 cells supplemented with exogenous GD3 and stained with anti-GD3 antibody. The surface of endolysosomes is reconstructed from the LAMP1 signal. (right) GD3 fluorescent signal is rendered as spheres with diameter proportionate to intensity, centered at the point of maximum intensity, colored according to whether it is inside (orange) or outside (yellow) LAMP1 + endolysosomes. Images shown are representative of two independent experiments with similar results.

Extended Data Fig. 10 Imaris analysis and 3D reconstruction of Airyscan microscopy images.

a, A small area within the fluorescence image of infected UGCG-KO1.3 cells recorded on the Zeiss-880 microscope with the Airyscan detector, showing the signals in individual channels for HAV capsid antibodies, K34C8 (green) and JC (red), and antibody to LAMP1 (magenta). b, Merged signals from panel (a). c, Merged signals from the two capsid antibodies, K34C8-Alexa488 and JC-Alexa594. To enhance the specificity of virus detection, only particles labelled simultaneously with both antibodies above threshold were included in the analysis. d, The positions of viral particles identified by labeling with both antibodies were modeled as spheres, with their center positioned at the point of maximum signal intensity, and their diameter proportional to the intensity and size of the fluorescence signal. e, Spherical representation of dually-labelled virus particles as in panel (d). f, Surface of LAMP1+ endolysosomes reconstructed from the LAMP1 antibody signal. g, Merged image of viral particles modeled as spheres and LAMP1+ endolysosomes. h, The Matlab extension in the IMARIS software package was used to analyze the coordinates of the virus particles and to segregate them into two groups: one in which the center was inside or on the surface of endolysosomes (yellow spheres), and another in which the center was outside of endolysosomes (blue spheres). i, Merged image after reconstruction and analysis. j, Full-field fluorescence images of (left) HAV capsids and (right) LAMP1+ endolysosomes with DAPI staining of nuclei in infected UGCG-KO cells. k, Results of the viral particle localization analysis showing segregation of particles into groups by color within the full-field image shown in panel (j). See also Extended Data Movie 1. Airyscan images and 3D reconstructions are representative of three independent experiments showing similar results.

Supplementary information

Supplementary information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Imaris 3D reconstruction of Airyscan microscopic images showing HAV particles within UGCG-KO1.3 cells 6 h after infection with naked virus. Virus particles, dually labelled with K34C8 and JC antibodies, are modelled as spheres, with their centre at the point of maximal fluorescence and diameter proportional to fluorescence intensity, and are segregated by colour according to their location either within (yellow) or outside (blue) endolysosomes, the surfaces of which were rendered by staining with antibodies against LAMP1 (magenta). Further details are provided in Fig. 4b and Extended Data Fig. 10.

Supplementary Video 2

Imaris 3D reconstruction of Airyscan microscopic images showing intracellular GM3-TopFluor (green) in supplemented UGCG-KO cells. Endolysosomes (magenta) were labelled with antibodies against LAMP1 and nuclei with DAPI (blue).

Supplementary Video 3

Imaris 3D reconstruction of Airyscan microscopic images showing intracellular GD3 detected by anti-GD3 antibodies in GD3-supplemented UGCG-KO cells. Endolysosomes (magenta) were labelled with antibodies against LAMP1 and nuclei with DAPI (blue). The GD3 signals are modelled as spheres, with their centre at the point of maximal fluorescence and diameter proportional to fluorescence intensity, and segregated by colour according to location either within or contacting (yellow), or outside (blue), LAMP1+ endolysosomes.

Supplementary Video 4

Imaris 3D reconstruction of Airyscan microscopic images showing endogenous GD1a (green) detected with anti-GD1a antibodies, modelled as spheres with their centre at the point of maximal fluorescence and diameter proportional to fluorescence intensity, in Huh-7.5 cells. Endolysosomes (magenta) were labelled with antibodies against LAMP1 and nuclei with DAPI (blue).

Source data

Source Data Fig. 1

MAGeCK output for combined CRISPR screen analysis related to Fig. 1c, and source data for all graphs.

Source Data Fig. 2

Source data for all graphs.

Source Data Fig. 3

Source data for all graphs.

Source Data Fig. 4

Source data for all graphs.

Source Data Extended Data Fig. 1

MAGeCK output for CRISPR screen 1 and 2 analyses related to Extended Data Fig. 1c, and source data for the graph in Extended Data Fig. 1e.

Source Data Extended Data Fig. 2

Source data for all graphs.

Source Data Extended Data Fig. 3

Source data for Extended Data Fig. 3d,e.

Source Data Extended Data Fig. 4

Source data for Extended Data Figs. 4d,e.

Source Data Extended Data Fig. 5

Source data for all graphs.

Source Data Extended Data Fig. 5

Uncut gel images for Extended Data Fig. 5f.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Das, A., Barrientos, R., Shiota, T. et al. Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus. Nat Microbiol 5, 1069–1078 (2020). https://doi.org/10.1038/s41564-020-0727-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-020-0727-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing