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Histo-blood group antigens of glycosphingolipids predict susceptibility of human intestinal enteroids to norovirus infection

Open AccessPublished:September 10, 2020DOI:https://doi.org/10.1074/jbc.RA120.014855
      The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene–dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.
      The successful generation of human mini-guts cultured ex vivo (
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      ). One remarkable finding is the ability of these nontransformed human cultures to support replication and recapitulate unique aspects of infection of previously noncultivatable pathogens, such as human noroviruses (
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      ). Exploiting this significant advancement, we have conducted a study of lipid and sphingolipid compositions of seven HIE cultures and characterized the interaction between their lipid membranes and human norovirus (HuNoV) virus-like particles (VLPs). These HIEs uniquely represent individuals with different ABO, Lewis, and secretor histo-blood group geno- and phenotypes with varying susceptibilities to human HuNoV infection (
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      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ).
      HuNoVs are the cause of 20% of all gastrointestinal infections worldwide and are responsible for 700 million infections each year (

      Centers for Disease Control and Prevention (2018) Norovirus Worldwide. https://www.cdc.gov/norovirus/trends-outbreaks/worldwide.html.

      ). The virus is highly contagious and thus constitutes a major societal challenge. In most cases, the infection is self-limiting, but the illness can be chronic, severe, or even fatal (
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      ). There is a large genetic and phenotypic variation of circulating strains of HuNoV, and the predominant GII.4 strains change through critical (epochal) mutations and appear as new, globally occurring, variants every 2–3 years (
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      ). The epidemiologically dominating GII strains typically infect secretors, persons who carry a functional secretor α1,2-fucosyltransferase coded for by the FUT2 allele (Fig. S1), whereas nonsecretors are homozygous for nonfunctional secretor (FUT2) alleles and are resistant to infection. The name “secretor” originally refers to the secretion of histo-blood group antigens (HBGAs) in saliva but, as recognized later, also reflects the expression of HBGA in the gastrointestinal epithelium. Surprisingly, few studies have successfully addressed the structural characterization of various glycoconjugates (i.e. glycolipids and glycoproteins carrying HBGAs of human intestinal tissues) (
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      ). Major reasons for this may be difficulties in obtaining representative material and sufficient amounts of tissues for chemical characterization and difficulties in establishing cultures of nontransformed intestinal cells that allow short- and long-term interaction studies with various pathogens and toxins.
      The introduction of an ex vivo infection model of HuNoV in HIEs has allowed for detailed studies aimed at revealing the virus-host cell interactions at the molecular and cellular levels, the understanding of which is crucial for the development of novel antiviral drugs and vaccines (
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      ,
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      Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.
      ). HIEs are derived from stem cells isolated from intestinal crypts in human intestinal tissue obtained from surgery or biopsy samples (
      • Sato T.
      • Stange D.E.
      • Ferrante M.
      • Vries R.G.
      • Van Es J.H.
      • Van den Brink S.
      • Van Houdt W.J.
      • Pronk A.
      • Van Gorp J.
      • Siersema P.D.
      • Clevers H.
      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      ). These cultures recapitulate the natural intestinal epithelium from where they are taken. HIEs are nontransformed and physiologically active and contain multiple epithelial cell types (enterocytes, goblet cells, and enteroendocrine and Paneth cells), and they may be grown as three-dimensional cultures in Matrigel or as monolayer cultures in collagen-coated wells (
      • Foulke-Abel J.
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      • Kovbasnjuk O.
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      • Ettayebi K.
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      • Hyser J.M.
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      • Crawford S.E.
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      • Estes M.K.
      • Donowitz M.
      Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract.
      ,
      • In J.G.
      • Foulke-Abel J.
      • Estes M.K.
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      Human mini-guts: new insights into intestinal physiology and host-pathogen interactions.
      ,
      • Yu H.
      • Hasan N.M.
      • In J.G.
      • Estes M.K.
      • Kovbasnjuk O.
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      • Donowitz M.
      The contributions of human mini-intestines to the study of intestinal physiology and pathophysiology.
      ,
      • Zachos N.C.
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      Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.
      ). HIEs thus provide excellent model systems to extend our knowledge on microbe host-cell interactions and specifically at the individual level, where glycoconjugates uniquely reflect individual genotypes of the donors (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ). Although HIEs are susceptible to HuNoV replication, the receptor is unknown, and possible attachment factors on HIEs have not been fully characterized. As a result, it is now important to characterize the lipid composition of a series of HIEs and their individual interactions with HuNoV VLPs as a model to compare the differences between susceptible and resistant enteroid cultures.
      In this study, we analyzed the lipid and sphingolipid composition of seven HIE lines generated from jejunal biopsies from six individuals. These HIEs were defined by their ABO, Lewis, and secretor HBGA geno- and phenotypes with genomic DNA sequencing and ELISA (Table 1) and by their susceptibility to HuNoV infections (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ). We performed lipid analyses of lower and upper phases from Folch partitionings and characterized phospholipids, cholesterol, and sphingolipids. We further performed kinetic studies of HuNoV VLPs binding to membrane-embedded glycosphingolipids (GSLs) isolated from individual HIEs. Our results establish that the HBGAs of GSLs match the ABO, Lewis, and secretor genotypes of individual HIEs and that this correlates with susceptibility to HuNoV GII.4 infection ex vivo. This strongly supports the concept that GSLs of human intestinal cells act as important attachment factors in the natural HuNoV infection process.
      Table 1Jejunal enteroid geno- and phenotypic HBGA characteristics
      HIE IDSecretorLewisHIE HBGAHuNoV Propagation (
      • Ettayebi K.
      • Crawford S.E.
      • Murakami K.
      • Broughman J.R.
      • Karandikar U.
      • Tenge V.R.
      • Neill F.H.
      • Blutt S.E.
      • Zeng X.L.
      • Qu L.
      • Kou B.
      • Opekun A.R.
      • Burrin D.
      • Graham D.Y.
      • Ramani S.
      • et al.
      Replication of human noroviruses in stem cell-derived human enteroids.
      ,
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      )
      FUT2 genotypePhenotypeFUT3 genotypePhenotypeABO genotypePhenotypeSummaryGII.4 SYD
      1JSe, se385Positivele202,314, le202,314NegativeOAAA+
      J. Nilsson, I. Rimkute, C. Sihlbom, V. R. Tenge, S. C. Lin, R. L. Atmar, M. K. Estes, and G. Larson, manuscript in preparation.
      J2Se, se428PositiveLe, LePositiveOBB LebB Leb+
      J4se428, se428NegativeLe, le202,314PositiveOOLeaLea
      J6Se, SePositiveLe, le59,508PositiveOAA LebA Leb+
      J8se428, se428NegativeLe, le202,314PositiveOOLeaLea
      J10se428, se428Negativele484,667, le59,508NegativeOAA
      See “Discussion.”
      A
      See “Discussion.”
      J4FUT2Se, se428, se428PositiveLe, le202,314PositiveOOLebLeb+
      FUT2, FUT3, and ABO genotypes were assessed for SNPs and deletions at known mutations sites as follows: for FUT2, A385T, G428A, C571T, and C628T; for FUT3, T59G, T202C, C314T, G484A, G508A, G667A, G808A, and T1067A; and for ABO, nucleotides 261 and 297 in exon 6 and 657, 703, 771, 796, 803, 829, and 930 in exon 7. Identified mutation sites are indicated by lowercase letters and in superscript type (e.g. se385). Secretor, Lewis, and HBGA phenotypes were obtained from ELISA. Se/se, secretor; Le/le, Lewis.
      * J. Nilsson, I. Rimkute, C. Sihlbom, V. R. Tenge, S. C. Lin, R. L. Atmar, M. K. Estes, and G. Larson, manuscript in preparation.
      ** See “Discussion.”

      Results

      Pheno- and genotypic histo-blood group status of HIEs

      HIE lines were phenotyped by ELISAs and genotyped by PCR amplification of ABO, FUT2, and FUT3 genes followed by DNA sequencing to determine their HBGA status. Phenotyping assays analyzed for the presence of Lea, Leb, A, B, and H antigens. Genotyping assessed SNPs and deletions at known mutation sites of the ABO, FUT2, and FUT3 genes, and the results are summarized in Table 1. Overall phenotyping results were in agreement with the genotyping findings. Of seven characterized HIEs, four were secretor-positive (SeSe, Sese, or Sesese; 1J, J2, J6, and J4FUT2), and three were secretor-negative (sese; J4, J8, and J10), whereas five were Lewis-positive (LeLe or Lele; J2, J4, J6, J8, and J4FUT2) and two were Lewis-negative (lele; 1J and J10). Only J2 and J6 were both secretor- and Lewis-positive, and only J10 was negative for both features. The J4FUT2 line was generated from J4 by overexpressing the FUT2 gene (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ). As recently shown, only the secretor-positive HIEs are susceptible to ex vivo HuNoV GII.4 Sydney infection (
      • Ettayebi K.
      • Crawford S.E.
      • Murakami K.
      • Broughman J.R.
      • Karandikar U.
      • Tenge V.R.
      • Neill F.H.
      • Blutt S.E.
      • Zeng X.L.
      • Qu L.
      • Kou B.
      • Opekun A.R.
      • Burrin D.
      • Graham D.Y.
      • Ramani S.
      • et al.
      Replication of human noroviruses in stem cell-derived human enteroids.
      ,
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ).

      Structural characterization of major lipids of HIEs

      The TLC and LC–MS analyses of Folch lower phases of the seven HIE lines identified their general lipid compositions (Fig. 1, A and B). The lipids were dominated by glycerophospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)), constituting around 80 mol % of the whole lipid composition and making up 49, 18, and 11 mol % of the total lipid content, respectively. Free cholesterol constituted about 12 mol % of the lipids, and the rest was made up of various sphingolipids (see below). The composition of all major phospholipids in all HIEs analyzed was essentially the same, including the dominance of PC over PE and PS. The fatty acid distributions of the different glycerolipids of individual HIEs are presented in Figs. S2 and S3.
      Figure thumbnail gr1
      Figure 1Lipid and sphingolipid content of HIEs. A, TLC of HIEs Folch lower-phase lipids with reference glycolipids enriched from an OSeLe meconium sample (
      • Karlsson K.A.
      • Larson G.
      Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.
      • Angström J.
      • Falk K.E.
      • Karlsson K.A.
      • Larson G.
      Chemical fingerprinting of non-acid glycosphingolipids in meconium of a human individual of blood group B Lea–b+ and secretor.
      • Angström J.
      • Falk K.E.
      • Karlsson K.A.
      • Larson G.
      Chemical fingerprinting of glycosphingolipids in meconium of a human blood group O Lea−b+ secretor.
      ). B, molar percentages of cholesterol and glycerolipids; C and D, sphingolipids of HIEs. E, TLC of alkaline-stable fractions of Folch lower-phase lipids of HIEs. F, TLC of alkaline-stable fractions of Folch lower-phase lipids of HIEs using borate-impregnated plate. GalCer, GlcCer, and HexCer from meconium (OLeSe), case 1 (ALeb) and 3 (OLea), represent references and previously published glycolipid extracts of human intestinal epithelium (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell surface antigens of fetal tissue: detailed analysis of glycosphingolipids of meconium of a human O Lea–b+ secretor.
      ,
      • Bjork S.
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Leffler H.
      Structures of blood group glycosphingolipids of human small intestine: a relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor.
      ). Single data points represent the raw data, horizontal lines the means, and error bars the S.D. for J2 (n = 5), J10 (n = 3), J4 (n = 2), 1J, J6, J8, and J4FUT2 (n = 1). Numbers beside TLCs correspond to the number of monosaccharides in GSLs. *, unknown structure colored pink with anisaldehyde staining. Sphingolipids marked with OH in brackets represent sphingolipids with hydroxy fatty acids. FC, free cholesterol.

      Structural characterization of sphingolipids of HIEs

      Sphingolipids constituted around 9 mol % of the measured lipids in all HIEs (Fig. 1, C–F). The major sphingolipids were sphingomyelin (SM (d18:1), 2.8 mol %) and dihydrosphingomyelin (DH-SM (d18:0), 1.4 mol %), free ceramides (Cer (d18:1) and DH-Cer (d18:0), 2.4 mol%), sulfatide (SHexCer (d18:1), 1.8 mol %), monohexosylceramide (HexCer (d18:1), 0.2 mol %), and GSLs with 4–7 monosaccharide residues. Some of these sphingolipids could be identified already in the rough lower-phase extracts (Fig. 1A), but mild alkaline hydrolysis efficiently removed the glycerolipids and exposed all of the sphingolipids on the TLC plates (Fig. 1E). Using a borate-impregnated TLC plate, and reference GalCer and GlcCer standards, it was possible to identify the major HexCer of all HIEs as GalCer with a complex composition of the ceramide part (Fig. 1F). The only major qualitative difference in sphingolipid composition for the different HIEs was in the region of GSLs with 4–7 glycan residues constituting the HBGA epitopes (Fig. 1, A and E). Sialic acid–containing GSLs were undetectable using the classical resorcinol staining of lipid extracts on TLC plates (not shown). Detailed structural characterization of fatty acids and long-chain bases of free ceramides, mono- and dihexosylceramides, and sphingomyelins is given in Figs. S4S6.

      Antibody staining of HBGA epitopes of lipid extracts from HIEs

      Anti-HBGA antibody chromatogram-binding assays (CBAs) of lower and upper phases extracted from HIEs were used to detect and characterize GSLs after separation on TLCs (Figure 2, Figure 3 and Fig. S8). When staining for Lewis a (Lea) and Lewis b (Leb), four major patterns were obtained (Fig. 2 (A and B) and Fig. S8 (B and C)). The J4 and J8 HIE lower-phase lipids were heavily stained for Lea but were negative for Leb, which matches well with their Lewis-positive, secretor-negative genotypes (Fig. 2 (A and B) and Table 1). The J2 and J6 lower-phase lipids were weakly stained for Lea but strongly stained for Leb (Fig. 2, A and B). This confirms their Lewis-positive, secretor-positive genotypes (Table 1). The appearance of weak bands of Lea in J2 and J6 is a parallel to the composition of HBGA GSLs found in meconium samples and in fetal gut (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell-surface antigens of human fetal tissue: meconium, a rich source of epithelial blood-group glycolipids.
      ) but is different from the composition found in adult small intestines, where Lea is not detected in secretor-positive samples (
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Larson G.
      • Leffler H.
      Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual.
      ). The 1J sample was negative for both antibodies, supporting the Lewis-negative genotype of this HIE (Fig. 2 (A and B) and Table 1). The J10 sample was negative for the anti-Leb antibody (Fig. 2B) but showed, surprisingly, in relation to its Lewis and secretor negativity (Table 1), a weak but distinct staining band with the anti-Lea antibody (Fig. 2A). The weak Lea-positive bands may be explained by the observation that not only the FUT3 but also the FUT5 gene encodes for an α1,3/1,4-fucosyltransferase that may be expressed in these cells and enable the biosynthesis of small amounts of Lea structures from the type 1 (Galβ1,3GlcNAc) precursor (Fig. S1).
      Figure thumbnail gr3
      Figure 3TLC-CBAs of Folch upper- and lower-phase lipids from J4 and J4FUT2 HIEs. A, anisaldehyde staining; B, anti-Lea antibody; C, anti-Leb antibody. Reference glycolipids are run on the first lanes and annotated in the margins. up, upper-phase lipids; lp, lower-phase lipids.
      Figure thumbnail gr2
      Figure 2TLC–CBA of HBGAs of GSLs from HIEs. HBGA epitopes were detected with anti-Lea (A), anti-Leb (B), anti-A (C), and anti-B (D) antibodies. AAS, anisaldehyde staining shows migration of reference GSLs also mounted on the first lane of all TLC-CBAs.
      Staining with the anti-A mAb (Fig. 2C), reactive to a terminal GalNAcα1,3 residue, showed the presence of an A active hexaglycosylceramide (A6-1) in the 1J extracts and of an A active heptaglycosylceramide (A7-1) in the J6 HIE extracts. This difference between these two HIEs in histo-blood group A expression is in agreement with both being A and secretor-positive but 1J being Lewis-negative and J6 being Lewis-positive (Table 1). Surprisingly, the lipid extract of J10, which was typed as Lewis- and secretor-negative, showed a distinct double band stained with the anti-A antibody. To further characterize this GSL, additional antibodies, reactive toward glycans with a terminal GalNAc α1,3 residue, were used. However, the GSL was not reactive when stained with an anti-Forssman antibody (Fig. S9B), nor was it stained when using another anti-A antibody, specific for the A type 2 chain structure (not shown), but it did stain with an anti-A6-1 mAb (Fig. S9C). Importantly, this clear reactivity could only be observed after mounting 20 times more J10 lipid extracts than usually used, which indicates that indeed small amounts of the A6-1 GSL were produced by the J10 HIEs. None of the lower-phase lipids of the HIEs were reactive with the anti-B antibody used for this study (Fig. 2D). However, only J2 was genotyped as B, and this antibody was reactive only toward the monofucosylated B (B6-1) and not toward the difucosylated B (B7-1; BLeb). BLeb is indeed expected to be the B-active HBGA GSL produced by J2 HIE, because this HIE has an OB, Lewis (FUT3; LeLe), and secretor (FUT2; Sese)-positive genotype. Thus, there would be no major bands detected with the anti-B antibody.
      The Folch upper- and lower-phase lipids of the HIEs J4 and J4FUT2, being the secretor-negative J4 line transduced with a functional FUT2 construct, were compared in more detail using the anti-Lea and anti-Leb antibodies (Fig. 3). As can be seen already with the chemical staining, the upper-phase lipids of both HIE cultures are devoid of ceramides, cholesterol, phosphoglycerolipids, and sphingomyelin but contain HBGA GSLs (Fig. 3A). CBA with anti-Lea reveals that a minor portion of the Lea pentaglycosylceramide (Lea-5), especially the more polar (slower-moving) ceramide bands, appears also in the upper phase of both HIEs (Fig. 3B). Overlay with anti-Leb does not stain the J4 extracts, but the J4FUT2 extracts are stained (Fig. 3C). The appearance of Leb in J4FUT2 extracts was expected because a functional FUT2 gene was transduced into the original J4 HIE culture, enabling the biosynthesis of the Leb epitope. A specific study of Lea, Leb, and A HBGA structures of the upper-phase GSL of all HIEs (Fig. S8) corroborated the results of the lower-phase CBAs (Fig. 2) and supported the almost perfect match between genotypes and HBGA phenotypes of the HIE GSLs.

      GII.4 Sydney VLPs bind to pure GSLs and lipid extracts of HIEs

      Sydney VLPs, representing one of the more recent human GII.4 HuNoV strains shown to replicate in HIEs (
      • Ettayebi K.
      • Crawford S.E.
      • Murakami K.
      • Broughman J.R.
      • Karandikar U.
      • Tenge V.R.
      • Neill F.H.
      • Blutt S.E.
      • Zeng X.L.
      • Qu L.
      • Kou B.
      • Opekun A.R.
      • Burrin D.
      • Graham D.Y.
      • Ramani S.
      • et al.
      Replication of human noroviruses in stem cell-derived human enteroids.
      ), were assayed for their binding pattern to reference type 1 chain GSLs (Fig. 4, A and B). As can be seen in Fig. 4B, this VLP is rather promiscuous in its binding to most of the HBGA structures tested. Although showing a preference for α1,2-fucosylated structures (H5-1, A6-1, B6-1, Leb, ALeb, BLeb), the VLPs also showed some binding to nonfucosylated Lc4Cer and to the α1,4-fucosylated Lea GSL.
      Figure thumbnail gr4
      Figure 4Binding of GII.4 Sydney VLPs to reference GSLs. A and B, TLC-CBA with GII.4 Syd NoV VLPs binding to type 1 chain reference GSLs of human meconium.
      With this in mind, we tested whether the Sydney VLP would show binding to vesicles made from the Folch lower-phase lipids of the HIEs, using a total internal reflection fluorescence (TIRF) microscopy–based assay. This assay relies on immobilizing VLPs at the bottom of a glass-bottom well and on imaging the binding and release of single fluorescent glycosphingolipid–containing liposomes to individual VLPs under equilibrium conditions (Fig. 5A). Analysis of the arrival rate of the vesicles yields information on the association behavior, where the rate of arrival is directly proportional to the association rate constant (kon). On the other hand, analysis of the vesicle's residence time can be used to estimate the dissociation rate constant (koff). Taken together, this assay therefore allows for the semiquantitative determination of the dissociation constant (KD = koff/kon) by independently measuring kon and koff (
      • Bally M.
      • Gunnarsson A.
      • Svensson L.
      • Larson G.
      • Zhdanov V.P.
      • Höök F.
      Interaction of single viruslike particles with vesicles containing glycosphingolipids.
      ). Here, this assay was carried out by forming supported lipid bilayers of pure B6-1 GSL in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids on a glass surface to immobilize the VLPs. To detect the interaction between surface-immobilized VLPs and Folch lower-phase lipids from HIEs, we used vesicles made of such lipids and containing small amounts of fluorescent lipids for visualization (Fig. 5A).
      Figure thumbnail gr5
      Figure 5Kinetic analysis of the binding of GII.4 Sydney VLP to HIE-vesicles. A, TIRF microscopy assay to probe the interaction between surface-immobilized HuNoV VLPs and HIE-vesicles. GII.4 Sydney VLPs were immobilized on the bilayer, and binding to VLPs was detected with 1% (w/w) rhodamine-labeled vesicles made of lower-phase lipid extracts. B, vesicle surface coverage. C, representative association curves displaying the number of newly arrived vesicles as a function of time for the different HIE-vesicles. D, representative normalized dissociation curves displaying the number of vesicles still bound with time. No dissociation events were recorded for J10. E, arrival rate (n+) obtained by fitting association data with a linear function. F, dissociation rate constant (koff) values obtained from fitting the decay functions with a monoexponential function. See for the fits. G, koff/n+ values. These values are proportional to the dissociation constant KD. In B and E–G, n = 6 for B6-1, n = 7 for J2 and J6, and n = 3 for all others. Single data points, raw data; horizontal lines, means; error bars, S.D. The statistical significance was determined using Welch's t test: *, p < 0.05; **, p < 0.01.
      As shown in Fig. 5B, vesicles from J2 and J6 HIEs exhibited excellent binding to the GII.4 Sydney VLPs. In comparison, binding to J4 and J8 vesicles was greatly reduced, whereas no VLP binding to J10 was observed. Negative controls (i.e. assays without VLPs) showed very little background binding and were similar in counts to the J10 experiments (Fig. S10). These results agree well with the binding pattern of the GII.4 Sydney strain deduced from the HBGA GSL content of the HIEs as well as the infection susceptibility of J2 and J6 and resistance of J4, J8, and J10. The weak interaction with J4 and J8 is in line with the weak binding of these VLPs to reference Lea (Fig. 4), the dominating HBGA GSL present in large amounts in these cells (Fig. 1A). These binding data further support the concept that HBGAs of the type 1 chain GSLs of human small-intestinal cells may indeed be important attachment factors for human GII.4 HuNoV infection.
      To further investigate the relationship between virus binding kinetics, affinity to the membranes, and susceptibility to virus infection, we characterized in detail the interaction kinetics of HuNoV VLPs to the various HIE membranes. Representative association and dissociation plots are shown in Fig. 5 (C and D), whereas the resulting kon, koff, and KD are compared semiquantitatively in Fig. 5 (E–G), respectively. It is noteworthy that dissociation rate constants were significantly lower for the two HIEs susceptible to HuNoV infection (J2 and J6), indicating that the ability of the virus to remain attached to the cell surface may be key in determining its susceptibility. The association rate only moderately correlates with susceptibility to virus infection. Whereas the association rate constant is clearly the highest for the J6 HIE, and accordingly KD the lowest for this case, it appears that J2, also susceptible to infection, is not distinctly different from the infection-resistant HIEs J4 and J8 in terms of their association behavior, indicating that the recruitment of the virus to the ligands may not be the major determinant in the context of virus infection. In line with their ability to replicate the virus, the two susceptible and permissive HIEs, J2 and J6, exhibit the lowest KD values, indicating that the affinity is the strongest for those cells that can be infected. This effect is, however, less pronounced for J2, suggesting that in this case, factors other than virus-glycolipid ligand interaction may be determining their ability to replicate the virus. In agreement with the very low vesicle surface coverage reported for J10 (Fig. 5B), the association rate for this HIE membrane was reduced to 0.01% of the value for J6. The dissociation events were thus too few to allow for data fitting and koff determination.

      Discussion

      The ex vivo culture system of HIEs has established a new niche toward understanding the physiology of the human gastrointestinal tract and the pathophysiology of human gastrointestinal diseases. Our aim was to link the biochemical nature of these cultures with in vivo pathophysiology of the digestive tract specifically related to HuNoV infection and propagation. Because individuals vary in their clinical susceptibility to different HuNoV strains, depending on their HBGA status, we analyzed the composition of lipids and glycosphingolipids of seven HIE lines established from individuals, varying in their ABO, Lewis, and secretor genes, and correlated HuNoV GII.4 Sydney VLP binding to lipid extracts of such HIE cultures to their ex vivo permissiveness to Sydney virus infection.
      Only a few attempts to characterize the lipid composition of human intestines have so far been reported (
      • Karlsson K.A.
      • Larson G.
      Potential use of glycosphingolipids of human meconium for blood group chemotyping of single individuals.
      ,
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell surface antigens of fetal tissue: detailed analysis of glycosphingolipids of meconium of a human O Lea–b+ secretor.
      ,
      • Bjork S.
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Leffler H.
      Structures of blood group glycosphingolipids of human small intestine: a relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor.
      ,
      • Holgersson J.
      • Strömberg N.
      • Breimer M.E.
      Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors.
      ,
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Larson G.
      • Leffler H.
      Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual.
      ,
      • McKibbin J.M.
      • Spencer W.A.
      • Smith E.L.
      • Mansson J.E.
      • Karlsson K.A.
      • Samuelsson B.E.
      • Li Y.T.
      • Li S.C.
      Lewis blood group fucolipids and their isomers from human and canine intestine.
      ), and to the best of our knowledge, this is the first to report on both the detailed glycero- and sphingolipid compositions of human intestinal epithelia and definitely the first comparing epithelia of so many individuals. Our studies are based on enteroids derived selectively from jejunal stem cells. Thus, differences between different parts of the intestines are not addressed in this study but are likely to appear (
      • Holgersson J.
      • Strömberg N.
      • Breimer M.E.
      Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors.
      ). Additionally, the culture conditions, as well as the lack of innervation and naturally surrounding cells and matrices, are not identical to the in vivo situation, which may affect the lipid composition of these cultures. For improving the general conclusions from the lipid analysis, however, we analyzed the composition of differentiated jejunal enteroids established from six different individuals and grown under identical, strictly controlled conditions. This means that the cultures are sterile and under no influence from any gut microflora, bile, gastric, or pancreatic juices or subjected to any shear forces caused by peristaltic movements. Finally, the progenitor stem cells, from which these cultures are established, were taken from biopsies of adult patients undergoing diagnostic or therapeutic enteroscopies and may not completely reflect the cellular composition of intestines of healthy newborns, children, or even healthy adults. The glycoprotein composition of the various HIEs will be reported separately,
      J. Nilsson, I. Rimkute, C. Sihlbom, V. R. Tenge, S. C. Lin, R. L. Atmar, M. K. Estes, and G. Larson, manuscript in preparation.
      and here we focus strictly on membrane-associated lipids and their interactions with HuNoV GII.4 infection.
      The major lipid components were essentially identical and quantitatively in the same range for all HIEs and also similar to what has been reported before for human meconium, which represents shed fetal intestinal epithelia (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell-surface antigens of human fetal tissue: meconium, a rich source of epithelial blood-group glycolipids.
      ). The complete cellular lipid composition of adult intestinal epithelia has not been studied before in molecular detail, although it is known to change during enterocyte differentiation and postnatal maturation, which probably affects the physical mucosal barrier, its selective permeability, and its functionality (
      • Alessandri J.M.
      • Arfi T.S.
      • Thieulin C.
      The mucosa of the small intestine: development of the cellular lipid composition during enterocyte differentiation and postnatal maturation].
      ,
      • van der Bijl P.
      • Lopes-Cardozo M.
      • van Meer G.
      Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells.
      ,
      • Danielsen E.M.
      • Hansen G.H.
      Lipid raft organization and function in brush borders of epithelial cells.
      ,
      • Bock J.
      • Liebisch G.
      • Schweimer J.
      • Schmitz G.
      • Rogler G.
      Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function.
      ). Because we studied the general lipid compositions of the HIE cultures, we could not define differences in asymmetries over basolateral or apical parts of the membranes or differentiate between lipids originating from the cellular plasma membranes, from endosomes, or from other subcellular organelles. However, because HIEs have been shown to support replication, and recapitulate unique aspects of infection, of previously noncultivatable pathogens other than HuNoV, such as cryptosporidium (
      • Heo I.
      • Dutta D.
      • Schaefer D.A.
      • Iakobachvili N.
      • Artegiani B.
      • Sachs N.
      • Boonekamp K.E.
      • Bowden G.
      • Hendrickx A.P.A.
      • Willems R.J.L.
      • Peters P.J.
      • Riggs M.W.
      • O'Connor R.
      • Clevers H.
      Modelling cryptosporidium infection in human small intestinal and lung organoids.
      ,
      • Wilke G.
      • Funkhouser-Jones L.J.
      • Wang Y.
      • Ravindran S.
      • Wang Q.
      • Beatty W.L.
      • Baldridge M.T.
      • VanDussen K.L.
      • Shen B.
      • Kuhlenschmidt M.S.
      • Kuhlenschmidt T.B.
      • Witola W.H.
      • Stappenbeck T.S.
      • Sibley L.D.
      A stem-cell-derived platform enables complete cryptosporidium development in vitro and genetic tractability.
      ) and Salmonella Typhi (
      • Nickerson K.P.
      • Senger S.
      • Zhang Y.
      • Lima R.
      • Patel S.
      • Ingano L.
      • Flavahan W.A.
      • Kumar D.K.V.
      • Fraser C.M.
      • Faherty C.S.
      • Sztein M.B.
      • Fiorentino M.
      • Fasano A.
      Salmonella Typhi colonization provokes extensive transcriptional changes aimed at evading host mucosal immune defense during early infection of human intestinal tissue.
      ), knowing the lipid composition of HIEs will probably be useful and of general significance for the field of host-pathogen interactions. Of special interest is the binding of enterotoxigenic Escherichia coli to histo-blood group A antigens on the apical surfaces of polarized small-intestinal enteroid monolayers (
      • Kumar P.
      • Kuhlmann F.M.
      • Chakraborty S.
      • Bourgeois A.L.
      • Foulke-Abel J.
      • Tumala B.
      • Vickers T.J.
      • Sack D.A.
      • DeNearing B.
      • Harro C.D.
      • Wright W.S.
      • Gildersleeve J.C.
      • Ciorba M.A.
      • Santhanam S.
      • Porter C.K.
      • et al.
      Enterotoxigenic Escherichia coli-blood group A interactions intensify diarrheal severity.
      ), which appears similar to the binding of HuNoV to fucosylated HBGAs of our HIEs (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ).
      For sphingolipids, there is biologically an asymmetric distribution over membrane bilayers, with a natural accumulation in the (apical) plasma membrane and with a majority, if not all, of the glycosphingolipids appearing in the outer leaflet of the bilayer (
      • Hansson G.C.
      • Simons K.
      • van Meer G.
      Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.
      ,
      • Nichols G.E.
      • Shiraishi T.
      • Allietta M.
      • Tillack T.W.
      • Young Jr, W.W.
      Polarity of the Forssman glycolipid in MDCK epithelial cells.
      ). This distribution is related both to the chemical properties of the sphingolipid long-chain bases and to the hydrophilic glycan parts of the GSLs extending toward the exterior water milieu. For GSLs, lateral clusters or microdomains within the plane of the plasma membrane have been called “glycosynapses” (
      • Si S.H.
      The glycosynapse.
      ), indicating a functional role for such microdomains. Both sphingomyelin and free ceramides as well as GSLs have been associated with the formation of microdomains (
      • Quinn P.J.
      A lipid matrix model of membrane raft structure.
      ), and with a prominent accumulation of sphingolipids in the outer layer of epithelial membranes, a structured organization is likely needed.
      We identified SM as the major sphingolipid, also being the dominant sphingolipid in isolated human epithelial cells (
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Larson G.
      • Leffler H.
      Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual.
      ). Indeed, sphingomyelin is often the most abundant sphingolipid in membranes and is highly interactive with cholesterol, another major component of the HIEs, setting the basic prerequisites for microdomain formations and possibly playing functional roles in cell signaling, trafficking, sorting, polarization, and apoptosis (
      • Simons K.
      • van Meer G.
      Lipid sorting in epithelial cells.
      ,
      • Simons K.
      • Ikonen E.
      Functional rafts in cell membranes.
      ). Most tissues contain sphingomyelin with 16:0, 18:0, 22:0, 24:0, and 24:1 acyl chains (
      • Ramstedt B.
      • Slotte J.P.
      Membrane properties of sphingomyelins.
      ). We also found C16:0 to be the dominant nonhydroxy fatty acid of sphingomyelin (SM d18:1) and dihydrosphingomyelin (DH-SM d18:0) in all HIEs (Figs. S4 and S6). In bilayer membranes, cholesterol favorably interacts with DH-SM (d18:0/16:0), and such membranes are more condensed than the ones with SM (d18:1/16:0) (
      • Kuikka M.
      • Ramstedt B.
      • Ohvo-Rekilä H.
      • Tuuf J.
      • Slotte J.P.
      Membrane properties of d-erythro-N-acyl sphingomyelins and their corresponding dihydro species.
      ).
      Another major component of the sphingolipids in the HIEs was the free ceramides showing a very complex composition of both long-chain bases (d18:0, d18:1 and t18:0) and hydroxy and nonhydroxy C16:0-24:1 fatty acids (Figs. S4S6). The biosynthesis of these different structures is highly regulated by different serine palmitoyltransferases, synthases, and desaturatases as well as by sphingomyelinase (Fig. S7) coded for by the corresponding genes SPTLC1, SPTLC2, CERS3, SGMS1, DES1, DES2, and ASM. Interestingly, the receptor protein for murine NoV (MNV) CD300lf is a type 1 integral membrane protein. It has been shown to bind to ceramide (and phosphatidylserine), and, by doing so, its protein epitopes are altered, suggesting a conformational change of the receptor complex and facilitating MNV infection (
      • Orchard R.C.
      • Wilen C.B.
      • Virgin H.W.
      Sphingolipid biosynthesis induces a conformational change in the murine norovirus receptor and facilitates viral infection.
      ). Ceramide has also been shown to affect porcine calicivirus infection (same family of Caliciviridae as HuNoV), although through another mechanism (
      • Murakami K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lin S.C.
      • Ramani S.
      • Ettayebi K.
      • Crawford S.E.
      • Zeng X.L.
      • Neill F.H.
      • Ayyar B.V.
      • Katayama K.
      • Graham D.Y.
      • Bieberich E.
      • Atmar R.L.
      • Estes M.K.
      Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.
      ,
      • Shivanna V.
      • Kim Y.
      • Chang K.O.
      Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses.
      ). Of particular interest is the recent finding that ceramide in the apical cell membrane plays an important role for HuNoV GII.3 replication in HIE cultures (
      • Murakami K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lin S.C.
      • Ramani S.
      • Ettayebi K.
      • Crawford S.E.
      • Zeng X.L.
      • Neill F.H.
      • Ayyar B.V.
      • Katayama K.
      • Graham D.Y.
      • Bieberich E.
      • Atmar R.L.
      • Estes M.K.
      Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.
      ).
      Ceramides are additionally important as biosynthetic precursors of all GSLs (Fig. S7), which, through the action of specific glycosyltransferases, are extended in a step-by-step process to acidic or neutral GSLs in linear or branched-glycan chains. The HBGA epitopes of human intestines are typically found on neutral GSLs with 5–12 monosaccharide residues as the result of α-glycosyltransferase activities, coded for mainly by the ABO, Lewis (FUT3), and secretor (FUT2) genes (
      • Bjork S.
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Leffler H.
      Structures of blood group glycosphingolipids of human small intestine: a relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor.
      ,
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Larson G.
      • Leffler H.
      Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual.
      ) (Fig. S1), also being responsible for the biosynthesis of HBGA epitopes on intestinal glycoproteins.
      After mild alkaline hydrolysis of lower-phase lipids and using an appropriate solvent for borate-impregnated TLC separation, we could establish GalCer as the major component of HexCers in all HIEs (Fig. 1F). The presence of GalCer in the HIE cultures may reflect a potentially important role in the cell membrane, as GalCer has been shown to bind to HuNoV VLPs, particularly in membrane domains of solid supported lipid bilayers (
      • Bally M.
      • Rydell G.E.
      • Zahn R.
      • Nasir W.
      • Eggeling C.
      • Breimer M.E.
      • Svensson L.
      • Höök F.
      • Larson G.
      Norovirus GII.4 virus-like particles recognize galactosylceramides in domains of planar supported lipid bilayers.
      ). However, the presence of GalCer also in the J10 cultures, which are not permissive to the virus, possibly indicates that GalCer may function as an additional attachment factor of importance for endosomal uptake and transfer but not as an individually selective plasma membrane factor.
      GalCer also serves as the natural precursor to sulfatide (3-O-Sulfo GalCer, SHexCer), which is another major sphingolipid component of HIEs and of human intestines, with both tissues showing a very heterogeneous ceramide part (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell surface antigens of fetal tissue: detailed analysis of glycosphingolipids of meconium of a human O Lea–b+ secretor.
      ,
      • Bjork S.
      • Breimer M.E.
      • Hansson G.C.
      • Karlsson K.A.
      • Leffler H.
      Structures of blood group glycosphingolipids of human small intestine: a relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor.
      ) (Figs. S4 and S5). The significance of the structural heterogeneities of the ceramides of GSLs has to await further functional studies of the HIEs. The glycan parts of the diHexCers were not resolved in this study; thus, the diHexCers could theoretically be lactosylceramide (GalGlcCer), as seen in adult intestine (
      • Karlsson K.A.
      • Larson G.
      Potential use of glycosphingolipids of human meconium for blood group chemotyping of single individuals.
      ), or digalactosylceramide (GalGalCer) (Fig. S7) or a combination of both, as seen in human meconium (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell surface antigens of fetal tissue: detailed analysis of glycosphingolipids of meconium of a human O Lea–b+ secretor.
      ).
      Because GSLs bearing HBGAs are enriched in the human intestinal epithelium, we hypothesized that individual differences of HIE GSL subsets would be related to the HBGA genotype of the individuals from which these cultures were established. Overall, this was also true (Table 1, Figure 2, Figure 3, and Figs. S1, S8, and S9). Thus, the 1J HIE, being secretor-positive, Lewis-negative, and of blood group A, strongly expressed A-6 type 1 chain. The J2 HIE, being secretor-positive, Lewis-positive, and of blood group B, expressed Leb and BLeb (chemical staining). The J4 HIE, being secretor-negative, Lewis-positive, and of blood group O, strongly expressed Lea. When transduced with a functional FUT2, the J4FUT2 HIE additionally expressed Leb. The J6 HIE, being secretor-positive, Lewis-positive, and of blood group A, expressed Leb and ALeb (A7-1). The J8 HIE, being secretor-negative, Lewis-positive, and of blood group O, strongly expressed Lea but not Leb. Finally, the J10 HIE, being secretor-negative, Lewis-negative, and of blood group A, did not express Leb but did surprisingly show reactivity with the general anti-A antibody. Using the same antibody, the phenotyping of the supernatant of J10 cultures, using the ELISA, also occasionally showed some “A” reactivity. This reactivity varied in intensity among different lipid preparations of the J10 HIEs. We could exclude the possibility that the GSL was the Forssman antigen or an A type 2 chain GSL; it was confirmed as an A type 1 chain structure using an anti-A6-1–specific mAb (Fig. S9). Interestingly, the expression of this GSL in the J10 line is very low and cannot be directly related to the FUT2 or FUT3 genotype of these cells. Clearly, the major HBGA-reactive GSLs of the HIEs were all of the type 1 chain. Importantly, we did not find any reactivity with two anti-Ley antibodies, indicating the lack of an α1,2-fucosylation of the terminal Gal of any type 2 chain GSLs.
      Our recent studies have shown that overexpression of FUT2 in the J4 secretor–negative HIE cells allows for HuNoV GII.4 infection (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ) and, as shown here, leads to a secretor-positive phenotype (Fig. 3C). We have shown in earlier studies that HuNoV VLPs bind to HBGAs of reference GSLs in a strain-specific manner (
      • Rydell G.E.
      • Dahlin A.B.
      • Höök F.
      • Larson G.
      QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics.
      ,
      • Rydell G.E.
      • Svensson L.
      • Larson G.
      • Johannes L.
      • Römer W.
      Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent α1,2-fucosylated glycosphingolipids.
      ,
      • Nasir W.
      • Bally M.
      • Zhdanov V.P.
      • Larson G.
      • Höök F.
      Interaction of virus-like particles with vesicles containing glycolipids: kinetics of detachment.
      ,
      • Nasir W.
      • Frank M.
      • Kunze A.
      • Bally M.
      • Parra F.
      • Nyholm P.G.
      • Höök F.
      • Larson G.
      Histo-blood group antigen presentation is critical for binding of norovirus VLP to glycosphingolipids in model membranes.
      ), and we now add GII.4 Sydney to this list (Fig. 4B). It is clear from HuNoV infections in HIEs that the GII.4 genotype prefers to replicate in secretor-positive cells (
      • Lupo J.
      • Morel-Baccard C.
      • Michard-Lenoir A.P.
      • Germi R.
      • Pothier P.
      • Ambert-Balay K.
      • Morand P.
      Fatal case of acute gastroenteritis with multiple viral coinfections.
      ). Our findings from overlay and TIRF microscopy assays confirm that GII.4 VLPs prefer to bind to type 1 chain GSL structures dependent on secretor status (FUT2 expression), which further supports GSLs as one of the important attachment factors for this virus.
      Beyond this, we further characterized the interaction kinetics between individual HuNoV VLPs and lipid bilayers made from lipids extracted from the different HIEs in an attempt at better understanding how affinity and interaction kinetics relate to susceptibility to virus infection. Whereas many efforts have been put into the identification of viral receptors, the interplay between the characteristics of the interaction between the virus and the receptor-containing membrane and infection is only poorly understood. It remains widely unknown how binding and release of the virus at the cell surface is modulated and whether the interaction should be strong or weak, fast or slow. The availability of lipid material from HIEs and the advent of methods allowing for the dynamic studies of interaction kinetics on a single-particle level, such as the TIRF assay used here, open new avenues to the study of such processes. Our study suggests that slower dissociation may correlate better with susceptibility, whereas the role of virus association to the membrane is less critical. It was also shown that, generally, the apparent affinity between the virus and the membrane is the highest (lowest KD) for membranes made of lipids of susceptible cells, although intermediate levels of binding were also observed for the extracts of nonsusceptible cells containing Lea. In the future, kinetic studies of virus-HIE membrane interactions, like the one presented here, are expected to extend our understanding of the role of cholesterol, glycerolipids, and sphingolipids (including HBGA active GSLs) in HuNoV infection. HIEs offer, in combination with the methods described here, a new model of looking at the human gut as a model system not only for HuNoV infection, but also for other pathogens and for dissecting various membrane components of the gut epithelium, to study other gastrointestinal diseases as well as the physiology and biochemistry of the intestines.

      Experimental procedures

      Establishment of human intestinal enteroids

      Establishment of HIE cells from patient tissues was originally described by Dr. Hans Clevers' group (
      • Sato T.
      • Stange D.E.
      • Ferrante M.
      • Vries R.G.
      • Van Es J.H.
      • Van den Brink S.
      • Van Houdt W.J.
      • Pronk A.
      • Van Gorp J.
      • Siersema P.D.
      • Clevers H.
      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      ,
      • Sato T.
      • Vries R.G.
      • Snippert H.J.
      • van de Wetering M.
      • Barker N.
      • Stange D.E.
      • van Es J.H.
      • Abo A.
      • Kujala P.
      • Peters P.J.
      • Clevers H.
      Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.
      ,
      • Sato T.
      • Clevers H.
      Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.
      ), and maintenance, expansion, and differentiation of cell cultures, resistant to or susceptible to HuNoV replication, has already been reported (
      • Zou W.Y.
      • Blutt S.E.
      • Crawford S.E.
      • Ettayebi K.
      • Zeng X.L.
      • Saxena K.
      • Ramani S.
      • Karandikar U.C.
      • Zachos N.C.
      • Estes M.K.
      Human intestinal enteroids: new models to study gastrointestinal virus infections.
      ,
      • Estes M.K.
      • Ettayebi K.
      • Tenge V.R.
      • Murakami K.
      • Karandikar U.
      • Lin S.C.
      • Ayyar B.V.
      • Cortes-Penfield N.W.
      • Haga K.
      • Neill F.H.
      • Opekun A.R.
      • Broughman J.R.
      • Zeng X.L.
      • Blutt S.E.
      • Crawford S.E.
      • et al.
      Human norovirus cultivation in nontransformed stem cell-derived human intestinal enteroid cultures: success and challenges.
      ). Cell pellets of differentiated jejunal HIEs from six separate individuals (named 1J, J2, J4, J6, J8, and J10) and J4 HIEs transduced with the FUT2 gene were collected from individual cultures and stored at −80 °C (
      • Ettayebi K.
      • Crawford S.E.
      • Murakami K.
      • Broughman J.R.
      • Karandikar U.
      • Tenge V.R.
      • Neill F.H.
      • Blutt S.E.
      • Zeng X.L.
      • Qu L.
      • Kou B.
      • Opekun A.R.
      • Burrin D.
      • Graham D.Y.
      • Ramani S.
      • et al.
      Replication of human noroviruses in stem cell-derived human enteroids.
      ,
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ). Ethical approvals were obtained from the Institutional Review Board of Baylor College of Medicine and Affiliated Hospitals.

      Production and characterization of secretor-negative J4 HIE cultures transduced to express functional FUT2

      The secretor-negative J4 line was genetically modified to express functional FUT2 following lentivirus transduction. The cDNA of FUT2 was obtained from mRNA of J2 by using the forward primer 5′-ATGGCCCACTTCATCCTC- 3′ and the reverse primer 5′-TTAGTGCTTGAGTAAGGGGGAC-3′. The cDNA was cloned into the lentiviral expression vector pLNSIN-IRES-puromycin or pLNSINIRES-hygromycin (
      • Haga K.
      • Fujimoto A.
      • Takai-Todaka R.
      • Miki M.
      • Doan Y.H.
      • Murakami K.
      • Yokoyama M.
      • Murata K.
      • Nakanishi A.
      • Katayama K.
      Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells.
      ) using an In-Fusin cloning kit (Takara-Clontech) according to the manufacturer's protocol. Lentiviruses were produced in HEK293T cells by transfecting four plasmids (pMDLg/pRRE, pMD2.G, pRSV-Rev, and pLVSIN-IRE-puro- FUT2) using polyethylenimine HCl Max MW 40000 (Polysciences) as a transfection reagent. The culture supernatant was harvested 60–72 h posttransfection. The supernatant was concentrated by a LentiX concentrator (Takara-Clontech) according to the manufacturer's protocol. HIEs were trypsinized for 10 min at 37 °C, and the trypsin was inactivated by CMGF−/10% FBS. After pelleting the cells by centrifugation (300 × g), the cells were plated with the lentivirus solution, and the plate was incubated with centrifugation (300 × g) for 1 h. After incubation, lentivirus solution was removed, and cells were washed in CMGF− and plated in CMGF+/Y-27632 (10 μm; Sigma) with Matrigel (Corning). Five days after lentivirus infection, cells were treated with puromycin (2 μg/ml) or hygromycin (300 μg/ml), and they were maintained until mock-treated cells were completely dead. Colonies that survived the selection were then cultivated as a modified HIE line (
      • Haga K.
      • Ettayebi K.
      • Tenge V.R.
      • Karandikar U.C.
      • Lewis M.A.
      • Lin S.C.
      • Neill F.H.
      • Ayyar B.V.
      • Zeng X.L.
      • Larson G.
      • Ramani S.
      • Atmar R.L.
      • Estes M.K.
      Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection.
      ).

      3′ characterization of HIE pheno- and genotypes

      Individual enteroid monolayers were propagated as described previously (
      • Zou W.Y.
      • Blutt S.E.
      • Crawford S.E.
      • Ettayebi K.
      • Zeng X.L.
      • Saxena K.
      • Ramani S.
      • Karandikar U.C.
      • Zachos N.C.
      • Estes M.K.
      Human intestinal enteroids: new models to study gastrointestinal virus infections.
      ,
      • Ettayebi K.
      • Crawford S.E.
      • Murakami K.
      • Broughman J.R.
      • Karandikar U.
      • Tenge V.R.
      • Neill F.H.
      • Blutt S.E.
      • Zeng X.L.
      • Qu L.
      • Kou B.
      • Opekun A.R.
      • Burrin D.
      • Graham D.Y.
      • Ramani S.
      • et al.
      Replication of human noroviruses in stem cell-derived human enteroids.
      ). Medium was replaced with PBS, and the cells were heated for 5 min. After clarification, the supernatants were analyzed by enzyme immunoassay for the presence of Lea, Leb, A, B, and H antigens, as described previously (
      • Reeck A.
      • Kavanagh O.
      • Estes M.K.
      • Opekun A.R.
      • Gilger M.A.
      • Graham D.Y.
      • Atmar R.L.
      Serological correlate of protection against norovirus-induced gastroenteritis.
      ). For genotyping, DNA was extracted from the monolayers and amplified by PCR to determine FUT2, FUT3, and ABO genotypes using primers specified in Table S1. The amplicons were purified using the GeneJET PCR Purification Kit (Thermo Scientific) and sequenced by GeneWiz (South Plainfield). The resulting chromatograms were assessed for SNPs and deletions at known mutation sites (for FUT2, A385T, G428A, C571T, and C628T; for FUT3, T59G, T202C, C314T, G484A, G508A, G667A, G808A, and T1067A; and for ABO, nucleotides 261 and 297 in exon 6 and 657, 703, 771, 796, 803, 829, and 930 in exon 7).

      Lipid extracts from HIEs

      The lipid extraction was performed using the Folch partition method, which has been described elsewhere (
      • Folch J.
      • Lees M.
      • Sloane Stanley G.H.
      A simple method for the isolation and purification of total lipides from animal tissues.
      ) and is well-established. Frozen cell pellets (1–2 × 106 cells) of HIEs were vigorously mixed with 3 ml of distilled water until the cell pellets dissolved. The cell suspensions were then transferred to Kimax tubes and mixed vigorously with 12 ml of chloloform/methanol (C/M) solution (2:1 by volume), so that the final proportions of chloroform/methanol/water (C/M/W) would be 8:4:3 (by volume). The extraction of lipids was conducted at 60 °C for 1 h. Following centrifugation at 400 × g for 10 min, the lower and upper phases were collected separately, solvents were evaporated under nitrogen, and extracts were weighed and dissolved in C/M (2:1 by volume). Lipid lower phases were used for lipidomics LC-MS and TLC analyses and stored at 4 °C.
      The upper phases of Folch partitions of lipids were desalted using Isolute Spe columns MFC18 (Biotage) under the Vac-Elut 15 mm Hg vacuum. Each column was first wetted with the practical upper phase of Folch partitions from C/M/W (8:4:3 by volume), and then the upper phases from lipid extracts were applied once onto the column without drying. The washing step was performed using the practical upper-phase fraction and then by drying the column under vacuum. Five ml of C/M (2:1 by volume) was applied to elute the lipids. Finally, the desalted upper-phase lipids from HIEs were dried under nitrogen and dissolved in C/M (2:1 by volume), used for TLC analyses, and stored at 4 °C.

      Mild alkaline hydrolysis

      Folch partition lower-phase lipids were treated with 0.2 m KOH in methanol for 3 h at room temperature. The hydrolysis was stopped by neutralization with 1 drop of acetic acid and diluted with chloroform and water, so that only one phase persisted. Samples were desalted using Isolute Spe columns MFC18 applying the protocol described above.

      Reference GSLs

      This study used the reference GSLs lactotetraosylceramide (Lc4Cer), H5-1, A6-1, A7-1, B6-1, Lea-5, and Leb-6 from type 1 chain HBGAs of pooled human meconia and three fractions of a blood group A, B, and O type meconia (AIII, BIII, and OIII) containing GSLs with 4–7 monosaccharides in the glycan chains. The GSLs were purified either from the meconium samples of single ABO blood group typed individuals or from the pooled meconium samples of individuals with identical ABO blood groups and were previously structurally characterized by MS and 1H NMR (
      • Karlsson K.A.
      • Larson G.
      Molecular characterization of cell surface antigens of fetal tissue: detailed analysis of glycosphingolipids of meconium of a human O Lea–b+ secretor.
      ,
      • Karlsson K.A.
      • Larson G.
      Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.
      ,
      • Angström J.
      • Falk K.E.
      • Karlsson K.A.
      • Larson G.
      Chemical fingerprinting of non-acid glycosphingolipids in meconium of a human individual of blood group B Lea–b+ and secretor.
      ). The reference Ley and Forssman GSLs were prepared from dog intestines and have been equally characterized (
      • McKibbin J.M.
      • Spencer W.A.
      • Smith E.L.
      • Mansson J.E.
      • Karlsson K.A.
      • Samuelsson B.E.
      • Li Y.T.
      • Li S.C.
      Lewis blood group fucolipids and their isomers from human and canine intestine.
      ,
      • Svensson L.
      • Hult A.K.
      • Stamps R.
      • Angström J.
      • Teneberg S.
      • Storry J.R.
      • Jorgensen R.
      • Rydberg L.
      • Henry S.M.
      • Olsson M.L.
      Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system.
      ).

      Production of HuNoV VLPs

      The HuNoV VLPs used in this study were produced in SF-9 insect cells using the baculovirus construct from the GII.4 Sydney strain as described elsewhere (
      • Jiang X.
      • Wang M.
      • Graham D.Y.
      • Estes M.K.
      Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.
      ,
      • Kou B.
      • Crawford S.E.
      • Ajami N.J.
      • Czakó R.
      • Neill F.H.
      • Tanaka T.N.
      • Kitamoto N.
      • Palzkill T.G.
      • Estes M.K.
      • Atmar R.L.
      Characterization of cross-reactive norovirus-specific monoclonal antibodies.
      ).

      TLC separation and detection of lipids and GSLs

      Pure GSLs (2 μg), meconium extracts (14 μg), or HIE lipid extracts (100 μg) were applied to alumina-backed silica gel 60 HPTLC plates (Merck) and chromatographed at room temperature with C/M/W (60:35:8 by volume) for 20 min. Two to three sets of GSLs were run in parallel on the same TLC plate, and the dried plate was then cut into two to three parts. GSLs on one of the plates were visualized by spraying with anisaldehyde/sulfuric acid/acetic acid (1:2:97 by volume) and heated at 180 °C for color development. The other plate parts were used for the CBA.
      For optimal separation and characterization of the monohexosylceramides (HexCer) GlcCer and GalCer, glass-backed silica gel 60 HPTLC plates were sprayed with sodium tetraborate, 1% solution in water, activated at 150 °C overnight, eluted with C/M/W (100:30:4 by volume) (
      • Karlsson K.A.
      • Samuelsson B.E.
      • Steen G.O.
      Separation of monoglycosylceramides (cerebrosides) of bovine kidney into subgroups and characterization by mass spectrometry.
      ) and stained with the anisaldehyde reagent.
      To chemically detect sialic acid–containing GSLs, HIE lipid extracts (upper and mild alkaline hydrolysis-treated lower phases) were applied on glass-backed silica gel 60 HPTLC plates and chromatographed at room temperature with C/M/W (60:35:8 by volume) for 20 min. Dry plates were visualized by spraying with resorcinol-HCl reagent (10 ml of 2% resorcinol in water, 40 ml of concentrated HCl, 0.125 ml of 0.1 m cupric sulfate) and heated at 150 °C for color development.

      Chromatogram-binding assay with antibodies (TLC-CBA)

      GSLs and lipid extracts were separated by TLC using C/M/W (60:35:8 by volume) as eluent. After chromatography, the plates were dried and immersed into a solution of 0.3% polyisobutylmethacrylate in diethylether:n-hexane (1:1 by volume) for 60 s, dried, and incubated in PBS, containing 3% BSA and 0.05% Tween 20 for 1 h, as described before (
      • Hansson G.C.
      • Karlsson K.A.
      • Larson G.
      • Samuelsson B.E.
      • Thurin J.
      • Bjursten L.M.
      Detection of blood group type glycosphingolipid antigens on thin-layer plates using polyclonal antisera.
      ).
      For anti-HBGA CBA, the TLC plates were overlaid with mouse monoclonal antibodies specific for either Lea (1:200) (catalog no. 4861, Gamma-Clone), Leb (1:100) (catalog no. 808407, SeraClone), A (1:100) (catalog no. 10008-1, Gamma-Clone), A-1 (1:4) (clone AH21, kind gift from Professor Henrik Clausen), Forssman (1:50) (kind gift from Dr. Lola Svensson, University of Gothenburg), Ley (1:200) (catalog no. Ab00492-1, Absolute Antibody), or Ley (1:100) (catalog no. 912501, BioLegend) (
      • Lloyd K.O.
      • Larson G.
      • Strömberg N.
      • Thurin J.
      • Karlsson K.A.
      Mouse monoclonal antibody F-3 recognizes the difucosyl type-2 blood group structure.
      ). The antibodies were all diluted in PBS with 0.5% BSA and left on the plate for 1 h 30 min. After removal of the primary antibody, the polyclonal goat anti-mouse detection antibody conjugated with alkaline phosphatase (catalog no. A0162, Sigma), diluted 1:500 in PBS with 0.5% BSA, was added for 1 h 30 min. After washing, the color reagent BCIP/NBT Fast (catalog no. B5665, Sigma) was applied. After washing, plates were dried and photographed. Plates were always washed three times with PBS and 0.05% Tween 20, and all steps were performed at room temperature.

      Chromatogram-binding assay with VLPs (TLC-CBA)

      GSLs were separated by TLC, and plates were prepared for the CBA as described above. For VLP CBAs, the TLC plates were then overlaid with GII.4 Sydney (1:100) in PBS and incubated for 2 h. After removal of unbound VLP, primary rabbit anti-HOV antibody (1:2500) (
      • Kou B.
      • Crawford S.E.
      • Ajami N.J.
      • Czakó R.
      • Neill F.H.
      • Tanaka T.N.
      • Kitamoto N.
      • Palzkill T.G.
      • Estes M.K.
      • Atmar R.L.
      Characterization of cross-reactive norovirus-specific monoclonal antibodies.
      ) incubation in dilution buffer (PBS with 0.5% BSA) was carried out for 1 h and followed with alkaline phosphatase–conjugated goat anti-rabbit antibody (1:5000) (catalog no. A3687, Sigma) incubation in dilution buffer for 1 h. Finally, staining with BCIP/NBT Fast was used to develop a color reaction (
      • Nilsson J.
      • Rydell G.E.
      • Le Pendu J.
      • Larson G.
      Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids.
      ). Plates were washed once, dried, and photographed. After each step, plates were washed three times with PBS and 0.05% Tween 20, and every step was performed at room temperature.

      Lipid analysis

      Lower-phase lipids, extracted using the Folch procedure (
      • Folch J.
      • Lees M.
      • Sloane Stanley G.H.
      A simple method for the isolation and purification of total lipides from animal tissues.
      ), were analyzed using a combination of direct infusion MS and ultra-HPLC tandem MS (UPLC-MS/MS).
      Phospholipids (i.e. PCs, PEs, PS, and SMs) were analyzed using a QTRAP 5500 mass spectrometer (Sciex) equipped with a robotic nanoflow ion source, TriVersa NanoMate (Advion BioSciences, Ithaca, NJ). The analysis was made using precursor ion scanning in negative (PC, PE, and PS) and positive (SM) ion mode according to previous work (
      • Brügger B.
      • Erben G.
      • Sandhoff R.
      • Wieland F.T.
      • Lehmann W.D.
      Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.
      ,
      • Ekroos K.
      • Chernushevich I.V.
      • Simons K.
      • Shevchenko A.
      Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer.
      ,
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      • Bonner R.
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      • Shevchenko A.
      Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.
      ), and the lipid species were quantified using heptadecanoyl (C17:0)-containing internal standards added during sample preparation.
      Sphingolipids (i.e. Cer, HexCer, and diHexCer), were analyzed using UPLC-MS/MS on a QTRAP 5500 mass spectrometer. Lipid species were separated using an Acquity BEH C8 column (2.1 × 100 mm with 1.7 μm particles; Waters) with water, acetonitrile, and isopropyl alcohol as mobile phases (
      • Amrutkar M.
      • Cansby E.
      • Nuñez-Durán E.
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      Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH.
      ). Quantification was made using external calibration curves with available reference substances.
      Free cholesterol was quantified using straight-phase HPLC coupled to evaporative light-scattering detection according to previous work (
      • Homan R.
      • Anderson M.K.
      Rapid separation and quantitation of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.
      ).

      Lipid vesicle preparation

      Bilayer and detection vesicles to be used in the TIRF microscopy–based assay were prepared by lipid hydration and extrusion. In brief, appropriate amounts of lipids dissolved in chloroform/methanol (2:1 by volume) were added to a round-bottom flask. The lipid solution was then dried under a light stream of nitrogen and subjected to vacuum for at least 90 min. After drying, the lipid film was hydrated in PBS buffer and vortexed several times before extruding the suspension by passing it several times through a polycarbonate membrane of appropriate pore size (Whatman) using a mini-extruder (Avanti Polar Lipids).
      Bilayer vesicles were prepared by extruding a vesicle suspension, consisting of 95% (w/w) POPC and 5% (w/w) B6-1 GSL, several times through a polycarbonate membrane with a pore size of 50 nm at room temperature. Passivation vesicles were prepared by extruding a POPC vesicle suspension through a polycarbonate membrane with pore size of 100 nm at room temperature. Detection vesicles were prepared by extruding a vesicle suspension consisting of 99% (w/w) Folch lower-phase lipids and 1% (w/w) N-(lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine through a polycarbonate membrane with a pore size of 100 nm. Lipid hydration and extrusion was carried out at 60 °C. The vesicles thus obtained all had similar size distributions as verified by nanoparticle-tracking analysis (see Fig. S11).

      HuNoV VLP binding to HIE lipids vesicles

      The surface of glass-bottom microtiter wells (96-well plate) (Mat Tek) was cleaned using 3.5% Hellmanex II (Hellma Analytics) in ultrapure water (18 megaohms) for 2 h and then thoroughly rinsed with ultrapure water. To form supported lipid bilayers containing 5% (w/w) of B6-1 GSL, 50 μl of vesicle suspension (total lipid concentration 0.1 mg/ml) was incubated for 30 min at room temperature. The wells were then washed several times with PBS, keeping the surface hydrated. HuNoV GII.4 Sydney VLPs were then added to the microtiter wells coated with the B6-1 GSL–presenting supported lipid bilayer and incubated for 45 min at room temperature. After an extensive washing with PBS, POPC vesicles were added to reach the final concentration of 10 μg/ml. The fluorescent vesicles for detection of VLP binding were added to the well at the final concentration of 1.3 × 1011 particles/ml in PBS, maintaining a total volume of 100 μl in the well. The number of vesicles in each sample was quantified using nanoparticle tracking analysis (Nanosight NS 300) and following the manufacturer's instructions for particle quantification. Time-lapse movies were recorded at room temperature with a TIRF microscope, 45 min after the injection of fluorescent vesicles.
      A Nikon Eclipse Ti2-E inverted microscope with a ×60 magnification (numerical aperture 1.49) oil immersion objective (Nikon Corp.) was used to acquire the time-lapse movies at a frame rate of 1 frame/s. The microscope was equipped with a solid-state light source (Spectra III light engine, Lumencor), a multi-band pass filter cube 86012v2 Dapi/FITC/TxRed/Cy5 (Nikon Corp.), and a Prime 95B sCMOS camera (Teledyne Photometrics). The images were further analyzed using an in-house Matlab (Mathworks) script (
      • Bally M.
      • Gunnarsson A.
      • Svensson L.
      • Larson G.
      • Zhdanov V.P.
      • Höök F.
      Interaction of single viruslike particles with vesicles containing glycosphingolipids.
      ) allowing for the determination of the vesicle surface coverage over time, the rate of arrival of the vesicles, and their residence time. The number of bound particles was measured in three separate spots in each well, with each spot being recorded for 350 frames. Here, any vesicle staying for three frames or more was considered. The number of bound particles was averaged over each time lapse, and the surface coverage was evaluated as the average number of bound particles in each spot. The association rate for each measurement was determined by summing together the arrival events on three different spots on the well followed by a linear fit, excluding the first 46 frames from it. The dissociation behavior was determined by fitting the dissociation curves obtained from the residence time (see Ref.
      • Bally M.
      • Gunnarsson A.
      • Svensson L.
      • Larson G.
      • Zhdanov V.P.
      • Höök F.
      Interaction of single viruslike particles with vesicles containing glycosphingolipids.
      for further details). Although vesicle binding to surface-immobilized VLPs has been previously reported to exhibit a multiexponential decay (
      • Bally M.
      • Gunnarsson A.
      • Svensson L.
      • Larson G.
      • Zhdanov V.P.
      • Höök F.
      Interaction of single viruslike particles with vesicles containing glycosphingolipids.
      ,
      • Nasir W.
      • Bally M.
      • Zhdanov V.P.
      • Larson G.
      • Höök F.
      Interaction of virus-like particles with vesicles containing glycolipids: kinetics of detachment.
      ), we here applied a single exponential fit as an approximation of the dissociation behavior (see Fig. S12).

      Data availability

      Lipidomics data are available upon request to Göran Larson ([email protected]), and VLP binding data are available upon request to Marta Bally ([email protected]). All other data are contained within the article.

      Acknowledgments

      Dr. Lola Svensson is gratefully acknowledged for the anti-Forssman antibody, Professor Henrik Clausen for kindly sharing the anti-A6-1 antibody (clone AH21), Professor Michael Breimer for access to glycolipids of adult human intestinal cells, and Associate Professor Niclas Karlsson for mass spectrometric analyses of GalCer and GlcCer references. We thank Professor B. V. Venkatar Prasad for fruitful discussions and Professor Mark Donowitz for a kind gift of the 1J line. All lipidomics MS analyses were performed at the Lipidomics Core Facility, BioMS national node, at Sahlgrenska Academy, University of Gothenburg.

      Supplementary Material

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