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. 2020 Mar 17;11(2):e00251-20.
doi: 10.1128/mBio.00251-20.

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Kei Haga  1 , Khalil Ettayebi  1 , Victoria R Tenge  1 , Umesh C Karandikar  1 , Miranda A Lewis  1 , Shih-Ching Lin  1 , Frederick H Neill  1 , B Vijayalakshmi Ayyar  1 , Xi-Lei Zeng  1 , Göran Larson  2 , Sasirekha Ramani  1 , Robert L Atmar  1   3 , Mary K Estes  4   3
Affiliations

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Kei Haga et al. mBio. .

Abstract

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.

Keywords: fucosyltransferase 2; glycobiology; histo-blood group antigens; isogenic enteroids; isogenic organoids; noroviruses; secretor status.

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Figures

FIG 1
FIG 1
HBGA expression phenotype by ELISA. Five-day differentiated HIEs were evaluated for HBGA expression with primary antibodies against either the Lea, Leb, A, or B epitope and HRP-conjugated secondary antibodies. Mean absorbance values from two ELISA replicates are plotted. Error bars denote standard deviations (SD). The threshold of detection is indicated by a dashed line at absorbance of 0.1.
FIG 2
FIG 2
A functional copy of FUT2 is needed for Fucα1,2Gal antigen expression on the apical surface (J2Fut2+/− and J4Fut2−/−/FUT2 images in panel A). Fucα1,2Gal antigen expression was not detected on the apical surfaces of cell lines with no functional FUT2 gene (J2Fut2−/− and J4Fut2−/− images in panel A). H antigen expression was analyzed by UEA-1 lectin (red) in HIE lines. (B and C) Specificity for UEA-1 lectin detection of Fucα1,2Gal antigen in all enteroid lines is demonstrated by the reduction of staining when UEA-1 was preincubated with either 10 mM (B) or 100 mM (C) l-fucose. (D to F) Graphical quantitation of fluorescence is shown below the image panels. Each data bar represents the mean fluorescence from six total wells collected from two experiments. Error bars denote SD. For each enteroid line, significant differences in fluorescence comparing no l-fucose pretreatment of UEA-1 (D) to 10 mM (E) or 100 mM (F) pretreatment were determined by Student’s t test (*, P < 0.05). In all image panels, the nuclei are marked with DAPI (blue), Fucα1,2Gal antigen was detected by UEA-1 lectin (red), and the brush border is indicated by actin expression using phalloidin (white). Bars, 20 μm.
FIG 3
FIG 3
Knocking out FUT2 prevents infection of GII.4, GII.17, and GI.1 HuNoV strains in J2 HIEs. HIE monolayers were inoculated with GII.4, GII.3, GII.17, or GI.1 HuNoV stool filtrate in 500 μM GCDCA-containing Intesticult medium for 1 h (GII.4, GII.3, and GII.17) or 2 h (GI.1) at 37°C. After two washes with CMGF(−) medium, the cells were cultured in the presence of GCDCA for 24 h at 37°C. Total well RNA was extracted, and genome equivalents (GEs) were determined by RT-qPCR. Each data bar represents the mean for three wells of inoculated HIE monolayers. Error bars denote SD. Each experiment was performed two or more times, with three technical replicates in each experiment. Data from a representative experiment are shown in this figure. Numbers above the bars indicate log10 fold change comparing GEs at 24 h postinfection (hpi) to 1 or 2 hpi. Significance was determined by two-way ANOVA with post hoc analysis using Tukey’s test (*, P < 0.05; n.s., not significant).
FIG 4
FIG 4
Knocking in FUT2 is sufficient for infection of J4 HIEs with all HuNoV strains tested. HIE monolayers were inoculated with GII.4, GII.3, GII.17, or GI.1 as described in the legend to Fig. 3. Total well RNA was extracted, and GEs were determined by RT-qPCR. Each data bar represents the mean for three wells of inoculated HIE monolayers. Error bars denote SD. Each experiment was performed two or more times, with three technical replicates in each experiment. Numbers above the bars indicate log10 fold change comparing GEs at 24 hpi to 1 or 2 hpi. Significance was determined by two-way ANOVA with post hoc analysis using Tukey’s test (*, P < 0.05; n.s., not significant).
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