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TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

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Science Immunology  13 May 2020:
Vol. 5, Issue 47, eabc3582
DOI: 10.1126/sciimmunol.abc3582
  • Fig. 1 VSV-SARS-CoV-2 infects human small intestinal enteroids.

    (A) Mouse small intestinal cells were analyzed by scRNA-seq and resolved into 20 clusters based on gene expression profiles (left). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Clusters 10 and 18: intraepithelial lymphocytes; clusters 1, 2, 5, 8, 9, 17, 19, and 20: enterocytes; cluster 3: goblet cells; cluster 4: enteroendocrine cells; cluster 7: Tuft cells; cluster 11: crypt stem cells; cluster 12: Paneth cells. Each dot represents a single cell. Note that Ace2high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 μm. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 105 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 105 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 μm. (E) Same as (C) except that virus titers were measured using a TCID50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, 80 μm. (G) Same as (D) except that human colon enteroids were used instead. Scale bars, 80 μm. For all figures except (A), experiments were repeated at least three times with similar results. (A) was performed once with small intestinal tissues pooled from three mice. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

  • Fig. 2 VSV-SARS-CoV-2 and wild-type SARS-CoV-2 replicate in ACE2+ human mature enterocytes.

    (A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to a TCID50 assay to measure the amount of infectious virus. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (D) Differentiated ileum enteroids in monolayer were apically or basolaterally infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (E) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bars, 32 μm. SARS-CoV-2–infected ACE2-positive cells are enlarged in the inset (yellow box). (F) SARS-CoV-2–infected duodenum monolayers were imaged along the Z stacks and sectioned for YZ planes (top) and reconstructed for 3D images (bottom). For all figures except (C) to (E), experiments were repeated at least three times with similar results. (C) to (E) were performed twice with technical duplicates in each experiment. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

  • Fig. 3 TMPRSS2 and TMPRSS4, but not ST14, mediate SARS-CoV-2 S–mediated entry.

    (A) Bulk RNA-seq results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, and HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left), or transfected with indicated plasmid combination for 24 hours (right), and infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with SARS-CoV-2 S and TMPRSS2 or TMPRSS4 for 48 hours. Cells were treated with trypsin at 0.5 μg/ml for 10 min. The levels of S and GAPDH were measured by Western blot. The intensity of bands was quantified by ImageJ and shown as percentage of the bottom band versus the top band in each lane. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8 × 105 PFU of VSV-SARS-CoV-2 on ice for 1 hour, washed with cold phosphate-buffered saline for three times, and shifted to 37°C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type (WT) or human ACE2-expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 for 24 hours. The red arrows highlight the formation of large syncytia. Scale bars, 100 μm. For all figures except (A), experiments were repeated at least three times with similar results. RNA-seq in (A) was performed once with duplicate samples. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

  • Fig. 4 TMPRSS2 and TMPRSS4 promote VSV-SARS-CoV-2 infection in enteroids.

    (A) Schematic diagram of SARS-CoV-2 infection of human mature enterocytes. SARS-CoV-2 particles, host proteins, and host cells are not proportional to the true sizes. On the basis of the scRNA-seq data (fig. S3A), TMPRSS4 is more highly expressed than TMPRSS2 on mature absorptive enterocytes and TMPRSS2 is more highly expressed on secretory cells than TMPRSS4. FPKM, fragments per kilobase per million mapped reads. (B) GFP-expressing HEK293 cells were mixed at 1:1 ratio and cocultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hours (right). Note the formation of cell-cell fusion (yellow), highlighted by black arrows. (C) Images in (B) were quantified based on the intensity of yellow signals. T2: TMPRSS2; T4: TMPRSS4. (D) Human duodenum enteroids in 3D Matrigel were transduced with lentiviruses encoding Cas9 and sgRNA against TMPRSS2 or TMPRSS4 (oligonucleotide information in table S1). Gene knockout (KO) enteroids were seeded into monolayers and infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Human duodenum, ileum, and colon enteroids were infected with 2.9 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The levels of indicated viral and host transcripts were measured by RT-qPCR and normalized to that of GAPDH. (F) Human duodenum enteroids seeded into collagen-coated 96-well plates were differentiated for 3 days; pretreated with SBTI (50 μg/ml), 10 μM camostat mesylate, or 10 μM E-64d for 30 min; and infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

  • Fig. 5 SARS-CoV-2 rapidly loses infectivity in the human GI tract.

    (A) SARS-CoV-2-mNeonGreen virus (2.5 × 105 PFU) was incubated with M199 medium, simulated human small intestinal (SI) fluid, or simulated human large intestinal (LI) fluid for indicated time points at 37°C. The virus was subsequently serially diluted and added to MA104 cells for 24 hours, and GFP signals were scanned by Typhoon 5. (B) Stool specimens from 10 patients with COVID-19 were collected and subjected to qPCR experiments to quantify the absolute levels of SARS-CoV-2 N gene. (A) was performed once in quadruplicate. (B) was performed once. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/47/eabc3582/DC1

    Fig. S1. ACE2 is highly expressed in the human intestine.

    Fig. S2. VSV-SARS-CoV-2 induces syncytia formation in cell culture and human intestinal cells.

    Fig. S3. TMPRSS2 and TMPRSS4 enhance VSV-SARS-CoV-2 infectivity.

    Fig. S4. Validation of CRISPR-Cas9–mediated knockout of TMPRSS2 and TMPRSS4 in human enteroids.

    Fig. S5. Rotavirus remains infectious in simulated human gastric and intestinal fluids.

    Table S1. qPCR and CRISPR deletion oligonucleotide information.

    Table S2. Bulk RNA-sequencing results of HEK293, Huh7.5, H1-Hela, and HT-29 cells and human ileum enteroids (Excel spreadsheet).

    Table S3. Raw data file (Excel spreadsheet).

    Movie S1. Formation of syncytia in 3D human duodenum enteroids infected by SARS-CoV-2 GFP chimeric virus.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. ACE2 is highly expressed in the human intestine.
    • Fig. S2. VSV-SARS-CoV-2 induces syncytia formation in cell culture and human intestinal cells.
    • Fig. S3. TMPRSS2 and TMPRSS4 enhance VSV-SARS-CoV-2 infectivity.
    • Fig. S4. Validation of CRISPR/Cas9 mediated knockout of TMPRSS2 and TMPRSS4 in human enteroids.
    • Fig. S5. Rotavirus remains infectious in simulated human gastric and intestinal fluids.
    • Table S1. QPCR and CRISPR deletion oligonucleotide information.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Bulk RNA-sequencing results of HEK293, Huh7.5, H1-Hela, HT-29 cells, and human ileum enteroids.
    • Table S3 (Microsoft Excel format). Raw data file.
    • Video S1 Formation of syncytia in 3D human duodenum enteroids infected by SARS-CoV-2 GFP chimeric virus.

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