TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes
GI bioinformatics cell biology clinical molecular biology
Authors: Zang, R. et al.
Link to paper: https://www.biorxiv.org/content/10.1101/2020.04.21.054015v1
Journal/ Pre-Print: bioRxiv
Tags: Bioinformatics, Cell Biology, Clinical, Gut, Molecular biology
1. SARS-CoV-2 pseudotype virus infects infects human duodenal, ileal and colonic enteroids
2. SARS-CoV-2 preferentially infects, and is release from, the apical membrane of human duodenal enteroids
3. Proteolytic processing of SARS-CoV-2 spike protein in human IECs is mediated by TMPRSS2 and TMPRSS4 and viral entry into the host cell mediated in combination with ACE2
The authors aim to characterise SARS-CoV-2 infection in human intestinal epithelial cells (IECs), given that some COVID-19 patients present with gastrointestinal symptoms. Genetically engineered SARS-CoV-2 reporter virus infected mature enterocytes in human enteroids. SARS-CoV-2 most successfully infected, and was released from, the apical membrane of human duodenal organoids. Using transfected HEK293 cells, they show TMPRSS2, TMPRSS4 and ACE2 are mediating virus entry. TMPRSS2 is shown to be expressed on secretory IECs and ACE2 and TMPRSS4 on neighbouring mature enterocytes. Virus entry and replication in enteroids was reduced upon CRISPR-Cas9 knockdown of TMPRSS2 and TMPRSS4. GI tract fluids inactivate SARS-CoV-2 in vitro.
Impact for SARS-CoV2/COVID19 research efforts
Understand the virology and/or cell biology of SARS-CoV2/COVID19
Clinical symptoms and pathogenesis of SARS-Cov2/COVID19
· In silico study / bioinformatics study (single-cell RNA-seq)
· In vitro study (HEK293 cell lines and ex vivo human intestinal organoids infected with SARS-CoV-2-S pseudotype virus)
Strengths and limitations of the paper
Novelty: 1. Mature enterocytes, expressing higher levels of ACE2, are infected by SARS-CoV2 reporter virus
2. SARS-CoV2 reporter virus infects, and is released from, the apical membrane of human duodenal enteroids
3. TMPRSS2, TMPRSS4 and ACE2 are important for viral entry into IECs
4. Paracrine interaction of TMPRSS2 expressing cells (secretory IECs) with neighbouring ACE2 and TMPRSS4 expressing cells (mature enterocytes) can drive cell fusion and viral infectivity in IECs
Standing in the field: Show viral shedding in fecal samples of COVID19 patients, which was previously reported. Mature enterocytes were shown to express higher ACE2 levels and higher viral infection, other publications also report ACE2 expression by mature enterocytes and successful infection of intestinal epithelial cells by SARS-CoV2 (https://doi.org/10.1101/2020.04.25.060350; https://doi.org/10.1101/2020.04.24.059667)
Appropriate statistics: Statistical analysis is not described throughout the paper. Error bars were used but type not stated.
Viral model used: vesicular stomatitis virus (VSV)- chimera GFP reporter virus, with its glycoprotein genetically replaced with the SARS-CoV-2 S protein
Main limitations: 1. Study uses genetically engineered VSV-reporter virus instead of WT virus, which might have an impact on viral replication
2. Study claims to show lack of infectious virus in fecal samples of Covid19 patients, however supportive data is missing also in the referenced paper. This is a big claim which is not fully supported.
3. Do not report number of replicates performed, which makes it hard to judge reproducibility of the data. Used only one human donor for duodenal organoid generation. Fecal samples of only 5 patients were used.
4. It isn’t clear which gene sets define the different cell clusters in their scRNAseq analysis of murine small intestinal epithelial cells
5. Inactivation of SARS-CoV-2 in GI tract fluids might not be physiologically accurate, as virus might be shielded from those fluids by microbiota in the intestinal lumen