TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Bestle, Dorothea; Heindl, Miriam Ruth; Limburg, Hannah; van, Thuy Van Lam; Pilgram, Oliver; Moulton, Hong M.; Stein, David A.; Hardes, Kornelia; Eickmann, Markus; Dolnik, Olga; Rohde, Cornelius; Becker, Stephan; Klenk, Hans‐Dieter; Garten, Wolfgang; Steinmetzer, Torsten; Böttcher‐Friebertshäuser, Eva

doi:10.1101/2020.04.15.042085

Cited by 117 publications

(88 citation statements)

References 67 publications

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“…To test this hypothesis, we co-expressed a C-terminally Strep-tagged full-length SARS-CoV-2 S protein in an HEK293 cell line that stably expresses ACE2 with or without additional introduction of TMPRSS2 or TMPRSS4. In mock cells, we readily observed the full-length S and a cleaved product that corresponded to the size of S1 fragment, presumably cleaved by furin protease that is ubiquitously expressed ( 35 ). Importantly, the expression of TMPRSS2 or TMPRSS4 enhanced S cleavage, as evidenced by the reduction of full-length S and increase of S2 levels (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Zang

Castro

McCune

et al. 2020

Preprint

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Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

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Section: Resultsmentioning

confidence: 99%

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

Zang

Castro

McCune

et al. 2020

Preprint

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“…In mock-transfected cells, we readily observed the full-length S and a cleaved product that corresponded to the size of S1 fragment ( Fig. 3C), presumably cleaved by furin protease that is ubiquitously expressed (39). Compared with the positive control exogenous trypsin treatment, expression of TMPRSS2 or TMPRSS4 promoted S cleavage, as evidenced by the reduction of full-length S and increased levels of S1 protein ( Fig.…”

Section: Tmprss2 and Tmprss4 But Not St14 Promote Sars-cov-2 Entrymentioning

confidence: 91%

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

et al. 2020

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Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2 + mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with COVID-19. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression. RESULTS SARS-CoV-2 infects human intestinal enteroidsIn the intestine, ACE2 functions as a chaperone for the sodiumdependent neutral amino acid transporter B 0 AT1 (encoded by SLC6A19) on IECs and regulates microbial homeostasis (21,22). ACE2 expression is substantially higher in the small intestine than in all the other organs, including the lung, in both humans and mice (fig. S1, A and B). Given these data, we assessed whether SARS-CoV-2 can infect IECs as a first step for understanding its implications for fecal-oral transmission. We performed single-cell RNA sequencing (scRNA-seq) to capture the global transcriptomics in all IEC subsets in the mouse small intestinal epithelium (Fig. 1A, left). ACE2 mRNA was predominantly seen in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes (Fig. 1A, right) (23, 24). In addition, bulk RNA-seq revealed that primary human ileum enteroids had substantially higher mRNA levels of all known CoV receptors, including ACE2, than the of 10 that Ace2 high 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 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 10 5 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 × 10 5 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 m. (E) Same as (C) except that virus titers were measured using a TCID 50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, ...

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“…The protein sequence between mouse and human is conserved, with 77% sequence identity suggesting structure and functional similarity 12 . Given the strong evidence that TMPRSS2 mediates coronavirus entry, when SARS-CoV-2 emerged it was soon demonstrated through loss-and gain-offunction experiments that TMPRSS2 is retained as a mediator of cell infection, and that this can be inhibited by camostat [13][14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

Shrimp

Kales

Sanderson

et al. 2020

Preprint

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SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic.Consequently, much research has gone into the development of pre-clinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites ("priming") that causes a conformational change allowing for viral and host membrane fusion. This fusion event is proceeded by release of viral RNA within the host cell. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. Additionally, knock-out studies in mice have demonstrated reduced infection in the absence of TMPRSS2 with no detectable physiological impact; thus, TMPRSS2 is an attractive target for therapeutic development. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat and gabexate, clinically approved agents in Japan for pancreatitis due to their inhibition of trypsin-like proteases.Nafamostat and camostat are currently in clinical trials against COVID19. The rank order potency for the three inhibitors is: nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM) and gabexate (IC50 = 130 nM). Further profiling of these three inhibitors against a panel of proteases provides insight into selectivity and potency.

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TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Cited by 117 publications

References 67 publications

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

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

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

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

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