key: cord-286001-pu1fetq7
authors: Zang, Ruochen; Castro, Maria F.G.; McCune, Broc T.; Zeng, Qiru; Rothlauf, Paul W.; Sonnek, Naomi M.; Liu, Zhuoming; Brulois, Kevin F.; Wang, Xin; Greenberg, Harry B.; Diamond, Michael S.; Ciorba, Matthew A.; Whelan, Sean P.J.; Ding, Siyuan
title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes
date: 2020-04-23
journal: bioRxiv
DOI: 10.1101/2020.04.21.054015
sha: 
doc_id: 286001
cord_uid: pu1fetq7

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.

Correspondence: Siyuan Ding, siyuan.ding@wustl.edu 22 infection of cultured cells (Fig. S2A) . We also observed multiple events of syncytia 139 formation between IECs in both 2D monolayer (Fig. S2B ) and in 3D Matrigel (Fig. S2C  140 and Video S1). The cell fusion and subsequent cytopathic effect may have important 141 implications regarding the common GI symptoms seen in COVID-19 patients (25) (26) (27) (28) (29) . TMPRSS4 and matriptase (encoded by ST14) shared a highly specific expression pattern 149 in human IECs (Fig. 3A) . Previous studies indicated that both TMPRSS4 and ST14 150 facilitate influenza A virus but neither play a role in SARS-CoV infection (31-34). Notably, 151 in our mouse scRNA-seq dataset, both TMPRSS4 and ST14 were found to be present in 152 mature enterocytes and had an increased co-expression pattern with ACE2 than 153 TMPRSS2 (Fig. S3A) . 154

To mechanistically dissect the entry pathway of SARS-CoV-2 in IECs, we set up an 156 HEK293 ectopic expression system to evaluate the SARS-CoV-2 chimera virus infectivity. 157

As reported (2, 5), ACE2 conferred permissiveness to SARS-CoV-2 infection ( Fig. 3B and 158 S3B). While TMPRSS2 alone did not mediate viral infection, co-expression of TMPRSS2 159 significantly enhanced ACE2-mediated infectivity (Fig. 3B) . Importantly, we found that 160 expression of TMPRSS4 but not ST14 also resulted in a significant increase in the levels of viral RNA and infectious virus titers in the presence of ACE2 ( Fig. 3B and S3C) . 162 TMPRSS4 and TMPRSS2 had an additive effect and mediated the maximal infectivity in 163 cell culture ( Fig. 3B and S3C-E) . 164 165 We reasoned that TMPRSS4 may function as a cell surface serine protease that 166 enhances S cleavage to promote viral entry. To test this hypothesis, we co-expressed a 167 C-terminally Strep-tagged full-length SARS-CoV-2 S protein in an HEK293 cell line that 168 stably expresses ACE2 with or without additional introduction of TMPRSS2 or TMPRSS4. 169

In mock cells, we readily observed the full-length S and a cleaved product that 170 corresponded to the size of S1 fragment, presumably cleaved by furin protease that is 171 ubiquitously expressed (35). Importantly, the expression of TMPRSS2 or TMPRSS4 172 enhanced S cleavage, as evidenced by the reduction of full-length S and increase of S2 173 levels (Fig. 3C) . 174 175 Based on these results, we hypothesize that TMPRSS serine proteases assist virus 176 infection by inducing S cleavage and exposing the fusion peptide for efficient viral entry. 177

Indeed, TMPRSS4 expression did not affect virus binding (data not shown) whereas the 178 endocytosis was significantly enhanced (Fig. 3D ). To examine S protein's fusogenic 179 activity, we examined SARS-CoV-2 S mediated cell-cell fusion in the presence of 180 absence of TMPRSS2 or TMPRSS4. Previous work with SARS-CoV and MERS-CoV 181 suggested S mediated membrane fusion takes place in a cell-type dependent manner 182 (36). We found that the ectopic expression of S alone was sufficient to induce syncytia 183 formation, independent of the virus infection (Fig. 3E ). This process was dependent on a 9 concerted effort from ACE2 and TMPRSS serine proteases. TMPRSS4 expression 185 triggered S-mediated cell-cell fusion, although to a lesser extent than TMPRSS2 (Fig. 3E) . 186

Collectively, we have shown that TMPRSS2 and TMPRSS4 activate SARS-CoV-2 S and 187 enhance membrane fusion and viral endocytosis into host cells. with SARS-CoV-2 S containing donor cells that were transfected with TdTomato. We 199 found that TMPRSS4 was able to function in cis, i.e. on the same cells as ACE2, and infection of primary human IECs, we used a CRISPR/Cas9-based method to genetically delete TMPRSS2 or TMPRSS4 in human duodenum enteroids. Efficient knockout was 208 confirmed by western blot (Fig. S4A) . Importantly, abrogating TMPRSS4 expression led 209 to a 4-fold reduction in SARS-CoV-2 chimera virus replication in human enteroid, even 210 more significant than TMPRSS2 knockout (Fig. 4B) , highlighting its importance in 211 mediating virus replication in primary cells. 212

In parallel to genetic depletion, we also tested the effect of pharmacological inhibition of 214 TMPRSS serine proteases on virus replication. We pre-treated enteroids with camostat 215 mesylate, a selective inhibitor of TMPRSS over other serine proteases including trypsin, 216 prostasin and matriptase (37). While camostat treatment significantly inhibited SARS-217

CoV-2 chimera virus infection, soybean trypsin inhibitor (SBTI) and E-64d, a cysteine 218 protease inhibitor that blocks cathepsin activity and has excellent inhibitory activity 219 against SARS-CoV-2 in vitro (3), did not had a major impact on virus replication in 220 enteroids (Fig. 4C) . Combined with the human enteroid results ( Fig. 1-2) , we hypothesize that the SARS-CoV-246 2 has the potential to and likely does replicate in human IECs but then get quickly 247 inactivated the GI tract. We collected stool specimens from a small group of COVID-19 248 patients. From 3 out of 5 fecal samples, we detected high RNA copy numbers of SARS-249

CoV-2 viral genome (Fig. 5B ). However, we were unable to recover any infectious virus 250 using a highly sensitive cell-based assay (6). 251

In this study we set out to address an important basic and clinically relevant question: Is 255 the persistent viral RNA seen in COVID-19 patients' stool infectious and transmissible? 256

We showed that despite SARS-CoV-2's ability to establish robust infection and replication 257 in human IECs ( Fig. 1-2) , the virus is rapidly inactivated by colonic fluids and we were 258 unable to detect infectious virus in fecal samples (Fig. 5) . Thus, the large quantities of integrity and give rise to GI pathology seen in COVID-19 patients. It is possible that in the 293 small intestine, whereas SARS-CoV-2 is relatively stable, additional proteases such as 294 trypsin likely enhance viral pathogenesis by triggering more robust IEC fusion (Fig. S2A) . 295

In this sense, although viruses are not released into the basolateral compartment ( were selected under 500 μg/ml G418. HEK293 cells stably expressing human ACE2 and 319 TMPRSS2 were selected under 500 μg/ml G418 and 5 μg/ml blasticidin. Table S1 ) in the presence of polybrene (8 µg/ml). At 48 357 hours post transduction, puromycin (2 µg/ml) was added to the maintenance media. 358

Puromycin was adjusted to 1 µg/ml upon the death of untransduced control enteroids. Table S1 ). Gene knockout enteroids were seeded into monolayers 502 and infected with 1.5X10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The 503 expression of VSV-N was measured by RT-qPCR and normalized to that of 504

(C) Human duodenum enteroids seeded into collagen-coated 96-well plates were 506 differentiated for 3 days, pre-treated with 50 μg/ml of soybean trypsin inhibitor 507 (SBTI), 10 μM of camostat mesylate, or 10 μM of E-64d for 30 minutes, and 508 infected with 1. 

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