SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

Abstract

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

“…The same difference in cleavage between WT and S686G pseudoviruses was observed for S2 ( Fig 2C, D). As expected based on earlier work (Mykytyn et al, 2021), SARS-CoV-2 pseudoviruses with MBCS mutations were more infectious on VeroE6 cells and less infectious on Calu-3 cells (Fig 3A-C). A similar trend was observed for the S686G mutant spike.…”

“…In order to establish culture conditions in which SARS-CoV-2 is genetically stable, we tested whether WT viruses would have a selective advantage on Calu-3 cells that possess serine protease mediated entry and little cathepsin-mediated entry (Mykytyn et al, 2021). For these experiments we first produced a Calu-3 P2 virus from the VeroE6 P1 stock ( Fig 5A).…”

“…Pseudoviruses containing MBCS mutations infected human airway organoids poorly (Mykytyn et al, 2021), indicating that these mutations could be prevented by virus propagation in these cells. To produce stocks in human airway organoids, we differentiated the organoids at 2D in transwell inserts at air-liquid interface for twelve weeks as described before ( Fig 7A) (Mykytyn et al, 2021). Apical cells, including ciliated cells, in these cultures expressed TMPRSS2 as shown by immunohistochemistry ( Fig 7B).…”

“…VeroE6 (ATCC® CRL 1586TM) wildtype and retrovirally-transduced cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Lonza) supplemented with 10% fetal bovine serum (FBS, Sigma, F7524, heat inactivated for 30 min at 56°C), HEPES, sodium bicabonate, penicillin (100 IU/mL) and streptomycin (100 IU/mL). VeroE6-TMPRSS2, VeroE6-GFP1-10, VeroE6-TMPRSS2-GFP1-10, and Calu-3-GFP1-10 cells were generated as described before (Mykytyn et al, 2021). Calu-3 and Calu-3-GFP1-10 cells were maintained in Eagle's modified Eagle's medium (EMEM, ATCC) supplemented with 10% FBS, penicillin (100 IU/mL) and streptomycin (100 IU/mL).…”

3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements

“…Yet the tissues used in this preprint were significantly smaller compared to the whole lung lobes used here. Importantly we now show the proteinaceous presence of the functional receptor ACE2 2 , and the essential restriction factor TMPRSS2 24–26 , in concert with NP detection. Importantly, we observed that in lung regions in which ACE2 and TMPRSS2 overlap, actual infection took place.…”