The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

Abstract

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33°C instead of 37°C. Even stronger preference for the upper airway temperature of 33°C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37°C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37°C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology.SIGNIFICANCE STATEMENTThe rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33°C) or lower (37°C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

“…This result was attributed to the cells containing low levels of CTSL but high levels of TMPRSS-2. 34 , 35 Also of note is a recent CRISPR knockout screen that identified essential host genes for SARS-CoV-2 infection. The authors suggested that TMPRSS-2, but not CTSL, was essential for viral infection of Calu-3 cells.…”

Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection

“…It is interesting to speculate as to why SARS-CoV-2 infection is productive in a monoculture of epithelial, but not endothelial cells. [12]. Endosomal entry may not only be inefficient, but it may also activate the cellular anti-viral response and thereby limit productive viral replication [43,44].…”

“…Cleavage at the S2' site can facilitate the fusion of viral and cellular membranes to deliver the viral RNA into the cytosol [1,11]. Alternative routes of virus uptake involving the endo-lysosomal pathway and a role for cathepsins in viral-host membrane fusion have also been reported in cells with low expression of TMPRSS2 [12]. Numerous studies suggest that endothelial cells express ACE2 [3,[13][14][15][16][17], Neuropilin receptors [18][19][20][21][22] and TMPRSS2 [23], suggesting that viral infection of endothelial cells is theoretically possible.…”

Pediatric nasal epithelial cells are less permissive to SARS-CoV-2 replication compared to adult cells

“…Following binding of the SARS-CoV-2 spike (S) protein to ACE2, the host surface protease TMPRSS2 cleaves the full-length S protein at its S2' site, facilitating the fusion of viral and cellular membranes to deliver the viral RNA into the cytosol (19). Low levels of TMPRSS2 trigger viral entry via a less efficient pathway, that is dependent on endosomes and cathepsin mediated spike cleavage (20). The nasal cavities of children express less TMPRSS2 than those of adults, which may explain the less frequent pediatric infections with SARS-CoV-2 (21).…”