SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

5 March 2020  | Cell.com


The present study provides evidence that host cell entry of SARS-CoV-2 depends on the SARS-CoV receptor ACE2 and can be blocked by a clinically proven inhibitor of the cellular serine protease TMPRSS2, which is employed by SARS-CoV-2 for S protein priming. Moreover, it suggests that antibody responses raised against SARS-CoV could at least partially protect against SARS-CoV-2 infection. These results have important implications for our understanding of SARS-CoV-2 transmissibility and pathogenesis and reveal a target for therapeutic intervention.
The finding that SARS-2-S exploits ACE2 for entry, which was also reported by Zhou and colleagues () while the present manuscript was in revision, suggests that the virus might target a similar spectrum of cells as SARS-CoV. In the lung, SARS-CoV infects mainly pneumocytes and macrophages (). However, ACE2 expression is not limited to the lung, and extrapulmonary spread of SARS-CoV in ACE2+ tissues was observed (). The same can be expected for SARS-CoV-2, although affinity of SARS-S and SARS-2-S for ACE2 remains to be compared. It has been suggested that the modest ACE2 expression in the upper respiratory tract () might limit SARS-CoV transmissibility. In light of the potentially increased transmissibility of SARS-CoV-2 relative to SARS-CoV, one may speculate that the new virus might exploit cellular attachment-promoting factors with higher efficiency than SARS-CoV to ensure robust infection of ACE2+ cells in the upper respiratory tract. This could comprise binding to cellular glycans, a function ascribed to the S1 domain of certain coronaviruses (). Finally, it should be noted that ACE2 expression protects from lung injury and is downregulated by SARS-S (), which might promote SARS. It will thus be interesting to determine whether SARS-CoV-2 also interferes with ACE2 expression.
Priming of coronavirus S proteins by host cell proteases is essential for viral entry into cells and encompasses S protein cleavage at the S1/S2 and the S2′ sites. The S1/S2 cleavage site of SARS-2-S harbors several arginine residues (multibasic), which indicates high cleavability. Indeed, SARS-2-S was efficiently cleaved in cells, and cleaved S protein was incorporated into VSV particles. Notably, the cleavage site sequence can determine the zoonotic potential of coronaviruses (), and a multibasic cleavage site was not present in RaTG13, the coronavirus most closely related to SARS-CoV-2. It will thus be interesting to determine whether the presence of a multibasic cleavage site is required for SARS-CoV-2 entry into human cells and how this cleavage site was acquired.
The S proteins of SARS-CoV can use the endosomal cysteine proteases CatB/L for S protein priming in TMPRSS2 cells (). However, S protein priming by TMPRSS2 but not CatB/L is essential for viral entry into primary target cells and for viral spread in the infected host (). The present study indicates that SARS-CoV-2 spread also depends on TMPRSS2 activity, although we note that SARS-CoV-2 infection of Calu-3 cells was inhibited but not abrogated by camostat mesylate, likely reflecting residual S protein priming by CatB/L. One can speculate that furin-mediated precleavage at the S1/S2 site in infected cells might promote subsequent TMPRSS2-dependent entry into target cells, as reported for MERS-CoV (). Collectively, our present findings and previous work highlight TMPRSS2 as a host cell factor that is critical for spread of several clinically relevant viruses, including influenza A viruses and coronaviruses (). In contrast, TMPRSS2 is dispensable for development and homeostasis () and thus constitutes an attractive drug target. In this context, it is noteworthy that the serine protease inhibitor camostat mesylate, which blocks TMPRSS2 activity (), has been approved in Japan for human use, but for an unrelated indication. This compound or related ones with potentially increased antiviral activity () could thus be considered for off-label treatment of SARS-CoV-2-infected patients.
Convalescent SARS patients exhibit a neutralizing antibody response that can be detected even 24 months after infection () and that is largely directed against the S protein. Moreover, experimental SARS vaccines, including recombinant S protein () and inactivated virus (), induce neutralizing antibody responses. Although confirmation with infectious virus is pending, our results indicate that neutralizing antibody responses raised against SARS-S could offer some protection against SARS-CoV-2 infection, which may have implications for outbreak control.
In sum, this study provided key insights into the first step of SARS-CoV-2 infection, viral entry into cells, and defined potential targets for antiviral intervention.

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