SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

Abstract

The SARS-Coronavirus-2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS-CoV-2-S) with the ACE2 receptor and activation by proteases, in particular TMPRSS2. Viruses can also spread through fusion of infected with uninfected cells. We therefore analyzed cell-cell fusion activity of SARS-CoV-2-S with regard to the requirements for ACE2 expression, proteolytic activation, and sensitivity to inhibitors and compared it to SARS-CoV-S. We compared S-protein-driven fusion with target cells recombinantly overexpressing ACE2, TMPRSS2, or both. SARS-CoV-2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while the reverse observation was made for SARS-CoV-S. TMPRSS2-mediated effects were inhibited by the serine protease inhibitor Camostat. Effector-target-cell fusion by SARS-CoV-2-S was only affected by Camostat when receptor expression was limiting or when the S1/S2 cleavage site was mutated. Mutational ablation of the SARS-CoV-2-S S2’ cleavage site abrogated any effects of TMPRSS2 on fusion. Mutation of the SARS-CoV-2-S S1/S2 cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting. When both factors were abundant, initial target-effector-cell fusion was unaltered, but syncytia remained smaller over time. Overall, its polybasic cleavage site renders SARS-CoV-2-S-mediated cell-cell fusion less dependent on TMPRSS2 activity on target cells. Unexpectedly, we observed enhancement of SARS-CoV-2-S-mediated fusion by Bromhexine, another TMPRSS2 inhibitor. This effect required intact proteolytic cleavage sites, suggesting interference of Bromhexine with proteolytic priming, but not in a therapeutically desired way. Infection with SARS-CoV-2-S-pseudotyped particles clearly differed in the requirements for proteolytic activation from cell-cell fusion. TMPRSS2 strongly enhanced infection, which was reversed by Camostat but not by Bromhexine.IMPORTANCECell-cell fusion allows the virus to infect additional target cells without the need to produce free virus. Fusion likely also contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2’ cleavage site is essential for activation by TMPRSS2 in trans and unravel important differences between SARS-CoV and SARS-CoV-2. Bromhexine, an inhibitor of the TMPRSS2 protease, is currently tested in clinical trials against COVID-19. Our results indicate that Bromhexine does not inhibit SARS-CoV-2-S-mediated particle entry and enhances fusion. We therefore caution against overly optimistic use of Bromhexine in higher dosage in clinical trials or as a therapy, at least until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similar to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on SARS-CoV-2-S-mediated fusion and may therefore currently represent a better choice in therapeutic regimens for COVID-19.

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein’s Receptor Binding Domain and Recombinant Human ACE2

“…However in a recent study, unlike Camostat, another known TMPRSS2 inhibitor; AMB and BHH did not reverse TMPRSS2-mediated enhancement of SARS-CoV-2 infection in 293Tcells 48 .…”

“…Our biochemical findings are most complementary to the report by Hornich, B.F. et. al., that observed inhibition of SARS-CoV-2-S-mediated fusion in the presence of 50 µM AMB 48 ; but observed a dose dependent enhancement of SARS-CoV-2-S-mediated fusion by BHH at the concentrations tested 48 . Furthermore, a computational transcriptomics study by Napolitano, F. et al 53 , found that AMB induced an opposite transcriptional profile compared to that induced by SARS-CoV-2 infection in vitro, suggesting that it may be playing a role in counteracting viral infection 53 .…”

“…Using a sensitive ELISA 42 , we found that AMB and its progenitor, BHH modulates the interaction of rhACE2 and S Our findings regarding the "paradoxical behavior" of BHH in vitro biochemical assay, are consistent with a recent report by Hörnich, B.F. et. al., that revealed that BHH enhanced SARS-CoV-2 S-mediated fusion in 293T cells at the concentrations tested 48 . Moreover, the unconventional dose-response curve that we observed in our interaction studies suggests that there may be more than one binding site for interaction of rhACE2 and Spike (RBD) protein for BHH and its metabolite-AMB, resulting in the potent inhibition of interaction at lower micromolar concentrations; compared to the impact at higher concentrations.…”

“…Suggesting that AMB and BHH, may have additional modes/sites of action 48 54 . This small sample-size study, revealed that early administration of oral BHH decreases the intensive care unit transfer, intubation, and the mortality rate in patients with COVID-19 54 .…”

“…The IC 50 values that we observed for the reference compounds are consistent with earlier reports [44][45][46][47] . While writing this manuscript, we found that other cellular studies tested BHH and AMB at certain or single concentrations [48][49][50] ; however, this is the first study to our knowledge to conduct the IC 50 evaluation for BHH and AMB against the novel SARS-CoV-2 infection induced CPE.…”