Tags

Type your tag names separated by a space and hit enter

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.
J Virol. 2021 04 12; 95(9)JV

Abstract

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.

Authors+Show Affiliations

Nachwuchsgruppe Herpesviren, Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany.Nachwuchsgruppe Herpesviren, Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany.Nachwuchsgruppe Herpesviren, Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany.Virologisches Institut, Universitätsklinikum Erlangen, Erlangen, Germany.Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany.Virologisches Institut, Universitätsklinikum Erlangen, Erlangen, Germany.Abteilung Infektionsmodelle, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany.Nachwuchsgruppe Herpesviren, Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany ahahn@dpz.eu.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

33608407

Citation

Hörnich, Bojan F., et al. "SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation." Journal of Virology, vol. 95, no. 9, 2021.
Hörnich BF, Groβkopf AK, Schlagowski S, et al. SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation. J Virol. 2021;95(9).
Hörnich, B. F., Groβkopf, A. K., Schlagowski, S., Tenbusch, M., Kleine-Weber, H., Neipel, F., Stahl-Hennig, C., & Hahn, A. S. (2021). SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation. Journal of Virology, 95(9). https://doi.org/10.1128/JVI.00002-21
Hörnich BF, et al. SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation. J Virol. 2021 04 12;95(9) PubMed PMID: 33608407.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation. AU - Hörnich,Bojan F, AU - Groβkopf,Anna K, AU - Schlagowski,Sarah, AU - Tenbusch,Matthias, AU - Kleine-Weber,Hannah, AU - Neipel,Frank, AU - Stahl-Hennig,Christiane, AU - Hahn,Alexander S, Y1 - 2021/04/12/ PY - 2021/01/05/received PY - 2021/02/14/accepted PY - 2021/2/21/pubmed PY - 2021/4/20/medline PY - 2021/2/20/entrez KW - COVID-19 KW - SARS-CoV-2 KW - TMPRSS2 KW - cell-cell fusion KW - spike KW - virus entry JF - Journal of virology JO - J Virol VL - 95 IS - 9 N2 - Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/33608407/SARS_CoV_2_and_SARS_CoV_Spike_Mediated_Cell_Cell_Fusion_Differ_in_Their_Requirements_for_Receptor_Expression_and_Proteolytic_Activation_ L2 - https://journals.asm.org/doi/10.1128/JVI.00002-21?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -