Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2
Tianling Ou, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan
PLOS Pathogens, doi:10.1371/journal.ppat.1009212
Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furincleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.
media containing 2% FBS. Same infection procedures were applied on other cell lines without the plate coating and transfection steps.
Cell surface expression and S protein analysis To measure surface TMPRSS2 expression of 293T-ACE2 transiently transfected with TMPRSS2 and the stable cell line 293T/ACE2/TMPRSS2, cells were detached by 1mM EDTA in PBS and then stained by 2 ug/ml of anti-Flag M2 antibody (Sigma-Aldrich, F1804) and 2 μg/ml of goat anti-mouse IgG (H+L) conjugated with Alexa 647 (Jackson ImmunoResearch Laboratories, #115-606-146). Flow cytometry analysis was done using Accuri C6 (BD Biosciences). To measure the endogenous TMPRSS2 expression of Vero, H1299, H1975 and Calu-3 cells, cells were permeabilized with PBS including 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min, and detected by 2 μg/ml monoclonal rabbit Anti-TMPRSS2 antibody [EPR3861] (Abcam, ab92323) and goat anti-rabbit IgG conjugated with HRP (Sigma-Aldrich, A0545). To determine the cleavage of S proteins, 293T cells were transfected with 2 μL of lipofectamine 2000 (Life Technologies) in complex with 1 μg plasmid expressing the indicated S protein variant. Cells were harvested for western blot analysis 48 hours post transfection. Cells were permeabilized with PBS including 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min, and detected by 1 μg/ml anti-Flag M2 antibody (Sigma-Aldrich, F1804) and goat anti-mouse IgG (Fab only) conjugated with HRP (Sigma-Aldrich, A9917).
..
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'abstract': '<jats:p>Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently '
'inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture '
'studies. However, human clinical trials of hydroxychloroquine failed to establish its '
'usefulness as treatment for COVID-19. This compound is known to interfere with endosomal '
'acidification necessary to the proteolytic activity of cathepsins. Following receptor binding '
'and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, '
'thereby activating membrane fusion for cell entry. The plasma membrane-associated protease '
'TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. '
'Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on '
'TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the '
'SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We '
'also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but '
'not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 '
'inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies '
'identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a '
'mechanistic explanation for the limited <jats:italic>in vivo</jats:italic> utility of '
'hydroxychloroquine as a treatment for COVID-19.</jats:p>',
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