Abstract: PLOS PATHOGENS RESEARCH ARTICLE Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2 Tianling Ou*, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan ID* Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, United States of America * tou@scripps.edu (TO); mfarzan@scripps.edu (MF) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Ou T, Mou H, Zhang L, Ojha A, Choe H, Farzan M (2021) Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2. PLoS Pathog 17(1): e1009212. https:// doi.org/10.1371/journal.ppat.1009212 Editor: Benhur Lee, Icahn School of Medicine at Mount Sinai, UNITED STATES Received: July 19, 2020 Accepted: December 3, 2020 Published: January 19, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.ppat.1009212 Copyright: © 2021 Ou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and the Supporting Information file. Funding: This work is supported by an emergency administrative supplement to NIH R01 AI129868. Abstract 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. Author summary The novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two..