Abstract: ARTICLE https://doi.org/10.1038/s42003-022-03841-8 OPEN Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture 1234567890():,; Zixuan Yuan 1,2,3,7, Mahmud Arif Pavel1,2,7, Hao Wang1,2,3, Jerome C. Kwachukwu 4, Sonia Mediouni5, Joseph Anthony Jablonski5, Kendall W. Nettles 4, Chakravarthy B. Reddy6, Susana T. Valente5 & Scott B. Hansen 1,2 ✉ Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ’s mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters—erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy. 1 Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA. 2 Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA. 3 Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL 33458, USA. 4 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, FL 33458, USA. 5 Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA. 6 Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA. 7These authors contributed equally: Zixuan Yuan, Mahmud Arif Pavel. ✉email: shansen@scripps.edu COMMUNICATIONS BIOLOGY | (2022)5:958 | https://doi.org/10.1038/s42003-022-03841-8 | www.nature.com/commsbio 1 ARTICLE C COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03841-8 oronavirus disease 2019 (COVID-19), a viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), recently emerged as a serious public health problem1,2. Currently, millions of people have been infected with SARS-CoV-2 worldwide. Proposed treatments for severe symptoms include a well-known FDA-approved antimalarial and anti-inflammatory agents chloroquine (CQ) and its derivative hydroxychloroquine (HCQ)3–7, but their mechanisms of action are poorly understood in human cells (and in particular in the presence of underlying conditions). A retrospective study claimed a benefit in particular with the macrolide antibiotic azithromycin8. However, their use is not without controversy9,10, and randomized control studies without an antibiotic appeared to have no..