TY - JOUR T1 - SARS-CoV-2 requires acidic pH to infect cells JF - bioRxiv DO - 10.1101/2022.06.09.495472 SP - 2022.06.09.495472 AU - Alex J.B. Kreutzberger AU - Anwesha Sanyal AU - Anand Saminathan AU - Louis-Marie Bloyet AU - Spencer Stumpf AU - Zhuoming Liu AU - Ravi Ojha AU - Markku T. Patjas AU - Ahmed Geneid AU - Gustavo Scanavachi AU - Catherine A. Doyle AU - Elliott Somerville AU - Ricardo Bango Da Cunha Correira AU - Giuseppe Di Caprio AU - Sanna Toppila-Salmi AU - Antti Mäkitie AU - Volker Kiessling AU - Olli Vapalahti AU - Sean P.J. Whelan AU - Giuseppe Balistreri AU - Tom Kirchhausen Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/06/14/2022.06.09.495472.abstract N2 - SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity.Significance Statement Infection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement.One sentence summary Detailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cellsCompeting Interest StatementTom Kirchhausen is a member of the Medical Advisory Board of AI Therapeutics, Inc. The other authors declare no competing interests. ER -