SARS-CoV-2 requires acidic pH to infect cells

Abstract: RESEARCH ARTICLE | MICROBIOLOGY OPEN ACCESS SARS-CoV-2 requires acidic pH to infect cells Alex J. B. Kreutzbergera,b, Anwesha Sanyala,b, Anand Saminathana,b, Louis-Marie Bloyetc , Spencer Stumpfc, Zhuoming Liuc , Ravi Ojhad, Markku T. Patjase , Ahmed Geneide, Gustavo Scanavachia,b, Catherine A. Doylef, Elliott Somervilleb, Ricardo Bango Da Cunha Correiaa,b, €kitiee, Volker Kiesslingi,j , Olli Vapalahtid,k,l , Sean P. J. Whelanc,1 , Giuseppe Balistrerid,k,m,1 Giuseppe Di Capriob,g, Sanna Toppila-Salmih, Antti Ma a,b,g,1 and Tom Kirchhausen , Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY; received June 07, 2022; accepted August 03, 2022 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 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. SARS-CoV-2 j live-cell imaging j virus entry j 3D imaging j infection route Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry begins with engagement at the cell surface and ends with deposition of the viral contents into the cytosol by membrane fusion. The first step is binding of the viral spike (S) protein with its cellular receptor angiotensin converting enzyme (ACE2) (1–4). The last step delivers the viral genomic RNA in association with the nucleocapsid protein (N), which is removed for translation of the input genome (5, 6). Proteolytic activation of S by additional host-cell factors is necessary for it to function as a fusogen. Cleavage of S by furin in producer cells (7) generates the S1 receptor binding subunit noncovalently associated with the S2 fusion subunit. The S protein is cleaved by cell surface or endosomal proteases during virion entry into host cells, which activate the viral fusion machinery (1, 8–10). This entry-associated proteolysis of S has led to the current model of two routes of infectious cell entry, as follows: fusion of viral and cellular membranes at the host-cell surface or fusion following endosomal uptake (6). The cellular proteases that are involved in processing S during entry include the transmembrane serine proteases TMPRSS2 or TMPRSS4 found at the cell surface (1, 8) and the endosomal cathepsins that require the acidic milieu of the compartments in which they are enriched (1, 10). Processing of S by TMPRSS proteases or by cathepsins, at a site designated S2’, depends on prior cleavage at the furin site in the producer cells (7, 11, 12). TMPRSS cleavage has been thought to result in infection from the plasma membrane and cathepsin cleavage, in cells lacking TMPRSS activity, with infection from endosomes (5,..