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Review 1: ""SARS-CoV-2 requires acidic pH to infect cells"

This paper investigates the role of acidic environments in cells for SARS-CoV-2 infection susceptibility using 3D single virion tracking microscopy. Reviewers find the study reliable but emphasize the need to validate the observations for the entry of authentic SARS-CoV-2 virus.

Published onJul 21, 2022
Review 1: ""SARS-CoV-2 requires acidic pH to infect cells"
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key-enterThis Pub is a Review of
SARS-CoV-2 requires acidic pH to infect cells

ABSTRACTSARS-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 StatementInfection 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 summaryDetailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.



In this manuscript, the authors describe the results obtained using a chimeric VSV in which SARS-CoV-2 S glycoprotein replaced the endogenous glycoprotein G, and the VSV phosphoprotein (P) was modified to include N-terminal eGFP. This chimeric virus depends on SARS-CoV-2 S glycoprotein for cell entry. In addition, labeling of the S protein with a fluorescent 95 dye, allowed visualization of the steps of cell entry.

Real-time 3D single virion tracking revealed that fusion and genome penetration requires virion exposure to a mild acidic milieu of pH 6.2-6.8. This process mainly occurs from endosomes, irrespective of the cell type tested and irrespective of the dependence of the virus on TMPRSS2 or cathepsin-mediated processing of S glycoprotein. The rationale behind this manuscript and the topic chosen are of evident interest; the experiments are informative and well performed and the manuscript is well written.

Main Comments
I am not an expert in the kind of microscopy used but the results seem timely and consistent, supporting the conclusions regarding the acidic-dependent entry of the VSV-SARS-CoV-2 chimeric viruses. Extension of these observations to the “authentic” SARS-CoV-2 virus entry is, for this reviewer, more speculative as this entry might be influenced by the different virus context (VSV membrane proteins in the chimeric virus). In this line, additional experiments using other inhibitors of endosomal acidification (i.e. NH4Cl, concanamycin A) may provide further support to this conclusion. In any case, a more detailed discussion of previous reports on the acidity dependence for SARS-CoV-2 cell entry/infection is required.

Other comments

The potential effect in the interpretation of the results, relative to in vivo physiological conditions, of the overexpression of TMPRSS2 in the studied cells, should be discussed. The origin and characteristics of the SVG-A cells used should be indicated.

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