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Review 2: "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 2: "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:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.



The S spike protein of SARS-CoV-2 mediates binding to the host cell and the fusion of the viral envelope with the cellular membrane. To trigger fusion, S has to be cleaved into subunits, S1 and S2, by furin in producer cells. Subsequently, a second site S2’ has to be processed by host cell proteases to release the still non-covalently attached S1 from S2 to subsequently trigger fusion by S2. 

The state of knowledge at the beginning of the study was that there are two pathways of infection of a host cell by SARS-CoV-2 depending on the given conditions: (i) endocytotic uptake of the virus and subsequent enzymatic cleavage by endosomal enzymes whose activity requires an acidic pH. Finally, cleavage triggers a conformational transformation of S  leading to the fusion of the envelope and endosomal membrane. (ii) Enzymatic cleavage of S  already occurs at the cell surface by the cellular transmembrane serine protease TMPRSS2, leading to the fusion of the envelope with the target membrane already at neutral pH—i.e., an acidic pH is not necessary. Both pathways lead to the release of the viral genome after fusion.  

The study reinvestigates the role of a low pH for successful infection by SARS-CoV-2 by tracking the attachment, cell entry, and fusion of the viral envelope with high temporal and spatial resolution 3D fluorescence microscopy. Various host cells differing in the expression level of TMPRSS2 are used. Apart from clinical SARS-CoV-2 samples mainly vesicular stomatitis virus for which its endogenous spike protein was replaced by the S spike protein of SARS-CoV-2 of either the original, Delta, or Omicron strains. To distinguish between the different phases of virus entry, the S1 subunit of S and the VSV internal structural protein P  were specifically labeled with different fluorophores with the first acting as a reporter for the fusion triggering a change of S and the second for the release of viral content upon fusion. The preprint is very well written and the description of the experiments, results, and conclusions is easy to understand and follow. The results are described in detail but concisely and are supported by clearly arranged figures. The experimental procedure is very well designed and convincing. The choice of different host cells, convincing control experiments, and an extensive quantitative evaluation provide a reliable basis for the results and conclusions.  

The main conclusion is that—not only for the endosomal pathway but also for fusion at the plasma membrane—a (mildly) acidic pH value is necessary for the successful release of the genome into the host cell. This acidic pH is not only necessary for the activity of endosomal enzymes,  but also for the release of S1 after cleavage of S2’ in both pathways. In accordance with ethical principles, the authors present experimental findings on test subjects showing that such a mild pH value also exists on the surface of the human nasal cavity. The necessity of a mild pH value for the activation of the S protein for fusion—in addition to the already known enzymatic processing— contributes significantly to the understanding of the mechanism of fusion and release of the SARS-CoV-2 genome. The view that fusion with the plasma membrane does not require that an acidic pH must be modified.


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