The Y498T499-SARS-CoV-2 Spike (S) protein interacts poorly with rat ACE2 and does not affect the rat lung.
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The rat is a useful laboratory model for respiratory disease, and SARS-CoV-2 proteins such as the spike (S) protein can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change described in the S-protein of the mouse adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lung. Using a real-time S-ACE2 quantitative fusion assay, ancestral and L452R S-protein fuses with human, but not rat ACE2 expressed on HEK293 cells. The QP-YT S-protein retains ability to fuse with human ACE2, and increases binding to rat ACE2. Although rat lower lung contains both ACE2 and TMPRSS2 target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration, or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2 and the QP-YT S-protein variant can bind to rat ACE2 but this does not result in infection or stimulate responses in the lung. Further amino acid changes in S-protein could enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.
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This is a study that would be of interest to the field and community.
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Comments to Author
Herein Green et al present an appealing in vivo Rat model to act as a surrogate for SARS-CoV-2 pathogenesis outside of PC3 containment. Overall it is an interesting approach but unfortunately largely negative data with respect to the system they aimed to set up. Whilst the system appeared to be working well in human Hek systems, it failed to work in the final animal model. As such, it would be good to see a few more controls and as such try and get this system to work. A couple of points for this: 1- The pseudotyping system presented appears to be the original system presented by the Bloom Laboratory. In the figures in this work from 2020, full length Spike is used from ref. 20. Additional studies using either VSV or lentiviral systems (see PMID: 32692348) used Spike glycoproteins with a 18 AA …
Comments to Author
Herein Green et al present an appealing in vivo Rat model to act as a surrogate for SARS-CoV-2 pathogenesis outside of PC3 containment. Overall it is an interesting approach but unfortunately largely negative data with respect to the system they aimed to set up. Whilst the system appeared to be working well in human Hek systems, it failed to work in the final animal model. As such, it would be good to see a few more controls and as such try and get this system to work. A couple of points for this: 1- The pseudotyping system presented appears to be the original system presented by the Bloom Laboratory. In the figures in this work from 2020, full length Spike is used from ref. 20. Additional studies using either VSV or lentiviral systems (see PMID: 32692348) used Spike glycoproteins with a 18 AA deletion from the cytoplasmic tail. This increased the ability of pseudotypes to transduce cells at levels significantly higher than that initially proposed by the Bloom laboratory. In this setting: a. Optimising the Spike by introducing the deletion in the cytoplasmic tail might help here. b. Using other systems (VSV backbone) that don't require lentriviral integration may also facilitate detection of viral Spike driven entry. It may also overcome restriction factors that limit the ability for lentiviruses to transduce cells. An additional thought would be to use a retroviral system pseudotyped with Spike, which maybe more suited to a RAT model. Overall the study is well done and has a great aim. If there was more positive data towards getting the model up and running, the manuscript would be a stronger contribution. If full length Spike is used, I would encourage the authors to try the systems listed above and hopefully that will enable observations in this promising animal model.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This is an interesting study that attempts to find the minimal requirements of SARS-CoV-2 Spike protein adaptation to rat ACE2 receptor. The goal is to create a tool with which the adapted Spike protein can be delivered via pseudovirus to challenge rats and thus understand its impact on driving inflammatory disease, without the influence of other viral proteins. The author's reveal correlative in vitro evidence that the QP498YT mutations that indicate the mutated Spike should bind to rat ACE2, however, the QP-YT pseudovirus is unable to enter rat lung cells and cause infection in vivo, or infect primary cells taken from the lungs (ex vivo). The majority of my critiques are relatively minor (albeit are a lengthy list), however I have three major concerns: 1. Is the rat infection data reproducible? …
Comments to Author
This is an interesting study that attempts to find the minimal requirements of SARS-CoV-2 Spike protein adaptation to rat ACE2 receptor. The goal is to create a tool with which the adapted Spike protein can be delivered via pseudovirus to challenge rats and thus understand its impact on driving inflammatory disease, without the influence of other viral proteins. The author's reveal correlative in vitro evidence that the QP498YT mutations that indicate the mutated Spike should bind to rat ACE2, however, the QP-YT pseudovirus is unable to enter rat lung cells and cause infection in vivo, or infect primary cells taken from the lungs (ex vivo). The majority of my critiques are relatively minor (albeit are a lengthy list), however I have three major concerns: 1. Is the rat infection data reproducible? Could the lack of infection be a problem with inoculation dose? Given the rat infection data goes against the in vitro findings and predicted computational interactions, reproducibility needs to be proven. 2. The methods used for attempted infection of primary cell isolates needs further optimization. The images show largely dead cells, indicating the conditions under which the experiment was performed were not optimal. 3. A lot of the microscopy interpretations are based on observations, yet the authors indicate differences in staining intensity. The authors should use relevant software to quantitate the mean fluorescence intensity to support the observation interpretations. My critiques, suggested edits and questions are below. Title: Consider changing the title to something simpler, as it is rather lengthy. Perhaps something like "SARS-CoV-2 Spike protein encoding known rodent mutations (QP498YT) does not infect rats." Line 72: the D in COVID-19 stands for "disease", therefore remove the word disease after the acronym. Line 94-97: The word "model" is used 3 times in one sentence - consider revising. Line 143: QPYT should be QP-YT. Other: Incucyte images - all appear to have a line at the bottom left-hand side. Is this a scale bar? If so, what is the scale? Alternatively, at what magnification was the image taken? Generally microscopic images refer to the magnification lens used, or some form of size reference, particularly when assessments are made on finding sites of cell clusters based on size. Figure 2: Was a gfp-only 293T control (i.e. not expressing S protein) also performed? This should be included in the figure to clearly show cell fusion is a result of S protein expression. Figure 2B should have statistical tests performed and displayed on the graph. Why were cherry red-HEK cells not used as per Figure 1? The visual confirmation of the clustering of cells due to fusion of 293T to HEK via change in colour in Figure 1 made it much easier to agree with the interpretations. Line 235: Given the syncytia were quantified, statistical analyses to show when significantly more syncytia was formed would add further integrity to the findings, particularly when commenting on one form of Spike caused more cell clustering over another. Line 241: VSV-G pseudotyped lentivirus was used as a positive control…..a positive control for what? This information should be included in the text. Line 242: The comments comparing the number of cells infected between the pseudoviruses bearing the different S-proteins would be better supported with accompanying quantification and statistics (if possible) to add integrity to the statements. I suggest graphing the data if it can be quantitated. Otherwise, point out that these statements are based upon visual observations - i.e. Observations of gfp positive sites align with fusion assay outcomes. Figure 4: "Representative images are shown at 20x magnification". This statement is incorrect. The images were taken using a 20x magnification lens and representative images are shown. Fluorescent is misspelled. The white line size should be given in the legend. No indication is given of whether these images are of the lower or other parts of the rat lungs. Line 253: "TMPRSS2 was relatively homogeneously expressed throughout the lower lung. In contrast ACE2, was found along the epithelium of the large airways." I found these sentences a little confusing. TMPRSS2 is homogenously expressed throughout the lower lung on what cells? How is ACE2 in contrast? Is ACE2 also only expressed in the lower lungs? The corresponding figure could benefit from a map, or labelling of what is considered large airways and alveoli - particularly as the TMPRSS2 images do not appear to have any large airways shown? Line 255: "ACE2 immunostaining was particularly high in specific cells attached to the surface or within the alveoli." Was there quantification of the mean fluorescence intensity of ACE2 staining performed? If not the statement "particularly high" should be revised. I'm also confused whether there are cells expressing ACE2 attached to the surface of other cells? Or does the statement mean that ACE2 is expressed at the apical surface of epithelial cells? Does 'within the alveoli' mean that ACE2 was detected throughout the cytoplasm of the alveoli, or they too expressed ACE2 at the surface? Also, the image given does not clearly show what staining is at the surface of the cell versus internal - consider providing zoomed in images to further support this statement. Line 256: The previous sentence is talking about images, then the next sentence is talking about inoculating an animal. The sentence is missing some wording - I would put the words "to anaesthetized rats" after "Ancestral or QP-YT S-protein pseudotyped lentivirus was then administered intratracheally". Figure 5: It is unclear whether the dot points are technical replicates, or pooled experimental data from repeated experiments. If they are technical replicates, why are the group sizes so varied? Other: No indication throughout the manuscript is given regarding whether the experiments were repeated to ensure biological relevance and integrity of the findings. This is particularly important for the rat inoculation experiment, where negative outcomes are being reported that disprove the hypothesis and are not what is expected given the in vitro data. All in vitro findings indicate that the QP-YT mutation should result in binding to rat ACE2 and thus infect in vivo or at least ex vivo. Therefore, the rat challenge experiment should have been performed more than once to confirm the findings are reproducible. Paragraph starting line 250: While there was no indication that there was any change in inflammation to the lungs (measured by BAL cell counts, viperin and CXCL10) or lung function, could the BAL cells not have been assessed for gfp to determine whether the pseudoviruses had bound or entered cells? Could the lung tissue have been harvested and digested as for Figure 6 then assessed for gfp expression? Figure 6: Line 384 "an example cell with intense ACE2 immunostaining" compared to what? It could be that other cells are slightly out of frame and so, appear duller compared to the one with the arrow. And why is the staining intensity important? What magnification lens was used to image B? The rounded morphology of the cells in A don't seem to match the elongated cells in B. After reading the results text, it seems that A were freshly isolated cells, and B were cells that were adhered to the plate overnight? However, this isn't reflected in the legend, which says both A and B are adherent cells. Figure 6B: The QP-YT panel seems to be largely dead cells leaving interpretations difficult to agree with. Paragraph starting line 266: I don't get why examining only adherent cells was necessary, particularly given gfp was detected in non-adherent cells? While it is discounted by the authors as potential autofluorescence due to cell death, it could be that the pseudovirus is causing rapid pyroptosis of cells at very low/undetectable infection rates. After creating the isolated lung cells, why not just co-incubate with the pseudovirus in suspension for a given time and then fix onto slides and image? Paragraph starting line 278: I am not an expert in computational docking and structural modelling, so I found this paragraph quite challenging to follow. I'm not sure what the take-home message of the interpretation of these results are trying to tell me. Given it is placed at the end of the results section, is it trying to explain why infection by the QP-YT pseudovirus failed in rats? Would this section be better at the beginning of the results section to give predictive evidence of potential interaction domains? The word "presumably" is used a couple of times, and the associated sentences seem to be trying to link the computational predictions with the in vitro findings, but leaves the reader feeling that the authors lack confidence in their own data - consider wording differently. Figure 7: The figure shows sequence alignments and predicted interactions, were there any predictions made for binding affinity/energies that could further support interpretation of the modelling? Line 403: "humanizing ACE2" - sounds like it is the ACE2 being humanized….I would say "without them being genetically modified to express human ACE2". Line 403: To my knowledge of the literature, wild-type mice are unable to be infected with the Delta VOC. Alpha and some of the Omicron isolates can productively infect wild-type mice. Consider re-wording this sentence (by removing mice), or cite the references that give evidence that all the VOCs mentioned can infect wild-type mice, as the one reference given only relates to infection of rats. Line 418: "Although humanising the mouse ACE2 at N31K and H353K increased infection". Instead of starting the sentence with "Although", I would start it with "Using an an ACE2-lentivirus system, intrapulmonary induction of modified ACE2 in mice revealed N31K and H353K increased infection……". Line 439: "Our structural analysis, predicted an interaction of Y498 in S-protein with H353 in rat ACE2 to form a presumptive favourable pi-pi ring stacking interaction." I would remove the word presumptive. You already indicate at the beginning of the sentence that the interaction is predicted. Line 450: If you are discussing differences in expression levels of ACE2 on cells to make your argument, then the fluorescence intensity should be quantified in the results section. Line 453: Remove the words "as discussed below". Line 501: Why is the QP-YT mutated Spike protein referred to as a mouse adapted variant here only? The study doesn't actually prove that these are the minimal Spike changes to cause infection of mice.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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