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    Reply to the reviewers

    Reviewer 1

    Summary: The authors used conventional confocal and super-resolution STED microscopy to characterize the actin filament network in response to SARS-CoV-2 infection in pulmonary cells. They demonstrate that, although total levels of actin are unchanged, F-actin polymerization increases upon infection, with the most significant changes occurring at 48 hours post infection. Notably, F-actin remodels from primarily stress-fiber architectures to circularized, F-actin nanostructures that tend to colocalize with viral M cluster rings at 48 hours post infection. Additionally, there is a significant increase in F-actin-associated filopodia-like structures, with an example of a possible cell-to-cell filopodia that could possibly be a mode of inter-cellular viral transmission. The authors complement their imaging-based experiments with RNAseq to profile the cellular gene expression of SARS-CoV-2 infected pulmonary cells, revealing an upregulation of RHO GTPases activate PKNs and alpha-actinins. They show that treatment of pulmonary cells with Rho/SFR and PKN inhibitors during infection decreases the size of viral M clusters and release to comparable levels as the known viral therapeutic, Remdesivir.

    Major comments:

    The majority of the author's conclusions are based off of qualitative and quantitative analysis of their fluorescence images. While they do mention briefly an ImageJ plug-in and the statistical tests performed, the description of their quantitative image-based analyses for each experiment is lacking. For example, how was viral M cluster and actin intensity measured? How was the signal intensity normalized to account for variations in antibody labeling or other cell-to-cell variations? For figures 3C&D, how did the authors calculate viral and actin ring diameter? It is necessary to expand on the details of the quantitative analysis for each parameter mentioned in the methods section and/or include a figure panel demonstrating the details of the analysis (similar to what is nicely displayed for M cluster size in Figure S1B). Response:

    We would like to thank reviewer suggestions to improve the material and methods section. We have incorporated all the suggested details for image analysis and also schematic where ever it is necessary in the figures and SI figures in the revised manuscript to clarify:

    • viral M cluster measurement (Figure S1A) ; no variation in M antibody labelling or in cells was observed per se. the pic of infection regarding M clusters was always 48h pi (maximum of M clusters intensity and area.
    • F-actin intensity was considered for each cells, labelling cells with Phalloidin (for at least 30 cells in each condition), imaging z-stack and then considering the whole F-actin content for each cell.
    • Intracellular viral and F-actin ring diameter was calculating using the scale bar on 3D STED images using ImagJ.

    In particular, the details regarding the F-actin orientation measurements is lacking. Is there a consistent reference point for the orientation of the actin filaments? When comparing across two different cells, it is unclear how the orientations are normalized. Perhaps it would be more informative to plot the difference or the range in angles? Or the distribution of the differences in angles? Another point that is a bit misleading is describing this analysis as "F-actin orientation" since the term "orientation" can has a specific meaning for polar filaments such as actin. For example, given resolution limitations of the imaging approaches used in this manuscript, the authors are reporting on the orientations of bundles/populations of actins and not orientations of individual filaments relative to one another within the bundle (e.g. anti-parallel vs parallel vs branched). The authors should clarify this in the text and also further expand on the utility of their F-actin orientation analysis and how it informs us on the mechanisms of actin-mediated viral infection.

    Response:

    To quantify F-actin rearrangements, we have analyzed the orientation angle of actin nano-fibers from STED images (as in Nature Communications. 8 (2017), doi:10.1038/ncomms14347).

    For this analysis all the images were imaged with STED 2D microscopy for better resolution (axial 60 nm resolution). From STED 2D microscopy images of F-actin, the orientation angle of nano-fibers were evaluated based on the structure tensor of each nano-fibers compares to its local neighborhood using the Java plugin for ImageJ “OrientationJ”. From the given images, the OrientationJ plugin computes the structure tensor for each pixel in the image by sliding the Gaussian analysis window over the entire image. The local angle of orientation properties encoded in color and it is also generating a distribution of angles for each nano-fibers for a given image. Here, in the STED images, it is considered the vertically elongated nano-fibers as the major orientation angle (as around +90 Deg and – 90 Deg from the cell edge) and others orientation angles were calculated accordingly. Area are normalized to the distribution curve of angles to compare the changes in distribution for infected and non-infected cell (as in Fig. 3B).

    We have incorporated above explanations in the material and method section (Image analysis section, Page 11) in the revised manuscript.

    For the majority of figures and findings, they report that between "22

    Response:

    We have incorporated the exact number of cells analyzed for each condition and details about data sets used for analysis in each figure legend in the revised manuscript.

    The actin filament network can assemble into different architectures that are dependent on subcellular location. For example, actin at the basal region of the cell closest to the coverslip often assembles into stress fibers, whereas the cortical actin network often forms astral, highly branched networks. It would be important to take this into account when comparing across different cellular conditions. It is unclear if the authors were consistent with the z-slice examined for the different cellular treatment/infection conditions. Were the analyses performed on individual z-stacks or max projection images?

    Response:

    We agree with the reviewer views on actin network in different planes. Thus, to ensure reasonable quantification and comparison among conditions, all images were taken with the same objective (63x oil N 1.4) and microscope settings (same gain, same laser power). For post-processing, we mainly choose individual cells, which are not in contact with others and individual z-stacks were taken. Z-stacks images with fixed 0.3 micrometer slices for each cells were taken to ensure the whole cell was in focus. The Z-projection images of individual cells were then performed and used to calculate the F-actin or viral M cluster or ER mean intensity in the whole cell. We have analyzed the mean intensity per individual cell using a Fiji/Image J.

    We have incorporated above details in material method (image analysis) section in the revised manuscript.

    Since a major impact of this paper is the first imaging-based characterization of actin filament assembly in response to infection, the authors should provide a more comprehensive display of the raw data images. For example, figure S2 provides a nice gallery of images of actin and viral M particles, however it should show separate image channels in gray scales and consistent scaling across all images. Furthermore, all figure panels showing distinct imaging experiments and quantitative results should be complemented with a supplemental figure showing a gallery of images. This would apply to actin nanostructure rings (Figure 3C/E), filopodia and cell-to-cell contacts (Figure 4A/D), treatment with remdesivir/PKN inihibitor (Figure 6B), and ER localization of M particles (Figure S5).

    Response:

    As the reviewer suggested, we have now created an image gallery for each figure panel (Figures 3, 4, 6 and S5, S3, S8, S9) including STED images that were added as supplemental figures.

    The results in Figure 3D are difficult to interpret. The images should be larger and labeled. Also, based on the 3D STED image in Figure 3D, it appears that the brightest actin signal is actually at the center portion of the viral M cluster. Does this contradict the TEM image and what is described in the text? For Figure 3E: a more relevant analysis might be line scans across multiple images showing how relative actin-M cluster intensity varies within the dimensions of the nanostructure to demonstrate more clearly a pattern of ring assembly of both M clusters and actin.

    Response:

    Since the virus “rings” were mostly found in intracellular places, far from membrane surface, some times during imaging we observed F-actin signal from the upper plane, which is possibly the reason for brightest F-actin signal appears at the center portion of the viral M cluster. Thus, for better clarity of the image and to support our statements we have now incorporated other new images in the Figure 3E (STED 3D images) showing that an heterogeneity of the F-actin labelling but strongly associated with intracellular viral M clusters.

    The authors should address the implications and significance of the described cellular morphological changes in the context of the more physiologically relevant tissue/organ system. How do the changes they observe upon infection in isolated cultured cells compare to when these cells are assembled into tissue/organs?

    Response:

    The significance of the cellular morphological changes upon SARS-CoV2 infection showing a contraction-like effect on the cells as well as higher cells and less contact area could account in a pulmonary tissue by the destructuration of the lung tissue, consistent with the lung damaged seen in the case of COVID19. A sentence in that sense was added in the Discussion section.

    For Figure 6 and S5, the authors infected and treated cells with an inhibitor at the same time point and demonstrate that M cluster size and release is reduced to somewhat comparable levels as treatment with Remdesivir. The authors should expand their analyses for this experiment to include the other quantitative parameters outlined in the paper: F actin/M cluster nanostructures, cellular morphology, filopodia formation, orientation of actin, etc. Additionally, it would be more informative to treat cells post-infection to more closely mimic cellular conditions of infection/treatment.

    Response:

    We have now included quantitative analysis for cellular morphological changes of cells with or without drug treatment (both in the presence of PKN inhibitor and Remdesivir upon SARS-CoV-2 infection) in the revised manuscript (Supplemental Figure S7). We observed a restoration of F-actin nanostructures as well as did not observe any filopodia-like structure formation upon treatment with PKN inhibitor in infected cells.

    Minor comments:

    The individual data points should be overlaid on the violin plots for better interpretability of the variability in the data. Response:

    We have incorporated new violin plots with overlaid data points in the revised version of the manuscript for each figure with quantitative data (Figures 1,2,5,6).

    For Figure 3E: the images look "stretched" with an altered relative aspect ratio.

    Response:

    For sake of clarity, we have incorporated new (better) 3D STED images for a better visualization of intracellular F-actin/M clusters “rings” in revised manuscripts (Figure 3E).

    1. The authors should include a cartoon model figure highlighting both (1) how their results contribute to our knowledge of actin-mediated viral assembly/replication and (2) unknown portions of the pathway that need to be further probed to better understand the mechanistic underpinnings of this process.

    Response:

    We have now included a model scheme figure summarizing our results in the revised manuscript, as a new figure 7.

    There have been several high resolution cellular imaging studies using other complementary 3D volumetric imaging approaches (e.g. cryo-electron tomography and FIB/SEM) to characterize the subcellular ultrastructure’s of SARS-CoV-2 infection. The authors should include a brief discussion on how their study complement or compare to these reports, in particular noting whether or not actin filament assemblies were observed in these data.

    Response:

    Thanks to the reviewers for this very pertinent remark, we have added in the Discussion (Page 7,8), a section commenting previous high-resolution cellular imaging studies (REFERENCES:* Mendonçà et al Nature Comm 12, 4629, 2021; Klein et al 2020*) comparing our 2D/3D STED imaging with complementary 3D EM or 3D cryo-ET or FIB/SEM of SARS-CoV-2 infected cells recently published.

    From Mendonca et al 2021, one can see some intracellular dense structure underneath the CoV-2 budding membrane area, but not able to see if F-actin filaments were present or not. It would be difficult to observe because the vRNP underneath the Spike decorating membrane are very dense. The study was focus on viral assembly and egress using cryo-ET/FIB but not on F-actin filament per se. We don’t know if their imaging conditions would preserve F-actin fibers on membranes. On the other side, when studying virus egress, then we can clearly see CoV-2 individual particles surfing on giant filopodia-like structures very much resembling our STED imaging of viruses on filopodia 48h pi. We can clearly see and recognize parallel F-actin filament bundles inside the enlarged filopodia (Figure 5 D/E) with viruses on it.

    Same results were observed using Cryo-EM tomography in another study (Klein et al 2020) where one can see viruses on filopodia for many cell types A549-hACE2, VeroE6, Calu3 infected cells.

    Reviewer 2

    The authors investigate the role of F-actin in infected human pulmonary alveolar A549-hACE2 cells. They investigate infection progression at different time points by the detection of the M protein by confocal microscopy and western blot. They compare the detection of M with S and N in western blot and with viral RNA detection by Q-PCR. The authors correlate M cluster formation to peak at 48h p.i. with particle assembly and particle release at 72h p.i. An increase in F-actin at 24h and 48h p.i. was monitored by confocal microscopy and z-stacks, whereas the overall amount of actin determined by western blot was not changed. Using 2D STED microscopy the authors identified F-actin rearrangement from stress fibers to filamentous protrusions at 24h-48h p.i. and conclude importance for particle assembly and release. By 3D STED microscopy M labeled intracellular organelles called "viral rings" surrounded by actin called "actin rings" are shown. By transmission electron microscopy (TEM) vesicular structures with budding particles were shown at intracellular membranes. The authors conclude from these findings that F-actin stabilizes assembly platforms at membranes or support the transport of virus loaded vesicles to the plasma membrane. The authors found more and longer filopodia in infected cells which were loaded with virus particles bridging cells suggesting role in cell-cell spread. At the plasma membrane they found bigger particles and at the filopodia smaller, suggesting release from the plasma membrane in packages.

    Transcriptom analysis of non infected and SARS-CoV-2 infected A549-hACE2 revealed upregulation of Rho-GTPases activated proteins like PKN and α-actinins upon infection. The levels of α-actinins in WB were 2-fold higher in infected cells. The authors show that inhibitors of Rho/SRF and PKN restored cell morphology, reduced M cluster formation and virus release. The PKN inhibitor blocked M in the ER. The authors conclude from this data a role of the alpha-actinins superfamily in SARS-CoV-2 assembly and egress.

    Major comments:

    The presented data are convincing but some figures may need improvements, see in minor comments. For some conclusions, more evidences like marker staining may be needed.

    Response:

    In accordance with the reviewer, we have significantly improve the figures in the revised manuscript. We identified that the intracellular compartment containing budding viruses were derived from the ER (gpr78 marker) – shown in revised Figure 6 - and not in lysosomes (Lamp1 marker) or extracellular vesicles (CD81 marker) – See new supplemental revised Figure S10. We have included all the new results and discussion in revised manuscripts.

    Minor comments:

    The authors conclude that F-actin stabilizes assembly platforms at membranes like ERGIC, but an ERGIC marker staining is not provided. The authors suggest that F-actin might also be involved in transport of virus loaded vesicles to the plasma membrane. Here a plasma membrane marker or native staining of particles may help to descriminate between intracellular Exosomes and extracellular particles. Co-staining with exosomal markers would also be more convincing.

    Response:

    Also as per reviewer suggestion we have identified that the intracellular compartment containing budding viruses were derived from the ER (gpr78 marker) – shown in revised Figure 6, Fig. S8. New quantitative analysis (including with PKN inhibitor) to support the data also included in the figures. Also we have used lysosomes marker LAMP1 and extracellular vesicles (EV) marker CD81 and we found that there was no colocalization with Viral M clusters ( Supporting Figure 10). we have tried the ERGIC marker grp53 without any success so far,

    Further, It is well documented on CryoFIB/SEM study of SARS-CoV-2-infected cells suggested the presence of “exit tunnels”, linking virion-rich intracellular vacuoles to the extracellular space (Mendonça, L. et al. Nature Communications 12, (2021)). The size of this vacuoles observed in the cell periphery was approximately 1 µm, which is well corelated with actin and viral ring we have observed from STED 2D images Also the author suggested that SARS-CoV-2 could possibly egress through these tunnels by a mechanism of exocytosis from these large intracellular vacuoles.

    We have now included all above results and discussions in revised manuscripts to support our claims.

    Figure S1 A. Align individual pictures in one line and do not overlap, scale bars not readable, Is in each picture the same magnification shown? Show representative pictures with the same area magnification!

    Response:

    Thanks to the reviewer to point out these imperfections, so we have improved the figures accordingly in the revised manuscript. Individual images are aligned properly, scale bars are readable, images are with the same magnification.

    Figure 3C and 3E for better orientation magnified areas should be indicated as squares, not in circles.

    Response:

    As suggested, we have modified the figure 3 accordingly in the revised manuscript.

    Figure S4 quality of pictures not appropriate to see differences.

    Response:

    We improved the figure quality in the revised manuscript (see new Figures 6 and S8)

    Fig S5 All pictures overlap in one? ER marker in blue very difficult to read.

    Response:

    We have modified the new figure S8 as such as the ER marker is visible (in magenta color) in the revised manuscript.

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  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors investigate the role of F-actin in infected human pulmonary alveolar A549-hACE2 cells. They investigate infection progression at different time points by the detection of the M protein by confocal microscopy and western blot. They compare the detection of M with S and N in western blot and with viral RNA detection by Q-PCR. The authors correlate M cluster formation to peak at 48h p.i. with particle assembly and particle release at 72h p.i. An increase in F-actin at 24h and 48h p.i. was monitored by confocal mircroskopy and z-stacks, whereas the overall amount of actin determined by western blot was not changed. Using 2D STED microscopy the authors identified F-actin rearrangement from stress fibers to filamentous protrusions at 24h-48h p.i. and conclude importance for particle assembly and release. By 3D STED microscopy M labeled intracellular organelles called "viral rings" surrounded by actin called "actin rings" are shown. By transmission electron microscopy (TEM) vesicular structures with budding particles were shown at intracellular membranes. The authors conclude from these findings that F-actin stabilizes assembly platforms at membranes or support the transport of virus loaded vesicles to the plasma membrane. The authors found more and longer filopodia in infected cells which were loaded with virus particles bridging cells suggesting role in cell-cell spread. At the plasma membrane they found bigger particles and at the filopodia smaller, suggesting release from the plasma membrane in packages. Transcriptom analysis of non infected and SARS-CoV-2 infected A549-hACE2 revealed upregulation of Rho-GTPaes activated proteins like PKN and α-actinins upon infection. The levels of α-actinins in WB were 2-fold higher in infected cells. The authors show that inhibitors of Rho/SRF and PKN restored cell morphology, reduced M cluster formation and virus release. The PKN inhibitor blocked M in the ER. The authors conclud from this data a role of the alpha-actinins superfamily in SARS-CoV-2 assembly and egress.

    Major comments:

    The presented data are convincing but some figures may need improvements, see in minor comments. For some conclusions more evidences like marker staining may be needed.

    Minor comments:

    The authors conclude that F-actin stabilizes assembly platforms at membranes like ERGIC, but an ERGIC marker staining is not provided. The autors suggest that F-actin migth also be involved in transport of virus loaded vesicles to the plasma membrane. Here a plasma membrane marker or native staining of particles may help to descriminate between intracellular Exosomes and extracellular particles. Co-staining with exosomal markers would also be more convincing.

    • Figure S1 A. Align individual pictures in one line and do not overlap, scale bars not readable, Is in each picture the same magnification shown? Show representative pictures with the same area magnification!
    • Figure 3C and 3E for better orientation magnified areas should be indicated as squares, not in circles.
    • Figure S4 quality of pictures not appropriate to see differences.
    • Fig S5 All pictures overlap in one? ER marker in blue very difficult to read.

    Significance

    The presented data provide a nice peace of work to the knoweledge on SARS-COV-2 replication in human pulmonary cells. The authors use advanced imaging and molecular biology methods for their experiments. The indentified cellular target may help to develop specific inhibitors for antiviral therapy.

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  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors used conventional confocal and super-resolution STED microscopy to characterize the actin filament network in response to SARS-CoV-2 infection in pulmonary cells. They demonstrate that, although total levels of actin are unchanged, F-actin polymerization increases upon infection, with the most significant changes occurring at 48 hours post infection. Notably, F-actin remodels from primarily stress-fiber architectures to circularized, F-actin nanostructures that tend to colocalize with viral M cluster rings at 48 hours post infection. Additionally, there is a significant increase in F-actin-associated filopodia-like structures, with an example of a possible cell-to-cell filopodia that could possibly be a mode of inter-cellular viral transmission. The authors complement their imaging-based experiments with RNAseq to profile the cellular gene expression of SARS-CoV-2 infected pulmonary cells, revealing an upregulation of RHO GTPases activate PKNs and alpha-actinins. They show that treatment of pulmonary cells with Rho/SFR and PKN inhibitors during infection decreases the size of viral M clusters and release to comparable levels as the known viral therapeutic, Remdesivir.

    Major comments:

    1. The majority of the author's conclusions are based off of qualitative and quantitative analysis of their fluorescence images. While they do mention briefly an ImageJ plug-in and the statistical tests performed, the description of their quantitative image-based analyses for each experiment is lacking. For example, how was viral M cluster and actin intensity measured? How was the signal intensity normalized to account for variations in antibody labeling or other cell-to-cell variations? For figures 3C&D, how did the authors calculate viral and actin ring diameter? It is necessary to expand on the details of the quantitative analysis for each parameter mentioned in the methods section and/or include a figure panel demonstrating the details of the analysis (similar to what is nicely displayed for M cluster size in Figure S1B).
    2. In particular, the details regarding the F-actin orientation measurements is lacking. Is there a consistent reference point for the orientation of the actin filaments? When comparing across two different cells, it is unclear how the orientations are normalized. Perhaps it would be more informative to plot the difference or the range in angles? Or the distribution of the differences in angles? Another point that is a bit misleading is describing this analysis as "F-actin orientation" since the term "orientation" can has a specific meaning for polar filaments such as actin. For example, given resolution limitations of the imaging approaches used in this manuscript, the authors are reporting on the orientations of bundles/populations of actins and not orientations of individual filaments relative to one another within the bundle (e.g. anti-parallel vs parallel vs branched). The authors should clarify this in the text and also further expand on the utility of their F-actin orientation analysis and how it informs us on the mechanisms of actin-mediated viral infection.
    3. For the majority of figures and findings, they report that between "22<n<50 cells" were analyzed. The authors should be more specific of the exact sample size for each experiment/figure panel displayed. In particular, it is unclear in a few figure panels showing exemplar images whether or not this is the full sample size (n=1) or just an exemplar image. I recommend reporting specifically in the figure legend and/or a supplemental table outlining the sample size and analysis used for each imaging experiment to add clarify to their quantitative analysis and strengthen their claims.
    4. The actin filament network can assemble into different architectures that are dependent on subcellular location. For example, actin at the basal region of the cell closest to the coverslip often assembles into stress fibers, whereas the cortical actin network often forms astral, highly branched networks. It would be important to take this into account when comparing across different cellular conditions. It is unclear if the authors were consistent with the z-slice examined for the different cellular treatment/infection conditions. Were the analyses performed on individual z-stacks or max projection images?
    5. Since a major impact of this paper is the first imaging-based characterization of actin filament assembly in response to infection, the authors should provide a more comprehensive display of the raw data images. For example, figure S2 provides a nice gallery of images of actin and viral M particles, however it should show separate image channels in gray scales and consistent scaling across all images. Furthermore, all figure panels showing distinct imaging experiments and quantitative results should be complemented with a supplemental figure showing a gallery of images. This would apply to actin nanostructure rings (Figure 3C/E), filopodia and cell-to-cell contacts (Figure 4A/D), treatment with remdesivir/PKN inihibitor (Figure 6B), and ER localization of M particles (Figure S5).
    6. The results in Figure 3D are difficult to interpret. The images should be larger and labeled. Also, based on the 3D STED image in Figure 3D, it appears that the brightest actin signal is actually at the center portion of the viral M cluster. Does this contradict the TEM image and what is described in the text? For Figure 3E: a more relevant analysis might be line scans across multiple images showing how relative actin-M cluster intensity varies within the dimensions of the nanostructure to demonstrate more clearly a pattern of ring assembly of both M clusters and actin.
    7. The authors should address the implications and significance of the described cellular morphological changes in the context of the more physiologically relevant tissue/organ system. How do the changes they observe upon infection in isolated cultured cells compare to when these cells are assembled into tissue/organs?
    8. For Figure 6 and S5, the authors infected and treated cells with an inhibitor at the same time point and demonstrate that M cluster size and release is reduced to somewhat comparable levels as treatment with Remdesivir. The authors should expand their analyses for this experiment to include the other quantitative parameters outlined in the paper: F actin/M cluster nanostructures, cellular morphology, filopodia formation, orientation of actin, etc. Additionally, it would be more informative to treat cells post-infection to more closely mimic cellular conditions of infection/treatment.

    Minor comments:

    1. The individual data points should be overlaid on the violin plots for better interpretability of the variability in the data.
    2. For Figure 3E: the images look "stretched" with an altered relative aspect ratio.
    3. The authors should include a cartoon model figure highlighting both (1) how their results contribute to our knowledge of actin-mediated viral assembly/replication and (2) unknown portions of the pathway that need to be further probed to better understand the mechanistic underpinnings of this process.
    4. There have been several high resolution cellular imaging studies using other complementary 3D volumetric imaging approaches (e.g. cryo-electron tomography and FIB/SEM) to characterize the subcellular ultrastructures of SARS-CoV-2 infection. The authors should include a brief discussion on how their study complement or compare to these reports, in particular noting whether or not actin filament assemblies were observed in these data.

    Significance

    Impact:

    This manuscript provides the first characterization of the architecture of the actin filament network upon SARS-CoV-2 infection. Since actin filament remodeling is a mechanism used by several other viruses, there is considerable interest in targeting these assemblies for the development of therapeutics to prevent and treat infection. This manuscript lays the groundwork for more detailed analysis probing the mechanisms mediating actin-mediated viral entry, replication, and release. Furthermore, it establishes some quantitative tools to standardize how this process is studied and analyzed in future studies.

    Audience:

    I anticipate that this work will motivate future studies aimed at further ultrastructural characterization of actin and other cytoskeletal filaments by complementary, high-resolution imaging techniques, as well as studies aimed at screening for small molecule drugs to inhibit actin-mediated viral infection.

    Field of expertise:

    cellular cryo-electron tomography, quantitative imaging, cytoskeletal-based motility, functional cytoskeleton-organelle interactions. Insufficient expertise to evaluate RNAseq experiments.

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  4. SciScore for 10.1101/2022.03.08.483451: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing with 5% milk in TBS-Tween, the membranes were incubated with HRP conjugated anti-mouse antibodies for N and S protein, and with HRP conjugated anti-rabbit antibody for M protein and alpha-actinins for 2h at room temperature, then washed in TBS-Tween buffer, incubated with ECL reagent (Amersham cat#RPN2236) and imaged using a Chemidoc Imager (Biorad)
    anti-mouse
    suggested: None
    anti-rabbit
    suggested: None
    Incubation with primary antibodies anti-SARS-CoV2 rabbit membrane (M) protein (1:100) was performed for 2 hours at room temperature.
    anti-SARS-CoV2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and infection: Human pulmonary Alveolar A549-hACE2 cells were obtained from original A549 (ECACC) transduced with a lentiviral vector expressing human ACE2 receptor (manufactured by FlashTherapeutics company, Toulouse, France) and sorted by cytometry for having more than 80% hACE2 on their surface.
    A549
    suggested: None
    The sorted A549-hACE2 cells were maintained in RPMI supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% sodium Pyruvate, 0.5% HEPES and antibiotics (penicillin/Streptavidin) and cultivated at 37°C with 5% CO2.
    A549-hACE2
    suggested: RRID:CVCL_A5KB)
    For virus production, VeroE6 cells were obtained from (ECACC) and maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) at 37°C with 5% CO2.
    VeroE6
    suggested: None
    Software and Algorithms
    SentencesResources
    Raw reads were visualized by FastQC to determine the quality of the sequencing.
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    High quality reads were mapped using with HISAT2 v2.1.0 with reads corresponding to the transcript with default parameters.
    HISAT2
    suggested: (HISAT2, RRID:SCR_015530)
    After mapping, Tag libraries were obtained with MakeTaglibrary from HOMER (default setting).
    HOMER
    suggested: (HOMER, RRID:SCR_010881)
    Read trimming was performed using trimmomatic (v 0.39) with the following parameters “ILLUMINACLIP:all_adapters_v0.38.fa:2:30:10 AVGQUAL:30 LEADING:0 TRAILING:0 SLIDINGWINDOW:6:30 MINLEN:38”.
    trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Trimmed reads where then aligned to the SARS-CoV-2 reference genome NC_045512.2.fasta (downloaded May 2021 from https://www.ncbi.nlm.nih.gov/nuccore/NC_045512) using the STAR (v 2.7.9a) aligner; STAR parameters where the following “--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --outSJfilterOverhangMin 12 12 12 12 --outSJfilterCountUniqueMin 1 1 1 1 --outSJfilterCountTotalMin 1 1 1 1 -- outSJfilterDistToOtherSJmin 0 0 0 0 --outFilterMismatchNmax 999 -- outFilterMismatchNoverReadLmax 0.04 --scoreGapNoncan -4 --scoreGapATAC -4 -- chimOutType WithinBAM HardClip --chimScoreJunctionNonGTAG 0 –alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 -- alignSJstitchMismatchNmax -1 -1 -1
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Samtools (v 1.12) was used to handle the alignments, and bedtools coverage was used to count reads in each viral feature (gene) using the genomic coordinates from GCF_009858895.2_ASM985889v3_genomic.gff (downloaded May 2021 from https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.gff.gz) only for protein coding features.
    Samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    bedtools
    suggested: (BEDTools, RRID:SCR_006646)
    All the images processed with ImageJ/Fiji.
    ImageJ/Fiji
    suggested: None
    Z stack was processed using ImageJ/Fiji, Imaris viewer.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Statistical analysis: Statistical tests were performed using Origin 2021 software.
    Origin
    suggested: (Origin, RRID:SCR_014212)
    Cell area and cell volume and height were calculated using 3D viewer plugin from Fiji image J.
    Fiji image J
    suggested: (ImageJ, RRID:SCR_003070)
    image
    suggested: (NIH Image, RRID:SCR_003073)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).


    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.


    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.


    Results from JetFighter: We did not find any issues relating to colormaps.


    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.


    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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