Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic

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   Review Commons

   Mar 15, 2023

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   I summarise the major findings of the work below. In my opinion the range and application of approaches has provided a broad evidence base that, in general, supports the authors conclusions. However, there are, in my opinion, particular failures to utilise and communicate this evidence. The manuscript may be much improved with attention in the following areas. In each case I will give general criticism with a few examples, but the principals of my comments could be applied throughout the work.

   1. Insufficient quantification. The investigation combines various sources of qualitative data (EM, …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   I summarise the major findings of the work below. In my opinion the range and application of approaches has provided a broad evidence base that, in general, supports the authors conclusions. However, there are, in my opinion, particular failures to utilise and communicate this evidence. The manuscript may be much improved with attention in the following areas. In each case I will give general criticism with a few examples, but the principals of my comments could be applied throughout the work.

   1. Insufficient quantification. The investigation combines various sources of qualitative data (EM, fluorescence microscopy, western blotting) to generate a reasonably strong evidence base. However, the work is over-reliant on representative images and should include more quantification from repeat experiments. When there are multiple fluorescence micrographs with intensity changes (not necessarily just representative images) (e.g. Figure 1 or 2) the authors should consider making measurements of these. Also the VLP production assays, which are assessed by western blotting would particularly benefit from a quantitative assessment (either by densitometry or, if samples remain, ELISA/similar approach).

   We have performed quantification of immunofluoresence, western blotting and VLP experiments from existing data. These quantification are presented in our revised manuscript. An overview of new quantification is shown below:

   Data shown

   Quantification now shown in

   Method

   Analysis

   Figure 1A

   Supp F1C

   IF

   HAE (-/+ SARS-CoV-2)

   • Tetherin total fluorescence intensity

   Figure 1D

   Supp F1E

   IF

   HeLa+ACE2 (-/+ SARS-CoV-2 )

   • Tetherin total fluorescence intensity

   Figure 2C

   Supp F2B

   IF

   A549+ACE2 (-/+ SARS-CoV-2)

   • Tetherin total fluorescence intensity

   Figure 2G

   Supp F2D

   IF

   T84 (-/+ SARS-CoV-2)

   • Tetherin total fluorescence intensity

   Supp F4A

   Supp F4B

   IF

   HeLa + ss-HA-Spike transients (-/+ HA stained cells) - Tetherin total fluorescence intensity

   Figure 4D

   Supp F4E

   IF

   HeLa + TetOne ss-HA-Spike stables (-/+ Dox)

   • Tetherin total fluorescence intensity

   Figure 4F

   Supp F4G

   W blot

   HeLa + TetOne ss-HA-Spike stables (-/+ Dox)

   – Tetherin abundance

   Figure 4G

   Supp F4I

   W blot – lysates

   Spike VLP experiments

   – tetherin abundance

   Figure 4G

   Supp F4J

   W blot - VLPs

   Spike VLP experiments

   • N-FLAG abundance

   Figure 6A

   Supp F7A

   W blot – lysates

   ORF3a VLP experiments

   – tetherin abundance

   Figure 6A

   Supp F7B

   W blot - VLPs

   ORF3a VLP experiments

   • N-FLAG

   For immunofluoresence anaysis, the mean, standard deviation, number of cells analysed and number of independent experiments are shown in the updated figure legends. Statistical analysis is also detailed in figure legends. Methods for the quantificaiton of fluoresence intensity is included in the Methods section.

   Densitometry was performed on western blots and VLP experiments as suggested. The mean, standard devisation and number of independent expreiments analysed are expressed in figure legends. Methods for densityometry quantification is now included.

   1. Insufficient explanation. I found some of the images and legends contained insufficient annotation and/or description for a non-expert reader to appreciate the result(s). Particularly if the authors want to draw attention to features in micrographs they should consider using more enlarged/inset images and annotations (e.g. arrows) to point out structures (e.g DMVs etc.). This short coming exacerbates the lack of quantification.

   Additional detail has been provided to the figure legends, and we have updated several figures to draw attention to features in micrographs. Black arrowheads have been added to Figures 1E, 2D, 2H to highlight plasma membrane-associated virions, and asterisks to highlight DMVs in Figures 1E, 2D and Supplemental Figures 2C, 2E. Similarly, typical Golgi cisternae are highlighted by white arrowheads micrographs in Figure 2E. These figure legends have also been modified to highlight these additions.

   1. Insufficient exploration of the data. I had a sense that some aspects of the data seem unconsidered or ignored, and the discussion lacks depth and reflection. For example the tetherin down-regulation apparent in Figures 1 and 2 is not really explained by the spike/ORF3a antagonism described later on, but this is not explicitly addressed.

   We have made changes throughout the manuscript, but the discussion especially has been modified. We now discuss the ORF3a data in more depth, discuss possible mechanisms by which ORF3a alone enhances VLP release, and discuss our ORF7a data in context to previous reports.

   The discussion has been updated to now include a better description of our data, and additional writing putting our work in to context with previously published work. See discussion section of revised manuscript.

   Also, Figure 6 suggests that ORF3a results in high levels of incorporation of tetherin in to VLPs, but I don't think this is even described(?). The discussion should also include more comparison with previous studies on the relationship between SARS-2 and tetherin.

   We have added a section to discuss how ORF3a may enhance VLP release,

   ‘We found that the expression of ORF3a enhanced VLP independently of its ability to relocalise tetherin (Figure 6A). This may be due to either the ability of ORF3a to induce Golgi fragmentation [38] which facilitates viral trafficking [39], or due to enhanced lysosomal exocytosis [37]. Tetherin was also found in VLPs upon co-expression with ORF3a (Figure 6A) which may also indicate to enhanced release via lysosomal exocytosis [37].

   The secretion of lysosomal hydrolases has been reported upon expression of ORF3a [31] and whilst this may in-part be due to enhanced lysosome-plasma membrane fusion, our data highlights that ORF3a impairs the retrograde trafficking of CIMPR (Supplemental Figures 6B, 6F, 6G), which may similarly increase hydrolase secretion.’ – (Line 625-654).

   The discussion has been developed to compare the relationship between SARS-CoV-2 and tetherin in previous studies,

   ‘SARS-CoV-1 ORF7a is reported to inhibit tetherin glycosylation and localise to the plasma membrane in the presence of tetherin [18]. We did not observe any difference in total tetherin levels, tetherin glycosylation, ability to form dimers, or surface tetherin upon expression of either SARS-CoV-1 or SARS-CoV-2 ORF7a (Figures 4A, 4B, 4C).

   Others groups have demonstrated a role for ORF7a in sarbecovirus infection and both SARS-CoV-1 and SARS-CoV-2 virus lacking ORF7a show impaired virus replication in the presence of tetherin [18,41]. A direct interaction between SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a and tetherin have been described [18,41], although the precise mechanism(s) by which ORF7a antagonises tetherin remains enigmatic. We cannot exclude that ORF7a requires other viral proteins to antagonise tetherin, or that ORF7a antagonises tetherin via another mechanism. For example, ORF7a can potently antagonise IFN signalling [42] which would impair tetherin induction in many cell types.’ – (Line 667-704).

   I have no minor comments on this draft of the manuscript.

   Reviewer #1 (Significance (Required)):

   Tetherin, encoded by the BST2 gene, is an antiviral restriction factor that inhibits the release of enveloped viruses by creating tethers between viral and host membranes. It also has a capacity for sensing and signalling viral infection. It is most widely understood in the context of HIV-1, however, there is evidence of restriction in a wide variety of enveloped viruses, many of which have evolved strategies for antagonising tetherin. This knowledge informs on viral interactions with the innate immune system, with implications for basic virology and translational research.

   This study investigates tetherin in the context of SARS-CoV-2. The authors use a powerful collection of tools (live virus, gene knock out cells, recombinant viral and host expression systems) and a variety of approaches (microscopy, western blotting, infection assays), which is, itself, a strength. The study provides evidence to support a series of conclusions: I) BST2/tetherin restricts SARS-CoV-2 II) SARS-CoV-2 ablates tetherin expression III) spike protein can modestly down-regulate tetherin IV) ORF3A dysregulates tetherin localisation by altering retrograde trafficking. These conclusions are broadly supported by the data and this study make significant contributions to our understanding of SARS-CoV-2/tetherin interactions.

   My enthusiasm is reduced by, in my opinion, a failure of the authors to fully quantify, explain and explore their data. I expect the manuscript could be significantly improved without further experimentation by strengthening these aspects.

   This manuscript will be of interest to investigators in virology and/or cellular intrinsic immunity. Given the focus on SARS-CoV-2 it is possible/likely that it will find a slightly broader readership.

   I have highly appropriate skills for evaluating this work being experienced in virology, SARS-CoV-2, cell biology and microscopy.

   We wish to thank Reviewer #1 for their comments which have helped us to improve the quality of our revised manuscript.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   BST2/tetherin can restrict the release and transmission of many enveloped viruses, including coronaviruses. In many cases, restricted viruses have developed mechanisms to abrogate tetherin-restriction by expressing proteins that antagonize tetherin; HIV-1 Vpu-mediated antagonism of tetherin restriction is a particularly well studied example. In this paper, Stewart et al. report their studies of the mechanism(s) underlying SARS-CoV-2 antagonism of tetherin restriction. They conclude that Orf3a is the primary virally encoded protein involved and that Orf3a manipulates endo-lysosomal trafficking to decrease tetherin cycling and divert the protein away from putative assembly sites.

   Major comments:- In my view some of the claims made by the authors are not fully supported by the data. For example, the bystander effect discussed in line 162 may suggest that infected cells can produce IFN but does not 'indicate' that they do

   This text has now been edited,

   ‘The levels of tetherin in uninfected HAE cells is lower than observed in uninfected neighbours in infected wells demonstrating that infected HAE cells are able to generate IFN to act upon uninfected neighbouring cells, enhancing tetherin expression.’ - (Lines 163-172).

   Most of the EM images show part of a cell profile, so statements such as (line 192) 'virus containing tubulovesicular organelles were often polarised towards sites of significant surface-associated virus' should be backed up with appropriate images, or indicated as 'not shown', or removed (the observation is not so important for this story). Line 196, DMVs can't be seen in these micrographs.

   The statement 'virus containing tubulovesicular organelles were often polarised towards sites of significant surface-associated virus' has been removed. The micrographs in Figure 1E have been re-cropped, and image iii replaced with an image showing DMVs and budding virions. Plasma membrane-associated virions are highlighted by black arrowheads, DMVs by black asterisks, and intracellular virion by a white arrow.

   Line 391, I can't see much change in CD63 distribution.

   CD63 reproducibly appears clustered towards the nuclei in ORF3a expressing cells, whilst CD63 positive puncta are abundant in the periphery of mock cells. CD63 puncta are also larger, and the staining of CIMPR and VPS35 also appears to be associated with larger organelles. We have amended the text to now read,

   ‘Expression of ORF3a also disrupted the distribution of numerous endosome-related markers including CIMPR, VPS35, CD63, which all localised to larger and less peripheral puncta (Supplemental Figure 6B), and the mixing of early and late endosomal markers’ - (Line 469).

   Quantification of the diameter of CD63 puncta indicate that they are larger in ORF3a expressing cells than in mock cells. Mock cells - 0.71μm (SD; 0.19), ORF3a - 1.15μm (SD;0.35). At least 75 organelles per sample, from 10 different cells. We have not included this data as we do not wish to labor this point but are happy to include this quantification if required to do so.

   Line 321, the authors show that ORF7a does not affect tetherin localization, abundance, glycosylation or dimer formation, but they don't show that it doesn't restrict SARS-CoV-2. Can they be sure that epitope tagging this molecule does not abrogate function (or the functions of any of the other tagged proteins for that matter), or that ORF7a works in conjunction with one of the other viral proteins?

   We are careful in the manuscript not to claim that ORF7a has no effect on tetherin. Our data indicate that ‘ORF7a does not directly influence tetherin localisation, abundance, glycosylation or dimer formation’ - (Line 361-362).

   We were unable to reproduce an effect of ORF7a on tetherin glycosylation. Our data conflicts with that presented by Taylor et al, 2015, where ORF7a impaired tetherin glycosylation and ORF7a localised to the plasma membrane in tetherin expressing cells. The experiments performed by Taylor et al used HEK293 cells and ectopically expressed tagged tetherin. The differences in results may be attributed to the differences between cell lines or due to differences between endogenous or ectopic / tagged tetherin.

   The study by Taylor et al uses SARS-CoV-1 ORF7a-HA from Kopecky-Bromberg et al., 2007 (DOI: 1128/JVI.01782-06), where the -HA tag is positioned at the C-terminus. Our ORF7a-FLAG constructs have a C-terminal epitope tag. While we cannot exclude the possibility that tagged proteins may act differently from untagged ones, the differences between our findings and previous work appear unlikely to be due to epitope tags.

   Our manuscript states that although we cannot find any effect of ORF7a on tetherin localisation, abundance, glycosylation, or dimer formation, we cannot exclude that ORF7a impacts tetherin by another mechanism. For example, ORF7a has been found to antagonise interferon responses. Tetherin is abundantly expressed in HeLa cells and expression does not require induction through interferon. None of our experiments above would be impacted by interferon antagonism yet this could impact other cell types besides infection in vivo. These possibilities may explain the reported differential impact of ORF7a by different labs. An addition comment has been added to the discussion to reflect this,

   ’We cannot exclude that ORF7a requires other viral proteins to antagonise tetherin, or that ORF7a antagonises tetherin via another mechanism. For example, ORF7a potently antagonises IFN signalling [38], which would impair tetherin induction in many cell types. - (Line 701-704).

   Note - Reference 38 has been added to the manuscript – Xia et al., Cell Reports DOI: 10.1016/j.celrep.2020.108234

   In the ORF screen, a number of the constructs are expressed at low level, is it possible they [the authors] are missing something?

   Some of the ORFs expressed in the miniscreen appear poorly expressed. We accept that in the use of epitope tagged constructs expression levels of individual viral proteins may impact upon a successful screen. However, this screen was performed to identify any potential changes in tetherin abundance or localisation, and the screen did successfully identify ORF3a, which we were able to follow-up and verify.

   Line 376, the authors refer to ORF3a being a viroporin. A recent eLife paper (doi: 10.7554/eLife.84477; initially published in BioRxiv) refutes this claim and builds on other evidence that ORF3a interacts with the HOPS complex. The authors should at least mention this work, especially in the discussion, as it would seem to provide a molecular mechanism to support their conclusions.

   This paper had not been peer reviewed at the time of our initial submission. We have now included the following text,

   ‘SARS-CoV-2 ORF3a is an accessory protein that localises to and perturbs endosomes and lysosomes [29]. It may do so by acting either as a viroporin [30] or by interacting with, and possibly interfering with the function of VPS 39, a component of the HOPS complex which facilitates tethering of late endosomes or autophagosomes with lysosomes [29,31]. Given ORF3a likely impairs lysosome function, the observed increased….’ - (Lines 444-449).

   Fig 3, the growth curves illustrated in Fig3 C and D do not have errors bars; how many times were these experiments repeated?

   These experiments require more repeats to include error bars. Infection and plaque assay (Figure 3C, 3D) are currently ongoing and we plan to complete them in the next 6-8 weeks and include them in the finalised manuscript.

   In the new experiments, infections will additionally be performed at MOI 0.01, in addition to the previous MOIs (1 and 5).

   Line 396, the authors show increased co-localization with LAMP1. As LAMP1 is found in late endosomes as well as lysosomes, they cannot claim the redistributed tetherin is specifically in lysosomes.

   We have altered the text to now say:

   ‘The ORF3a-mediated increase in tetherin abundance within endolysosomes could be due to defective lysosomal degradation.’ - (Line 475).

   There seems to be a marked difference in the anti-rb555 signal in the 'mock' cells in panels 5H and Suppl 6E. Is there a good reason for this, or does this indicate variability between experiments?

   Antibody uptake experiments in Figure 5H and Supp Figure 6E were performed and acquired on different days. Relatively low levels of signal are available in these antibody uptake experiments, and the disperse labelling seen in the mocks does not aid this.

   Fig 6a, why is there negligible VLP release from cells lacking BST2 and ORF3a-strep? How many times were these experiments performed? Is this a representative image? I think it confusing to refer to the same protein by two different names in the same figure (i.e. BST2 and tetherin). Do the authors know how the levels of ORF3a expressed in cells in these experiments compares to those seen in infected cells?

   We have changed the blot in Figure 6A for one with clearer FLAG bands. Three independent experiments were performed for Figure 6A. Quantification of VLPs is now included in Supplemental Figure 7B.

   We have changed ‘Bst2’ to ‘tetherin’ in all previous figures relating to protein; Figure 4G, Figure 6A, B, C.

   We have no current information to compare ORF3a levels in these experiments versus in infected cells. We can investigate quantifying this if necessary.

   My final point is, perhaps, the trickiest to answer, but nevertheless needs to be considered. As far as we know, SARS-CoV-2 and at least some other coronaviruses, bud into organelles of the early secretory pathway, often considered to be ERGIC. In the experiments shown here the authors provide evidence that ORF3a can influence tetherin recycling, but the main way of showing this is through its increased association with endocytic organelles. Do the authors have any evidence that Orf3a reduces tetherin levels in the ERGIC or whether the tetherin cycling pathway(s) involve the ERGIC?

   This is an interesting point, and as the reviewer concedes, this is tricky to answer. Expression of ORF3a causes the redistribution or remodeling of various organelles (Figures 1E, 2D, 2F, Supp Figures 2C, 2E, 3E, 6B, 6C, 6D). We have been unable to test the direct involvement of ERGIC, despite attempts with a number of commercial antibodies. Given the huge rearrangements of organelles during SARS-CoV-2 infection, it is unclear exactly what will happen to the distribution of ERGIC.

   Minor comments: Line 53, delete 'shell' its redundant and confusing when the authors have said coronaviruses have a membrane.

   Deleted.

   Line 61, delete 'the'

   Deleted.

   Line 72, delete 'enveloped'; coronaviruses already described as enveloped viruses (line 53)

   Deleted.

   Lines 93 - 100, lop-sided discussion of the viral life cycle; this paragraph is mostly about entry, which is not relevant to this paper, and does not really deal with the synthesis and assembly side of the cycle.

   We have now added the following text,

   ‘….liberating the viral nucleocapsid to the cytosol of the cell. Upon uncoating, the RNA genome is released into the host cytosol and replication-transcription complexes assemble to drive the replication of the viral genome and the expression of viral proteins. Coronaviruses modify host organelles to generate viral replication factories - so-called DMVs (double-membrane vesicles) that act as hubs for viral RNA synthesis [10]. SARS-CoV-2 viral budding occurs at ER-to-Golgi intermediate compartments (ERGIC) and newly formed viral particles traffic through secretory vesicles to the plasma membrane where they are released to the extracellular space.’ - (Lines 95-104).

   Line 103, why are the neighbouring cells 'naive'?

   ‘naïve’ removed.

   Line 112 - 113, delete last phrase; tetherin is described as an IFN stimulated gene in line 111; to be accurate, the beginning of the sentence should be 'Tetherin is expressed from a type 1 Interferon stimulated gene ...'

   Amended.

   Line 118 - 119, should say 'For tetherin-restricted enveloped viruses' as not all enveloped viruses are restricted by tetherin.

   Amended.

   Line 131, coronaviruses are not the only family of tetherin-restricted viruses that assemble on intracellular membranes, e.g. bunyaviruses.

   This has been modified and now reads,

   ‘In order for tetherin to tether coronaviruses, tetherin must be incorporated in the virus envelope during budding which occurs in intracellular organelles.’ - (Lines 133-135).

   Line 192, there is no EM data in Supplemental Fig 1C.

   This has now been removed.

   Line 251, 'a synchronous infection event' should be 'synchronous infection' as there will be multiple infection events.

   This has been changed.

   Page 13 (and elsewhere), unlike Southern, 'Western' should not have a capital letter, except at the start of a sentence.

   These have been updated throughout the manuscript (Lines 183, 341, 3549, 356, 392, 509, 763, 1330, 1399).

   Lines 330 and 352, can the authors quantitate S protein-induced reduction in cell surface tetherin rather than using the somewhat subjective 'mild'?

   These are now changed to,

   ‘Transient transfection of cells with ss-HA-Spike caused a 32% decrease in tetherin as observed by immunofluorescence (Supplemental Figure 4A, 4B), with…’ – (Line 370).

   ‘To explore whether the Spike-induced tetherin downregulation altered virus release, we performed experiments with virus like particles (VLPs) in HEK293T …’ – (Line 399).

   Line 379, OFR, should be ORF.

   Yes, changed.

   Line 448, 'Tetherin retains the ability' - did it ever loose it?

   This has been rephrased to,

   ‘Tetherin has the ability to restrict a number of different enveloped viruses that bud at distinct organelles.’ - (Line 547).

   Line 451, 'luminal' is confusing in this context.

   This has been modified to,

   ‘Tetherin forms homodimers between opposing membranes (e.g., plasma membrane and viral envelope) that are linked via disulphide bonds.’ - (Line 549).

   Line 453, the process of virus envelopment is likely to be more than a 'single step'

   This now reads,

   ‘…virus during viral budding, which occurs in modified ERGIC organelles.’ - (Line 552).

   Line 457, in my view the notion that Vpu abrogation of tetherin restriction is just due to redistribution of tetherin to the TGN is somewhat simplistic and disregards a lot of other work.

   We have removed mention of mechanisms of tetherin antagonism by other viruses. The key point we wish to make here is that tetherin is lost from the budding compartment. This now reads,

   ‘Many enveloped viruses antagonise tetherin by altering its localisation and removing it from the respective site of virus budding.’ – (Line 552-553).

   Line 472, what is meant by 'resting states'?

   This should have been ‘in the absence of stimulation’ and have now been re-written,

   ‘Tetherin is an IFN-stimulated gene (ISG) [13], and many cell types express low levels of tetherin in the absence of stimulation.’ - (Line 577).

   Line 1204, how were 'mock infected cells .......... infected'?

   This has now been re-written,

   ‘Differentiated nasal primary human airway epithelial (HAE) cells were embedded to OCT….’ - (Line 1385).

   Reviewer #2 (Significance (Required)):

   This study builds on published work supporting the notion that SARS-Cov-2 ORF3a is an antagonist for the restriction factor tetherin. Importantly, it provides insights to the the mechanism of ORF3a mediated tetherin antagonism, specifically to ORF3a inhibits tetherin cycling, diverting the protein to lysosomes and away from compartment(s) where virions assemble. Overall, the authors provide good supporting evidence for these conclusions, however there are issues that the authors need to address.

   We wish to thank Reviewer #2 for their insightful comments and suggestions for improving this work.

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   Restriction factors are major barriers against viral infections. A prime example is Tetherin (aka BST2), which is able to physically tether budding virions to the plasma membrane preventing release of the infectious particles. Of note, tetherin has broad anti-viral activity and has been established as a crucial innate immune defense factor against HIV, IAV, SARS-CoV-2 and other important human pathogens. However, successful viruses like SARS-CoV-2 evolved strategies to counteract restriction factors and promote their replication. Important restriction factors, such as tetherin, may often be targeted by multiple viral strategies to ensure complete suppression of their anti-viral activities by the pathogen. Of note, it was previously published that the accessory protein ORF7a of SARS-CoV-2 binds to (Petrosino et al, Chemistry Europe, 2021) and antagonizes it (Martin-Sancho et al, Molecular Cell, 2021). Previous data on SARS-CoV also revealed that ORF7a promotes cleavage of tetherin (Taylor et al, 2015, J Virol). In this manuscript, the authors show that tetherin restricts SARS-CoV-2 by tethering virions to the plasma membrane and propose that tetherin is targeted by two proteins of SARS-CoV-2. Whereas the Spike protein promotes degradation of tetherin, the accessory protein ORF3a redirects tetherin away from newly forming SARS-CoV-2 virions. While the overall findings that both S and ORF3a are additionally targeting tetherin is both novel and intriguing, additional evidence is needed to support this. In addition, the authors show that in their experimental setups ORF7a does not induce cleavage of tetherin. This is in direct contrast to previously published data both on SARS-CoV(-1) and -2 (Taylor et al, 2015, J Virol; Petrosino et al, Chemistry Europe, 2021; Martin-Sancho et al, Molecular Cell, 2021). From my point of view that needs further experimental confirmation. While the authors state that the impact of Spike on tethrin is mild, the experiments should still allow the conclusion whether there is a (mild) effect or not. The mechanism of ORF3a is fortunately more robustly assessed and provides some novel insights. Unfortunately, the whole manuscript suffers from a striking lack of quantifications. In addition, it is not clear whether and how many times experiments were repeated to the same results. Overall, the data in this manuscript seem very speculative and preliminary and thus do not support the authors conclusions.

   Major:

   Much of the data seems like it was only done once. As I am sure that this is a writing issue, please clearly state how many times the individual assays were repeated, provide the quantification graphs and appropriate statistics. Some experiments may need additional quantification and confirmation by other methods to be convincing.

   Quantification is provided throughout the revised manuscript. Figure legends have also been updated to provide information on quantification and statistical analysis.

   For example, Figure 1A, C and D: Please quantify the levels of tetherin and use an alternative readout, e.g. Western blotting of infected cells.

   Quantification has been performed and included in our revised manuscript in Supplemental Figures 1C, 1E. Tetherin is not shown in Figure 1C.

   A table is provided (above) to highlight the additional quantification.

   Figure 2A: Please quantify.

   We are not sure we understand this point. The western blot shown in Figure 2A demonstrates the ectopic expression of ACE2 in our A549 cell line. A549 cells have been used by many labs to study SARS-CoV-2 infection, but express negligible ACE2.

   Fig 3A: Please show and confirm successful tetherin KO in the cell lines that are used not only in microscopy.

   A new blot is now shown in Figure 3A, including a blot demonstrating tetherin loss in both KO lines.

   Figure 4C: Please quantify

   Currently flow cytometry experiments have been performed twice each and this is now detailed in the figure legends. The data shown in each panel is representative and the data has been explored using analogous approaches. For example, Figure 4C is complemented by Figures 4A and 4B, Figures 4E is complemented by 4D and 4F. We do not feel that repeating these flow cytometry analysis will significantly improve the manuscript.

   Figure 4D: Please quantify the effects are not obvious from the images provided.

   Quantification is now provided in Supplemental Figure 4E.

   Figure 4E, F Please provide a quantification of multiple independent repeats, the claimed differences are neither striking nor obvious.

   Quantification of 4F is now provided in Supplemental Figure 4G. Tetherin levels were quantified to be reduced by 25% (SD: 8%) by addition of Doxycycline and induction of ss-HA-Spike. Information for quantification is provided in figure legends.

   Figure 5A: Please quantify

   These experiments have currently been performed twice and this is now described in the figure legends. Data shown is representative. We can perform one more repeat of these experiments to quantify if neccessary, but do not feel it will significantly alter the manuscript.

   Figure 3C and D: At timepoint 0 the infection input levels are different. The initial infection levels have to be the same to draw the conclusion that tetherin KO affects virion release and not the initial infection efficiency. Can the authors either normalize or ensure that the initial infection is the same in all conditions and that variations in the initial infection efficiency do not correlated with the impact of tetherin on replication/release ? How often were those experiments repeated? Are the marginal differences in infectious titre significant? Overall the impact of tetherin on SARS-CoV-2 is very underwhelming but that may be due to efficient viral tetherin-counteraction strategies. Why is the phenotype inverted at 72 h?

   Equal amounts of virus, as measured by plaque-forming units (PFU), were used for both HeLa cell lines and thus at 0 hpi the variation seen is within the parameters of the assay used. It remains possible that tetherin affects virus entry but this is unlikely and this assay was not designed to investigate that effect.

   Growth curve assays are currently being repeated using an MOI of 0.01, 1 and 5. We are removing the 72 hpi sample from future experiments. At this time point, we find that the extensive cell death caused by viral replication (especially at higher MOIs) makes it difficult to accurately separate the released from intracellular fractions and conclusions cannot be accurately drawn from the data.

   Additional repeats of these experiments are in progress and will be included in the finalised manuscript.

   Figure 4B and C: Can the authors provide an explanation why SARS-CoV ORF7a is not inducing cleavage/removes glycosylation of tetherin. To show that the assays work, an independent positive control needs to be included. The FACS data in C is unfortunately not quantified.

   See above comments (Reviewer #2) regarding discussion on ORF7a. Additional text has been included to discuss ORF7a data,

   ‘SARS-CoV-1 ORF7a is reported to inhibit tetherin glycosylation and localise to the plasma membrane in the presence of tetherin [18]. We did not observe any difference in total tetherin levels, tetherin glycosylation, ability to form dimers, or surface tetherin upon expression of either SARS-CoV-1 or SARS-CoV-2 ORF7a (Figures 4A, 4B, 4C).

   Others groups have demonstrated a role for ORF7a in sarbecovirus infection and both SARS-CoV-1 and SARS-CoV-2 virus lacking ORF7a show impaired virus replication in the presence of tetherin [18,41]. A direct interaction between SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a and tetherin have been described [18,41], although the precise mechanism(s) by which ORF7a antagonises tetherin remains enigmatic. We cannot exclude that ORF7a requires other viral proteins to antagonise tetherin, or that ORF7a antagonises tetherin via another mechanism. For example, ORF7a can potently antagonise IFN signalling [42] which would impair tetherin induction in many cell types.’ – (Line 667-704).

   Fig 4G: The rationale and result of this experiment are not clear.

   The rationale for Spike VLP experiments is explained at Line 403. Given that Spike caused a reproducible decrease in cellular tetherin, we examined whether this downregulation was sufficient to antagonise tetherin and increase VLP yield.

   Fig 6: What is the benefit of doing the VLP assays as opposed to genuine virus experiments? To me it rather seems to be making the data unnecessarily complex. Again, no quantifications or repeats are provided.

   VLPs are used to separate the budding and release process from the replication process of RNA viruses. VLPs have been used in a number of SARS-CoV (DOI: 1002/jmv.25518) and HIV-1 (DOI: https://doi.org/10.1186/1742-4690-7-51) studies to analyse the impact of tetherin (and tetherin mutants) on release.

   VLP experiment quantification are now included throughout.

   Minor: Fig 1D: How do the authors explain the mainly intracellular Spike staining?

   We do not understand this point. Spike staining is intracellular, whether expressed alone or in the context of infected cells.

   Please add statistical analyses on the data e.g. Fig. 3 C and D

   Additional repeats of these experiments are in progress and will be included in the finalised manuscript.

   Fig. 4B and F: Why do the annotated sizes of tetherin differ between the blots?

   Figures 4B and 4F are run in non-reduced and reduced conditions respectively. In order to best show the dimer deficient C3A-Tetherin, blots are typically run in non-reduced conditions to exemplify dimer formation and to highlight any defects in dimer formation. The rest of the blots in the manuscript are run in denaturing conditions to aid blotting of other proteins. (Lines 957-958) and now (Lines 1356-1357).

   Fig. 5A: What is ORF6a? Do the authors mean ORF6?

   Yes, this has been changed.

   An MOI of 1 is NOT considered a low or relevant MOI. Can the authors either rephrase or repeat experiments with an actual low or relevant MOI i.e. 0.01 ?

   We are currently repeating these experiments and are including MOIs of 0.01, 1 and 5.

   Why were the cell models switched between Figure 1 and 2 and essentially the same experiments repeated?

   HeLa cells express high levels of tetherin at steady state, whilst A549 cells require IFN stimulation. HeLa cells demonstrate that tetherin downregulation occurs via an IFN-independent manner. A549 and T84 cells are more physiologically relevant cell types for SARS-CoV-2 infection. These points are stated in Lines 230 and 261.

   The manuscript may benefit a lot from streamlining and removing unessential deviations from the main message (e.g. discussions why multistep/single step growth curves are used/not relevant; why are they shown if the authors conclude that a single step is not relevant?). The discussion is extremely lengthy and does not provide sufficient discussion of the presented data.

   The multistep/single step growth curve text will be adapted, but it will be re-written after additional infection experiments.

   We have removed from the Discussion a small section discussing ORF7a mutants, given that the emphasis of our manuscript is not on ORF7a.

   We have also removed a small section describing the rearrangements of intracellular organelles by SARS-CoV-2 as it does not directly relate to the central message of our manuscript.

   According to my opinion, the current manuscript does not provide significant advancement for the field. While the intention was to update and expand our existing knowledge about tetherin restriction by SARS-CoV-2, the experiments do not support this yet. However, the general premise and approach/concept of the manuscript would be appealing to a broader audience. I especially like the notion that multiple proteins of SARS-CoV-2 could synergistically counteract an important innate immune defense factor, tetherin. My expertise is on SARS-CoV-2 and the interplay between the virus and host cell restriction factors.

   Reviewer #3 (Significance (Required)):

   According to my opinion, the current manuscript does not provide significant advancement for the field. While the intention was to update and expand our existing knowledge about tetherin restriction by SARS-CoV-2, the experiments do not support this yet. However, the general premise and approach/concept of the manuscript would be appealing to a broader audience. I especially like the notion that multiple proteins of SARS-CoV-2 could synergistically counteract an important innate immune defense factor, tetherin. My expertise is on SARS-CoV-2 and the interplay between the virus and host cell restriction factors.

   We thank Reviewer#3 for their comments and suggestions for improving this work.

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   Referee #3

   Evidence, reproducibility and clarity

   Restriction factors are major barriers against viral infections. A prime example is Tetherin (aka BST2), which is able to physically tether budding virions to the plasma membrane preventing release of the infectious particles. Of note, tetherin has broad anti-viral activity and has been established as a crucial innate immune defense factor against HIV, IAV, SARS-CoV-2 and other important human pathogens. However, successful viruses like SARS-CoV-2 evolved strategies to counteract restriction factors and promote their replication. Important restriction factors, such as tetherin, may often be targeted by multiple viral strategies to …

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   Referee #3

   Evidence, reproducibility and clarity

   Restriction factors are major barriers against viral infections. A prime example is Tetherin (aka BST2), which is able to physically tether budding virions to the plasma membrane preventing release of the infectious particles. Of note, tetherin has broad anti-viral activity and has been established as a crucial innate immune defense factor against HIV, IAV, SARS-CoV-2 and other important human pathogens. However, successful viruses like SARS-CoV-2 evolved strategies to counteract restriction factors and promote their replication. Important restriction factors, such as tetherin, may often be targeted by multiple viral strategies to ensure complete suppression of their anti-viral activities by the pathogen. Of note, it was previously published that the accessory protein ORF7a of SARS-CoV-2 binds to (Petrosino et al, Chemistry Europe, 2021) and antagonizes it (Martin-Sancho et al, Molecular Cell, 2021). Previous data on SARS-CoV also revealed that ORF7a promotes cleavage of tetherin (Taylor et al, 2015, J Virol).

   In this manuscript, the authors show that tetherin restricts SARS-CoV-2 by tethering virions to the plasma membrane and propose that tetherin is targeted by two proteins of SARS-CoV-2. Whereas the Spike protein promotes degradation of tetherin, the accessory protein ORF3a redirects tetherin away from newly forming SARS-CoV-2 virions.

   While the overall findings that both S and ORF3a are additionally targeting tetherin is both novel and intriguing, additional evidence is needed to support this. In addition, the authors show that in their experimental setups ORF7a does not induce cleavage of tetherin. This is in direct contrast to previously published data both on SARS-CoV(-1) and -2 (Taylor et al, 2015, J Virol; Petrosino et al, Chemistry Europe, 2021; Martin-Sancho et al, Molecular Cell, 2021). From my point of view that needs further experimental confirmation. While the authors state that the impact of Spike on tethrin is mild, the experiments should still allow the conclusion whether there is a (mild) effect or not. The mechanism of ORF3a is fortunately more robustly assessed and provides some novel insights. Unfortunately, the whole manuscript suffers from a striking lack of quantifications. In addition, it is not clear whether and how many times experiments were repeated to the same results. Overall, the data in this manuscript seem very speculative and preliminary and thus do not support the authors conclusions.

   Major

   Much of the data seems like it was only done once. As I am sure that this is a writing issue, please clearly state how many times the individual assays were repeated, provide the quantification graphs and appropriate statistics. Some experiments may need additional quantification and confirmation by other methods to be convincing. For example, Figure 1A, C and D: Please quantify the levels of tetherin and use an alternative readout, e.g. Western blotting of infected cells. Figure 2A: Please quantify. Fig 3A: Please show and confirm successful tetherin KO in the cell lines that are used not only in microscopy. Figure 4C: Please quantify. Figure 4D: Please quantify the effects are not obvious from the images provided. Figure 4E,F Please provide a quantification of multiple independent repeats, the claimed differences are neither striking nor obvious. Figure 5A: Please quantify.

   Figure 3C and D: At timepoint 0 the infection input levels are different. The initial infection levels have to be the same to draw the conclusion that tetherin KO affects virion release and not the initial infection efficiency. Can the authors either normalize or ensure that the initial infection is the same in all conditions and that variations in the initial infection efficiency do not correlated with the impact of tetherin on replication/release ? How often were those experiments repeated? Are the marginal differences in infectious titre significant? Overall the impact of tetherin on SARS-CoV-2 is very underwhelming but that may be due to efficient viral tetherin-counteraction strategies. Why is the phenotype inverted at 72 h? Figure 4B and C: Can the authors provide an explanation why SARS-CoV ORF7a is not inducing cleavage/removes glycosylation of tetherin. To show that the assays work, an independent positive control needs to be included. The FACS data in C is unfortunately not quantified.

   Fig 4G: The rationale and result of this experiment are not clear.

   Fig 6: What is the benefit of doing the VLP assays as opposed to genuine virus experiments? To me it rather seems to be making the data unnecessarily complex. Again, no quantifications or repeats are provided.

   Minor

   Fig 1D: How do the authors explain the mainly intracellular Spike staining?

   Please add statistical analyses on the data e.g. Fig. 3 C and D

   Fig. 4B and F: Why do the annotated sizes of tetherin differ between the blots?

   Fig. 5A: What is ORF6a? Do the authors mean ORF6?

   An MOI of 1 is NOT considered a low or relevant MOI. Can the authors either rephrase or repeat experiments with an actual low or relevant MOI i.e. 0.01 ?

   Why were the cell models switched between Figure 1 and 2 and essentially the same experiments repeated? The manuscript may benefit a lot from streamlining and removing unessential deviations from the main message (e.g. discussions why multistep/single step growth curves are used/not relevant; why are they shown if the authors conclude that a single step is not relevant?). The discussion is extremely lengthy and does not provide sufficient discussion of the presented data.

   According to my opinion, the current manuscript does not provide significant advancement for the field. While the intention was to update and expand our existing knowledge about tetherin restriction by SARS-CoV-2, the experiments do not support this yet. However, the general premise and approach/concept of the manuscript would be appealing to a broader audience. I especially like the notion that multiple proteins of SARS-CoV-2 could synergistically counteract an important innate immune defense factor, tetherin. My expertise is on SARS-CoV-2 and the interplay between the virus and host cell restriction factors.

   Significance

   According to my opinion, the current manuscript does not provide significant advancement for the field. While the intention was to update and expand our existing knowledge about tetherin restriction by SARS-CoV-2, the experiments do not support this yet. However, the general premise and approach/concept of the manuscript would be appealing to a broader audience. I especially like the notion that multiple proteins of SARS-CoV-2 could synergistically counteract an important innate immune defense factor, tetherin.

   My expertise is on SARS-CoV-2 and the interplay between the virus and host cell restriction factors.

   Read the original source
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   Referee #2

   Evidence, reproducibility and clarity

   BST2/tetherin can restrict the release and transmission of many enveloped viruses, including coronaviruses. In many cases, restricted viruses have developed mechanisms to abrogate tetherin-restriction by expressing proteins that antagonize tetherin; HIV-1 Vpu-mediated antagonism of tetherin restriction is a particularly well studied example. In this paper, Stewart et al. report their studies of the mechanism(s) underlying SARS-CoV-2 antagonism of tetherin restriction. They conclude that Orf3a is the primary virally encoded protein involved and that Orf3a manipulates endo-lysosomal trafficking to decrease tetherin cycling and divert …

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   Referee #2

   Evidence, reproducibility and clarity

   BST2/tetherin can restrict the release and transmission of many enveloped viruses, including coronaviruses. In many cases, restricted viruses have developed mechanisms to abrogate tetherin-restriction by expressing proteins that antagonize tetherin; HIV-1 Vpu-mediated antagonism of tetherin restriction is a particularly well studied example. In this paper, Stewart et al. report their studies of the mechanism(s) underlying SARS-CoV-2 antagonism of tetherin restriction. They conclude that Orf3a is the primary virally encoded protein involved and that Orf3a manipulates endo-lysosomal trafficking to decrease tetherin cycling and divert the protein away from putative assembly sites.

   Major comments:

   In my view some of the claims made by the authors are not fully supported by the data. For example, the bystander effect discussed in line 162 may suggest that infected cells can produce IFN but does not 'indicate' that they do. Most of the EM images show part of a cell profile, so statements such as (line 192) 'virus containing tubulovesicular organelles were often polarised towards sites of significant surface-associated virus' should be backed up with appropriate images, or indicated as 'not shown', or removed (the observation is not so important for this story). Line 196, DMVs can't be seen in these micrographs. Line 391, I can't see much change in CD63 distribution.

   Line 321, the authors show that ORF7a does not affect tetherin localization, abundance, glycosylation or dimer formation, but they don't show that it doesn't restrict SARS-CoV-2. Can they be sure that epitope tagging this molecule does not abrogate function (or the functions of any of the other tagged proteins for that matter), or that ORF7a works in conjunction with one of the other viral proteins? In the ORF screen, a number of the constructs are expressed at low level, is it possible they are missing something?

   Line 376, the authors refer to ORF3a being a viroporin. A recent eLife paper (doi: 10.7554/eLife.84477; initially published in BioRxiv) refutes this claim and builds on other evidence that ORF3a interacts with the HOPS complex. The authors should at least mention this work, especially in the discussion, as it would seem to provide a molecular mechanism to support their conclusions.

   Fig 3, the growth curves illustrated in Fig3 C and D do not have errors bars; how many times were these experiments repeated?

   Line 396, the authors show increased co-localization with LAMP1. As LAMP1 is found in late endosomes as well as lysosomes, they cannot claim the redistributed tetherin is specifically in lysosomes.

   There seems to be a marked difference in the anti-rb555 signal in the 'mock' cells in panels 5H and Suppl 6E. Is there a good reason for this, or does this indicate variability between experiments?

   Fig 6a, why is there negligible VLP release from cells lacking BST2 and ORF3a-strep? How many times were these experiments performed? Is this a representative image? I think it confusing to refer to the same protein by two different names in the same figure (i.e. BST2 and tetherin). Do the authors know how the levels of ORF3a expressed in cells in these experiments compares to those seen in infected cells?

   My final point is, perhaps, the trickiest to answer, but nevertheless needs to be considered. As far as we know, SARS-CoV-2 and at least some other coronaviruses, bud into organelles of the early secretory pathway, often considered to be ERGIC. In the experiments shown here the authors provide evidence that ORF3a can influence tetherin recycling, but the main way of showing this is through its increased association with endocytic organelles. Do the authors have any evidence that Orf3a reduces tetherin levels in the ERGIC or whether the tetherin cycling pathway(s) involve the ERGIC?

   Minor comments:

   Overall, the manuscript should be carefully edited to ensure the text reads clearly. A few examples of thing that need to be fixed are:-

   Line 53, delete 'shell' its redundant and confusing when the authors have said coronaviruses have a membrane.

   Line 61, delete 'the'

   Line 72, delete 'enveloped'; coronaviruses already described as enveloped viruses (line 53)

   Lines 93 - 100, lop-sided discussion of the viral life cycle; this paragraph is mostly about entry, which is not relevant to this paper, and does not really deal with the synthesis and assembly side of the cycle.

   Line 103, why are the neighbouring cells 'naive'?

   Line 112 - 113, delete last phrase; tetherin is described as an IFN stimulated gene in line 111; to be accurate, the beginning of the sentence should be 'Tetherin is expressed from a type 1 Interferon stimulated gene ...'

   Line 118 - 119, should say 'For tetherin-restricted enveloped viruses' as not all enveloped viruses are restricted by tetherin.

   Line 131, coronaviruses are not the only family of tetherin-restricted viruses that assemble on intracellular membranes, e.g. bunyaviruses.

   Line 192, there is no EM data in Supplemental Fig 1C.

   Line 251, 'a synchronous infection event' should be 'synchronous infection' as there will be multiple infection events

   Page 13 (and elsewhere), unlike Southern, 'Western' should not have a capital letter, except at the start of a sentence.

   Lines 330 and 352, can the authors quantitate S protein-induced reduction in cell surface tetherin rather than using the somewhat subjective 'mild'?

   Line 379, OFR, should be ORF.

   Line 448, 'Tetherin retains the ability' - did it ever loose it?

   Line 451, 'luminal' is confusing in this context.

   Line 453, the process of virus envelopment is likely to be more than a 'single step'

   Line 457, in my view the notion that Vpu abrogation of tetherin restriction is just due to redistribution of tetherin to the TGN is somewhat simplistic and disregards a lot of other work.

   Line 472, what is meant by 'resting states'?

   Line 1204, how were 'mock infected cells .......... infected'?

   Significance

   This study builds on published work supporting the notion that SARS-Cov-2 ORF3a is an antagonist for the restriction factor tetherin. Importantly, it provides insights to the the mechanism of ORF3a mediated tetherin antagonism, specifically to ORF3a inhibits tetherin cycling, diverting the protein to lysosomes and away from compartment(s) where virions assemble. Overall, the authors provide good supporting evidence for these conclusions, however there are issues that the authors need to address.

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   Referee #1

   Evidence, reproducibility and clarity

   I summarise the major findings of the work below. In my opinion the range and application of approaches has provided a broad evidence base that, in general, supports the authors conclusions. However, there are, in my opinion, particular failures to utilise and communicate this evidence. The manuscript may be much improved with attention in the following areas. In each case I will give general criticism with a few examples, but the principals of my comments could be applied throughout the work.

   1. Insufficient quantification. The investigation combines various sources of qualitative data (EM, fluorescence microscopy, western blotting) …

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   Referee #1

   Evidence, reproducibility and clarity

   I summarise the major findings of the work below. In my opinion the range and application of approaches has provided a broad evidence base that, in general, supports the authors conclusions. However, there are, in my opinion, particular failures to utilise and communicate this evidence. The manuscript may be much improved with attention in the following areas. In each case I will give general criticism with a few examples, but the principals of my comments could be applied throughout the work.

   1. Insufficient quantification. The investigation combines various sources of qualitative data (EM, fluorescence microscopy, western blotting) to generate a reasonably strong evidence base. However, the work is over-reliant on representative images and should include more quantification from repeat experiments. When there are multiple fluorescence micrographs with intensity changes (not necessarily just representative images) (e.g. Figure 1 or 2) the authors should consider making measurements of these. Also the VLP production assays, which are assessed by western blotting would particularly benefit from a quantitative assessment (either by densitometry or, if samples remain, ELISA/similar approach).
   2. Insufficient explanation. I found some of the images and legends contained insufficient annotation and/or description for a non-expert reader to appreciate the result(s). Particularly if the authors want to draw attention to features in micrographs they should consider using more enlarged/inset images and annotations (e.g. arrows) to point out structures (e.g DMVs etc.). This short coming exacerbates the lack of quantification.
   3. Insufficient exploration of the data. I had a sense that some aspects of the data seem unconsidered or ignored, and the discussion lacks depth and reflection. For example the tetherin down-regulation apparent in Figures 1 and 2 is not really explained by the spike/ORF3a antagonism described later on, but this is not explicitly addressed. Also, Figure 6 suggests that ORF3a results in high levels of incorporation of tetherin in to VLPs, but I don't think this is even described(?). The discussion should also include more comparison with previous studies on the relationship between SARS-2 and tetherin.

   I have no minor comments on this draft of the manuscript.

   Significance

   Tetherin, encoded by the BST2 gene, is an antiviral restriction factor that inhibits the release of enveloped viruses by creating tethers between viral and host membranes. It also has a capacity for sensing and signalling viral infection. It is most widely understood in the context of HIV-1, however, there is evidence of restriction in a wide variety of enveloped viruses, many of which have evolved strategies for antagonising tetherin. This knowledge informs on viral interactions with the innate immune system, with implications for basic virology and translational research.

   This study investigates tetherin in the context of SARS-CoV-2. The authors use a powerful collection of tools (live virus, gene knock out cells, recombinant viral and host expression systems) and a variety of approaches (microscopy, western blotting, infection assays), which is, itself, a strength. The study provides evidence to support a series of conclusions: I) BST2/tetherin restricts SARS-CoV-2 II) SARS-CoV-2 ablates tetherin expression III) spike protein can modestly down-regulate tetherin IV) ORF3A dysregulates tetherin localisation by altering retrograde trafficking. These conclusions are broadly supported by the data and this study make significant contributions to our understanding of SARS-CoV-2/tetherin interactions.

   My enthusiasm is reduced by, in my opinion, a failure of the authors to fully quantify, explain and explore their data. I expect the manuscript could be significantly improved without further experimentation by strengthening these aspects.

   This manuscript will be of interest to investigators in virology and/or cellular intrinsic immunity. Given the focus on SARS-CoV-2 it is possible/likely that it will find a slightly broader readership.

   I have highly appropriate skills for evaluating this work being experienced in virology, SARS-CoV-2, cell biology and microscopy.

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5. 

   Version published to 10.1101/2021.01.06.425396v2 on bioRxiv

   Dec 22, 2022
6. 

   Rapid Reviews Infectious Diseases

   Aug 13, 2021

   Chin-Tien Wang

   Review 1: "SARS-CoV-2 spike downregulates tetherin to enhance viral spread"

   This preprint demonstrates that host cells employ a known pathway in which interferon-induced tetherin “tethers” SARS-CoV-2 to the inner plasma membrane to restrict viral exit. Reviewers recognize the data as reliable with minor revisions needed.

   Read the original source
7. 

   Rapid Reviews Infectious Diseases

   Aug 13, 2021

   Barry Milavetz

   Review 2: "SARS-CoV-2 spike downregulates tetherin to enhance viral spread"

   This preprint demonstrates that host cells employ a known pathway in which interferon-induced tetherin “tethers” SARS-CoV-2 to the inner plasma membrane to restrict viral exit. Reviewers recognize the data as reliable with minor revisions needed.

   Read the original source
8. 

   Rapid Reviews Infectious Diseases

   Aug 13, 2021

   Strength of evidence

   Reviewers: Chin-Tien Wang | 📗📗📗📗◻️
   Barry Milavetz (University of North Dakota) | 📘📘📘📘📘

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9. 

   ScreenIT

   Jan 7, 2021

   SciScore for 10.1101/2021.01.06.425396: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cells were found free of mycoplasma by immunofluorescence (DAPI), electron microscopy and were tested using MycoAlert Mycoplasma Detection Kit (Lonza)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche,	FLAG suggested: (Advanced Targeting Systems Cat# AB-450-1000, RRID:AB_10584603) anti-DYKDDDDK suggested: None ant…

   SciScore for 10.1101/2021.01.06.425396: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cells were found free of mycoplasma by immunofluorescence (DAPI), electron microscopy and were tested using MycoAlert Mycoplasma Detection Kit (Lonza)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche,	FLAG suggested: (Advanced Targeting Systems Cat# AB-450-1000, RRID:AB_10584603) anti-DYKDDDDK suggested: None anti-HA suggested: None
   3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000),	anti-tetherin suggested: None anti-SARS-CoV-2 suggested: None anti-TGN46 suggested: (Abcam Cat# ab50595, RRID:AB_2203289) anti-ZFPL1 suggested: (Sigma-Aldrich Cat# HPA014909, RRID:AB_1859055) anti-Beta2microglobulin suggested: None ACE2 suggested: (Proteintech Cat# CL488-66699, RRID:AB_2883371)
   anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic	anti-human tetherin suggested: None
   Secondary antibodies used in this study were: Goat anti-Mouse IgG Alexa488/555 and Goat anti-rabbit IgG Alexa 488/555 (ThermoFisher) secondaries were used for confocal microscopy.	anti-Mouse IgG suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160) anti-rabbit IgG suggested: None
   Goat IRDye 680 anti-mouse, anti-rabbit and Goat IRDye 800 anti-mouse, anti-rabbit antibodies (Li-Cor) were used for Western blotting.	anti-mouse , suggested: None anti-rabbit suggested: (Rockland Cat# KFB001, RRID:AB_828189) anti-mouse suggested: None
   Coverslips were inverted over drops of either rabbit anti-tetherin or rabbit anti-spike antibodies diluted in 1% BSA/PBS.	anti-spike suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: A549 cells were a gift from Dr Brian Ferguson, University of Cambridge, UK and were cultured in DMEM supplemented with 10% fetal bovine serum, L-glutamine, and Penicillin/Streptomycin, in 5% CO2 at 37 °C.	A549 suggested: None
   T84 cells were purchased from ATCC and were cultured in DMEM: F-12 medium containing 5% fetal bovine serum, L-glutamine, and Penicillin/Streptomycin, in 5% CO2 at 37 °C.	T84 suggested: None
   SARS-CoV-2 ORF miniscreen: HeLa cells were transiently transfected with 2xStrep-tagged SARS-CoV-2 ORF DNA (27) using HeLa Monster (Mirus Bio).	HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
   Generation of stable cell lines: Lentiviral constructs: ACE2 stable HeLa and A549 cell lines were generated using the lentiviral pLVX-ACE2-Blasticidin construct from Dr Yohei Yamauchi (University of Bristol).	ACE2 suggested: RRID:CVCL_DR94)
   Retroviral constructs: pQCXIH-SARS-CoV-1-ORF7a-FLAG and pQCXIH-SARS-CoV-2-ORF7a-FLAG were transfected to HEK293 cells with the packing plasmids pMD.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   SARS-CoV-2 infections: HeLaWT+ACE2, HeLaBst2KO+ACE2, A549+ACE2 or T84 cells were infected with isolate BetaCoV/ Australia/VIC01/2020 (19), which had been passaged once on Vero cells following receipt from Public Health England.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Virus Growth Curves: Multiple subconfluent T25 flasks of WT HeLa +ACE2 and KO HeLa +ACE2 cells were each infected with a single stock of SARS-CoV-2, at an MOI of 5 and 1, for the one-step and multi-step growth curves, respectively.	KO HeLa +ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   All cloning was verified by Sanger sequencing (GeneWiz).	GeneWiz suggested: (GENEWIZ, RRID:SCR_003177)
   Immunofluorescence colocalisation analysis: Appropriate threshold values were manually applied to each channel and the Manders’ overlap coefficient between two channels was quantified using the JACoP plugin of ImageJ Fiji software (39).	ImageJ suggested: (ImageJ, RRID:SCR_003070)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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   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.

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   Read the original source
10. 

    Version published to 10.1101/2021.01.06.425396v1 on bioRxiv

    Jan 6, 2021