Morphology-dependent entry kinetics and spread of influenza A virus

  1. Sarah Peterl
  2. Carmen M. Lahr
  3. Carl N. Schneider
  4. Janis Meyer
  5. Xenia Podlipensky
  6. Vera Lechner
  7. Maria Villiou
  8. Larissa Eis
  9. Steffen Klein
  10. Charlotta Funaya
  11. Elisabetta Ada Cavalcanti-Adam
  12. Frederik Graw
  13. Christine Selhuber-Unkel
  14. Karl Rohr
  15. Petr Chlanda

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Abstract

Influenza A viruses (IAV) display a broad variety of morphologies ranging from spherical to long filamentous virus particles. These diverse phenotypes are believed to allow the virus to overcome various immunological and pulmonary barriers during entry into the airway epithelium and influence the viral entry pathway. Remarkably, lab-adapted IAV strains lost this morphological variance and exhibit preferred spherical morphology. However, it remains unclear which factors lead to this lab-adapted preference and which pulmonary defense factors are responsible for the preferred filamentous morphology in physiological settings. In this study, we established fluorescent reporter viruses with spherical or filamentous morphology but with the same surface glycoproteins. We developed a correlative fluorescence and scanning electron microscopy workflow to analyze the impact of viral morphology on cell-to-cell spread and identify conditions under which IAV with either spherical or filamentous morphology confer an advantage. Our findings demonstrate that filamentous IAV cell-to-cell spread is significantly slower in various cell lines, which can explain the predominant spherical morphology in lab-adapted strains. This observation is consistent with delayed entry kinetics of filamentous viruses structurally analyzed by cellular cryo-electron tomography. We found that cellular junction integrity and mucin do not exert morphology-dependent inhibition of IAV cell-to-cell spread. On the other hand, filamentous virions confer an advantage under the pressure exerted by neutralizing antibodies against hemagglutinin.

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    Reply to the reviewers

    Response to reviewers

    We sincerely thank all reviewers for taking the time to review our manuscript and for providing insightful comments and suggestions. Your feedback has been invaluable in improving the quality and clarity of our work.

    Reviewer #1

    Evidence, reproducibility and clarity

    This manuscript by Peterl and colleagues seeks to understand the long-standing observation that influenza A virus generally exhibits a filamentous phenotype in vivo which is lost upon serial passaging in vitro or in embryonated chicken eggs. In addressing this question, the authors perform a detailed quantitative comparison of how filamentous and spherical strains of influenza spread in cell culture in the presence or absence of perturbations including neutralizing antibodies, mucin, and disruption of cell-cell junctions.

    The manuscript reports several observations that will be of interest to researchers in the area of influenza virus morphology and spread. Using a combination of imaging modalities, the authors convincingly demonstrate that spherical strains of influenza virus produce larger plaques than filamentous strains that are isogenic except for mutations in M1. The authors show that this is at least partly attributable to differences in entry kinetics. The authors also recapitulate a prior finding that filamentous viruses are more resistant to neutralizing antibodies than spherical ones. In most cases, the authors' claims are supported by the data presented. A few partial exceptions are noted below.

    The paper would be strengthened by a clearer description of some of the experimental approaches which lack important details in some instances. The scope of the paper is also limited somewhat by the use of immortalized cell lines that lack physiological features of the airway epithelium. Although this limitation is understandable from a technical standpoint, a discussion of these limitations should be included. Specific comments are listed below.

    Major Points

    In Figure 4, it is not stated at what time the cell density is measured in panel B, and how this might change across the time points sampled in panel C. This would make the experiment difficult to reproduce. This could be a very important consideration if the cells reach confluency soon after the infection is initiated, since the plaque sizes seem statistically similar out to 24hpi in 4B.

    Thank you for your comment on cell densities in Figure 4 B. We agree that the quantification of cell confluency across the time points is crucial in this context. Furthermore, we recognize that counting the number of nuclei within a well is not the most accurate method for comparing the two cell lines. We now provide measurements of relative cell density based on plasma membrane staining for uninfected MDCK-WT and MDCK-α-Catenin-KO cells at 24h and 48h for three biological replicates (Figure 4 A and B). These data show that MDCK-α-Catenin-KO have lower confluency (area=229.69 µm2) at 48 h compared to MDCK-WT cells (area=361.24 µm2). While the confluency of MDCK-WT cells was > 95% at both time points, MDCK-a-Catenin-KO cells did not reach 70% confluency, which reflects the lack of adherens junctions in these cells.

    In Figure 4F, it appears that plaque sizes for M1Ud are less affected by mucin than M1WSN plaques at all concentrations tested. However, the authors conclude that "mucin did not show any IAV morphology-dependent inhibitory effect as indicated by the slopes of linear fits of the plaque diameters" (Line 265). I understand that the authors are looking for dose-dependent effects, but it is not clear to me why an analysis based on the slope is preferable, especially when the response to mucins may not be linear. How does the availability of IAV receptors in the porcine gastric mucin used here compare to human airway mucins? Finally, the authors should clarify the number of replicates for this experiment.

    Thank you for pointing out that the data representation of IAV WSN and WSN-M1Udorn plaque growth in the presence of mucin (Figure 4 C) lacked clarity. We agree and removed the regression fitting and, instead, show all individual plaque sizes (Extended Figure 4 B). We now provide relative reduction of plaque sizes compared between WSN and WSN-M1Udorn plaques at each mucin concentration using 3 or 4 independent experiments (Figure 4 E). This did not reveal that there was a significant reduction in plaque size change between WSN and WSN-M1Udorn in the absence or presence of mucin. We changed our conclusion: "mucin did not show an IAV morphology-dependent inhibitory effect as indicated by the relative plaque size decrease of WSN-M1Udorn compared to WSN across the mucin concentrations" (Line 278).

    We have included information on the mucus composition and receptor availability in the discussion: "Notably, we used porcine gastric mucin, which might differ structurally and in the sialic acid linkage types compared to human mucins (Nordman et al., 2002, doi: 10.1042/bj3640191; Zhang et al., 2021, doi: 10.1007/s10719-021-10014-y). However, both in the porcine stomach and human airway, MUC5AC molecules are the predominant gel-forming mucins." (Graigner et al., 2006, 10.1007/s11095-006-0255-0) (Line 436).

    One key difference between the cells used here and the airway epithelium is the presence of multiciliated cells that could alter viral transport in ways that depend on morphology and may be difficult to predict. I appreciate that this concept is outside the scope of the current work, but it is an important point that warrants mention.

    We have now included fluorescence microscopy data using anti-MUC5AC antibody to assess mucin production in Calu-3 cells. Importantly, we could demonstrate that Calu-3 cells used in our study express mucins (Figure 4 D). We acknowledge that the absence of multiciliated cells is a limitation and plan to address this in future studies by using air-liquid interface cultures and by incorporating primary human bronchial cells. We established a transwell Calu-3 cell culture under air-liquid interface (ALI) conditions, which allowed for cell polarization. The apical surface of Calu-3 cells grown in an ALI culture contains more mucin than in liquid-covered unpolarized cultures. We plan to adapt and further develop a correlative imaging workflow to be able to assess spread in transwells in a separate study, as this is technically more challenging. We have included this in the discussion (Line 440-444).

    Minor Points

    It is somewhat unclear what is being captured in the data in Figure 5D-I. I assume that the cell surfaces that are imaged here are from infected cells within the plaque. If this is the case, it is difficult to tell whether the particles that are being quantified are incoming viruses or viruses that are currently budding. MEDI8852 is a stalk-binding antibody which would not be expected to inhibit viral attachment. This is unlikely to change the interpretation since the data shows differences between spherical and filamentous strains. However, a clearer description of this data would be helpful.

    We appreciate your constructive feedback. Figure 5 captures the effect of HA-stalk-binding MEDI8852 antibodies on IAV spread and morphology. While this antibody does not prevent receptor binding, it blocks membrane fusion and exerts pressure on the viruses, which, based on our hypothesis, can be overcome by increasing the number of HA on the surface of filamentous viruses. This is now also confirmed in Figure 5B showing that entry of spherical viruses is more sensitive to MEDI8852 than entry of filamentous viruses above concentration of 5 nM.

    SEM images of IAV plaques in MDCK cells in the presence of 1 nM MEDI8852 antibody show that viral morphology is not altered by antibody pressure. We agree that this method provides information on IAV morphology but does not allow us to distinguish between incoming or budding viruses. However, virus entry is fast, and IAV release from plasma membrane is slow as obvious from transmission electron microscopy studies showing large quantities of budding virions connected to plasma membrane by budding neck (example: DOI: 10.1099/vir.0.036715-0). Hence, it can be assumed that the majority of viruses captured by SEM on the cell surface are budding viruses. We have included this in the discussion (Line 409-414).

    Nevertheless, to further address this limitation, we now provide a more robust analysis of IAV particle numbers and morphologies from supernatants of serial passaging in MDCK cells under MEDI8852 antibody pressure, using cryo-EM (Fig. 5 D, E). In accordance with the SEM data, we did not observe morphological changes of IAV in the presence of the antibody.

    For experiments in Calu-3 cells, is trypsin added to the culture media following infection? If not, what percentage of HA is proteolytically cleaved? I would expect these cells to express activating proteases, but if activation is less efficient, this could favor the filamentous strain (as discussed in ref 49).

    Thank you for this comment. Yes, trypsin was added to the medium of Calu-3 cells during infection. We included this in the methods section.

    The schematic in Figure 4D illustrates mucins as tethered to the cell surface. This does not reflect the experiments in Figure 4E and F, where secreted mucins are added to the overlay media.

    We agree, and we removed the schematic representation of mucins in Figure 4D, instead we show data on mucin production in Calu-3 cells (Figure 4 D).

    There are a few small typos. Line 61: "to results in" and Line 111: "neutralizing antibodies against hemagglutinin are more effectively blocking virions with spherical morphology."

    We corrected the typo in line 61 and changed the phrasing of lines 111-112 for more clarity.

    Significance

    A strength of this manuscript is the quantitative rigor of the approaches used, which reveal interesting differences in the spread of filamentous and spherical influenza. These differences are compelling, but are limited somewhat in their significance by the difficulty of evaluating whether or not some of the observations would be preserved in differentiated airway epithelial cells. The authors do not over-generalize their conclusions, but more detailed discussion of these potential limitations is warranted.

    As mentioned above, we agree that a differentiated airway is important; however, assessing determining factors responsible for inhibition might be difficult due to the high complexity of the culture composed of different cells. The presented methods allow quantitatively assessing individual factors, which provides benefits. Hence, both approaches are valid and important.

    Reviewer #2

    Evidence, reproducibility and clarity

    Summary: This manuscript by Peteryl and colleagues explores the question of why some influenza viruses (typically those that have been recently isolated from animals, though also the Udorn strain) produce filamentous particles, while influenza viruses that have been adapted to eggs or cell culture form spherical particles. This is a long standing question in the influenza field, and the authors have used a nice set of new tools and approaches to shed light on this question. They created mScarlet labelled viruses that produce spherical (WSN) or predominantly filamentous (WSN with an M segment from Udorn) virions, but share the same glycoproteins. While this approach is not novel (the fact that the segment 7 of Udorn drives a filamentous phenotype has been previously demonstrated), the authors used these viruses in an elegant series of experiments to look at the rate of cell to cell spread within a plaque to show that the spherical viruses spread more quickly. The authors then explored the effect of cell density, inhibitors designed to inhibit different routes of viral entry, and the presence of neutralizing antibody. The experiments are thoughtfully designed, and the electron microscopy in particular is beautifully done. In general, the conclusions are supported by the data, though the specific claim that filamentous viruses have an advantage in viral entry in the presence of neutralizing antibody would be significantly strengthened by performing the specific entry assay the authors employ earlier in the manuscript.

    Major comments: The key conclusions are largely convincing, though the authors should perform the entry assays they employ in figure 3 (measuring the kinetics of entry and the efficiency of entry) to determine whether the delay in cell to cell spread they observe for spherical viruses in the presence of neutralizing antibody is due specifically to the effect on entry. I also am concerned about the method used to determine that the antibody treatment in Fig 5D-H results in a difference in the number of virions produced. While I appreciate that SEM is time consuming and difficult to quantify, counting the number of virions seen in a single field of view from 7 or 12 cells does not provide a robust foundation to support the central claim of the paper, that the difference in speed of filamentous and spherical viral spread is due to a difference in their ability to support viral entry in the presence of neutralizing antibody . If the authors wish to count virions produced by the WSN/WSN M-Udorn viruses in the presence/absence of neutralizing antibody it would be sensible to perform a synchronized high MOI infection and measure infectious titer by plaque assay (as this would be able to quickly and easily measure millions of virions produced by hundreds of thousands of cells).

    Thank you very much for the suggestion to perform an entry assay in the presence of a neutralizing antibody to determine whether the antibody acts at the level of viral entry. We now provide data on the entry efficiency of WSN and WSN-M1Udorn in the presence of increasing MEDI8852 concentrations (Figure 5 B). The results show that entry of the WSN spherical viruses are more affected by MEDI8852 at 5 nM and 10 nM, compared to WSN-M1Udorn, suggesting that the reduced plaque growth presented in Figure 5 C reflects an inhibition of IAV entry.

    We agree that the quantification of virions at the surface of 7-12 cells in SEM images is not a robust method. Therefore, we removed the quantification as it is technically very time-consuming to obtain a large enough dataset or to perform statical power analysis on how many cells would need to be screened. We additionally performed a serial passaging experiment of WSN and WSN-M1Udorn under antibody pressure, providing a more robust analysis of IAV particle numbers and morphologies from supernatants using cryo-EM (Fig. 5 D, E). By quantifying the length/diameter ratio of at least 80 virions per condition, we observed that both IAV morphologies remained stable in the presence of the antibody after five passages.

    The two entry assays could be done in parallel, and I anticipate them to take ~3 days per replicate (a day to seed, a day to infect/add NH4Cl at the indicated time points and fix, a day to image and analyze data). Similarly, infected cells at high MOI in the presence/absence of nAb, collecting viral supernatants, and tittering by plaque assay should take ~one week. The reagents to perform these experiments are already in hand, and as the costs will be limited to standard tissue culture reagents, using a microscopy set up the authors already possess. The experiments throughout the paper are well described, with appropriate methodological detail and statistical analysis.

    Minor comments: • Viruses without the mScarlet spread faster, the WSN-Udorn has more viruses with mScarlet than the WSN does so how do we know that some of the difference isn't down to that?

    Thank you for this important question. It is correct that viruses without mScarlet spread faster. We used WSN mScarlet viruses for CLSEM and live cell imaging of Calu-3 cells. To ensure that the observed differences in viral spread kinetics were not attributable to the presence or absence of mScarlet but to viral morphology, we conducted additional immunofluorescence staining for viral nucleoprotein (NP) or matrix protein 2 (M2) (Extended Figure 1 H-I). This allowed us to account for all viral plaques, including those that were not mScarlet-positive. This way we obtained data for our experiments with MDCK-α-Catenin-KO cells, mucin, zanamivir and MEDI8852 (Figure 4 and 5).

    • While Calu3 cells are reported to make mucus the authors should verify the expression of relevant mucus proteins in their hands, and this phenotype can be variable depending on culture conditions.

    Thank you for highlighting this important point. We verified the expression of MUC5AC in Calu-3 cells grown on cover slips and observed MUC5AC expression in distinct puncta (Figure 5 D).

    • In 5F and I does 'mock' mean no antibody or no virus?

    We apologize for the imprecise nomenclature in Figure 5 F and changed the Figure description.

    • The authors should either include data to support the claim in line 410: "Our data provide further evidence that IAV filamentous morphology is lost to accelerate cell-to-cell spread by faster entry kinetics and to achieve higher entry efficiency" or reword this sentence, since at present this manuscript does not include experiments demonstrating the loss of filamentous morphology in tissue culture of the WSN-M1 Udorn virus.

    Thank you, we agree and modified the sentence.

    Significance

    The data and conclusions presented in this manuscript are exciting and novel, and should be of high interest to virologists and cell biologists. The work builds on (and appropriately references) prior work in the field of influenza particle shape by the Lamb, Barclay, Garcia-Sastre, Vahey, Fletcher and Ivanovic groups. It provides new information and techniques to show that spherical virions spread faster than filamentous virions within plaques, and this advantage is not negated by cell density, the presence of mucus, or different entry inhibitors but is significantly reduced in the presence of neutralizing antibodies. It also includes other useful observations to the field (the fact that infected Calu3 cells migrate to the center of infected plaques, the fact that the entry kinetics and success rate of filaments is lower compared to spheres). Expertise: virology, influenza, virion morphology, cell biology

    __Reviewer #3 __

    Evidence, reproducibility and clarity:

    The manuscript by Peterl et al. deals with the still interesting question of why influenza A viruses are filamentous in natural isolates but adopt a spherical phenotype in cell culture. The authors generated recombinant IAV reporter viruses that display identical antigenic (HA and NA) surfaces but differ in their morphology due to expression of an M1 protein that confers a spherical or filamentous phenotype. The data show that spherical viruses exhibit increased entry kinetics and spread faster in cell culture compared to filamentous viruses and that this is also the case in the presence of mucins and at a low cell density. Interestingly, the authors found that spherical viruses are more efficiently blocked by neutralizing HA antibodies than filamentous viruses, providing an interesting advantage for the filamentous phenotype of natural IAV isolates due to antibody pressure. The manuscript is of the usual excellent quality of the working group of Petr Chlanda and the data are very interesting. The experiments are well thought out and the results are comprehensible, convincing and visually very clear. The fact that a current preprint also describes that neutralizing antibodies drives filamentous virus formation (as mentioned by the authors in the discussion) does not diminish the message and quality of this work. There were a few minor open questions that came to mind that could be included in the discussion: The authors found that the filamentous morphology was stable throughout multiple rounds of infection during plaque formation. Is this still the case even with multiple passages (e.g 10x) in cell culture or does the number of spherical particles increase at some point?

    Thank you for your positive feedback and this suggestion. We performed serial passaging of WSN and WSN-M1Udorn in MDCK cells in the presence of 1 nM MEDI8852 antibody and harvested supernatants from passage 1 and 5. Supernatants were plunge-frozen, and virion counts and morphologies were determined by cryo-electron microscopy. Data from at least 80 analyzed virions per condition showed that the overall number of spherical and filamentous virions was reduced after passage 5 under antibody pressure (Fig 5 D). However, both morphologies remained stable throughout five passages in the presence of MEDI8852 (Fig. 5 E). We did not observe an increase in spherical particles after five passages.

    The filamentous virus spreads slower in cell culture. Does NA play a role here? NA is probably distributed differently on the surface of filamentous viruses (at the tips) than on spherical viruses?

    Thank you for this comment. As correctly pointed out, NA is enriched on one side/tip of filamentous (Calder et al., 2010, doi:10.1073/pnas.1002123107) or spherical IAV as now highlighted in Figure 1 D and E (white arrowheads). This asymmetric NA distribution and the HA-NA balance have been reported to be crucial for the release of newly formed virions and their spread through the mucus layer in the airway epithelium (De Vries et al., 2019, doi: 10.1016/j.tim.2019.08.010). Additionally, we compared the role of NA in the spread of spherical and filamentous IAV by performing fluorescent plaque assays in the presence of Zanamivir, a potent NA inhibitor. Analysis of plaque growth in the presence of increasing Zanamivir concentrations showed that the spread of both IAV morphologies was inhibited to a comparable extent (Figure 4 F and extended Figure 4 C). This result suggests that the inhibition of NA enzymatic activity does not influence the IAV morphology-dependent spread. We have included this information in the results (Line 281-285) and discussion (Line 465-468).

    Reviewer #3 (Significance (Required)):

    The manuscript is of the usual excellent quality of the working group of Petr Chlanda and the data are very interesting. The experiments are well thought out and the results are comprehensible, convincing and visually very clear.

  2. Review Commons

    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 #3

    Evidence, reproducibility and clarity

    The manuscript by Peterl et al. deals with the still interesting question of why influenza A viruses are filamentous in natural isolates but adopt a spherical phenotype in cell culture. The authors generated recombinant IAV reporter viruses that display identical antigenic (HA and NA) surfaces but differ in their morphology due to expression of an M1 protein that confers a spherical or filamentous phenotype. The data show that spherical viruses exhibit increased entry kinetics and spread faster in cell culture compared to filamentous viruses and that this is also the case in the presence of mucins and at a low cell density. Interestingly, the authors found that spherical viruses are more efficiently blocked by neutralizing HA antibodies than filamentous viruses, providing an interesting advantage for the filamentous phenotype of natural IAV isolates due to antibody pressure. The manuscript is of the usual excellent quality of the working group of Petr Chlanda and the data are very interesting. The experiments are well thought out and the results are comprehensible, convincing and visually very clear. The fact that a current preprint also describes that neutralizing antibodies drives filamentous virus formation (as mentioned by the authors in the discussion) does not diminish the message and quality of this work. There were a few minor open questions that came to mind that could be included in the discussion: The authors found that the filamentous morphology was stable throughout multiple rounds of infection during plaque formation. Is this still the case even with multiple passages (e.g 10x) in cell culture or does the number of spherical particles increase at some point? The filamentous virus spreads slower in cell culture. Does NA play a role here? NA is probably distributed differently on the surface of filamentous viruses (at the tips) than on spherical viruses?

    Significance

    The manuscript is of the usual excellent quality of the working group of Petr Chlanda and the data are very interesting. The experiments are well thought out and the results are comprehensible, convincing and visually very clear.

  3. Review Commons

    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 #2

    Evidence, reproducibility and clarity

    Summary:

    This manuscript by Peteryl and colleagues explores the question of why some influenza viruses (typically those that have been recently isolated from animals, though also the Udorn strain) produce filamentous particles, while influenza viruses that have been adapted to eggs or cell culture form spherical particles. This is a long standing question in the influenza field, and the authors have used a nice set of new tools and approaches to shed light on this question. They created mScarlet labelled viruses that produce spherical (WSN) or predominantly filamentous (WSN with an M segment from Udorn) virions, but share the same glycoproteins. While this approach is not novel (the fact that the segment 7 of Udorn drives a filamentous phenotype has been previously demonstrated), the authors used these viruses in an elegant series of experiments to look at the rate of cell to cell spread within a plaque to show that the spherical viruses spread more quickly. The authors then explored the effect of cell density, inhibitors designed to inhibit different routes of viral entry, and the presence of neutralizing antibody. The experiments are thoughtfully designed, and the electron microscopy in particular is beautifully done. In general, the conclusions are supported by the data, though the specific claim that filamentous viruses have an advantage in viral entry in the presence of neutralizing antibody would be significantly strengthened by performing the specific entry assay the authors employ earlier in the manuscript.

    Major comments:

    The key conclusions are largely convincing, though the authors should perform the entry assays they employ in figure 3 (measuring the kinetics of entry and the efficiency of entry) to determine whether the delay in cell to cell spread they observe for spherical viruses in the presence of neutralizing antibody is due specifically to the effect on entry. I also am concerned about the method used to determine that the antibody treatment in Fig 5D-H results in a difference in the number of virions produced. While I appreciate that SEM is time consuming and difficult to quantify, counting the number of virions seen in a single field of view from 7 or 12 cells does not provide a robust foundation to support the central claim of the paper, that the difference in speed of filamentous and spherical viral spread is due to a difference in their ability to support viral entry in the presence of neutralizing antibody . If the authors wish to count virions produced by the WSN/WSN M-Udorn viruses in the presence/absence of neutralizing antibody it would be sensible to perform a synchronized high MOI infection and measure infectious titer by plaque assay (as this would be able to quickly and easily measure millions of virions produced by hundreds of thousands of cells).

    The two entry assays could be done in parallel and I anticipate them to take ~3 days per replicate (a day to seed, a day to infect/add NH4Cl at the indicated time points and fix, a day to image and analyze data). Similarly, infected cells at high MOI in the presence/absence of nAb, collecting viral supernatants, and tittering by plaque assay should take ~one week. The reagents to perform these experiments are already in hand, and as the costs will be limited to standard tissue culture reagents, using a microscopy set up the authors already possess. The experiments throughout the paper are well described, with appropriate methodological detail and statistical analysis.

    Minor comments:

    • Viruses without the mScarlet spread faster, the WSN-Udorn has more viruses with mScarlet than the WSN does so how do we know that some of the difference isn't down to that?
    • While Calu3 cells are reported to make mucus the authors should verify the expression of relevant mucus proteins in their hands, and this phenotype can be variable depending on culture conditions.
    • In 5F and I does 'mock' mean no antibody or no virus?
    • The authors should either include data to support the claim in line 410: "Our data provide further evidence that IAV filamentous morphology is lost to accelerate cell-to-cell spread by faster entry kinetics and to achieve higher entry efficiency" or reword this sentence, since at present this manuscript does not include experiments demonstrating the loss of filamentous morphology in tissue culture of the WSN-M1 Udorn virus.

    Significance

    The data and conclusions presented in this manuscript are exciting and novel, and should be of high interest to virologists and cell biologists. The work builds on (and appropriately references) prior work in the field of influenza particle shape by the Lamb, Barclay, Garcia-Sastre, Vahey, Fletcher and Ivanovic groups. It provides new information and techniques to show that spherical virions spread faster than filamentous virions within plaques, and this advantage is not negated by cell density, the presence of mucus, or different entry inhibitors but is significantly reduced in the presence of neutralizing antibodies. It also includes other useful observations to the field (the fact that infected Calu3 cells migrate to the center of infected plaques, the fact that the entry kinetics and success rate of filaments is lower compared to spheres).

    Expertise: virology, influenza, virion morphology, cell biology

  4. Review Commons

    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

    This manuscript by Peterl and colleagues seeks to understand the long-standing observation that influenza A virus generally exhibits a filamentous phenotype in vivo which is lost upon serial passaging in vitro or in embryonated chicken eggs. In addressing this question, the authors perform a detailed quantitative comparison of how filamentous and spherical strains of influenza spread in cell culture in the presence or absence of perturbations including neutralizing antibodies, mucin, and disruption of cell-cell junctions.

    The manuscript reports several observations that will be of interest to researchers in the area of influenza virus morphology and spread. Using a combination of imaging modalities, the authors convincingly demonstrate that spherical strains of influenza virus produce larger plaques than filamentous strains that are isogenic except for mutations in M1. The authors show that this is at least partly attributable to differences in entry kinetics. The authors also recapitulate a prior finding that filamentous viruses are more resistant to neutralizing antibodies than spherical ones. In most cases, the authors' claims are supported by the data presented. A few partial exceptions are noted below.

    The paper would be strengthened by a clearer description of some of the experimental approaches which lack important details in some instances. The scope of the paper is also limited somewhat by the use of immortalized cell lines that lack physiological features of the airway epithelium. Although this limitation is understandable from a technical standpoint, a discussion of these limitations should be included. Specific comments are listed below.

    Major Points

    In Figure 4, it is not stated at what time the cell density is measured in panel B, and how this might change across the time points sampled in panel C. This would make the experiment difficult to reproduce. This could be a very important consideration if the cells reach confluency soon after the infection is initiated, since the plaque sizes seem statistically similar out to 24hpi in 4B.

    In Figure 4F, it appears that plaque sizes for M1Ud are less affected by mucin than M1WSN plaques at all concentrations tested. However, the authors conclude that "mucin did not show any IAV morphology-dependent inhibitory effect as indicated by the slopes of linear fits of the plaque diameters" (Line 265). I understand that the authors are looking for dose-dependent effects, but it is not clear to me why an analysis based on the slope is preferable, especially when the response to mucins may not be linear. How does the availability of IAV receptors in the porcine gastric mucin used here compare to human airway mucins? Finally, the authors should clarify the number of replicates for this experiment.

    One key difference between the cells used here and the airway epithelium is the presence of multiciliated cells that could alter viral transport in ways that depend on morphology and may be difficult to predict. I appreciate that this concept is outside the scope of the current work, but it is an important point that warrants mention.

    Minor Points

    It is somewhat unclear what is being captured in the data in Figure 5D-I. I assume that the cell surfaces that are imaged here are from infected cells within the plaque. If this is the case, it is difficult to tell whether the particles that are being quantified are incoming viruses or viruses that are currently budding. MEDI8852 is a stalk-binding antibody which would not be expected to inhibit viral attachment. This is unlikely to change the interpretation since the data shows differences between spherical and filamentous strains. However, a clearer description of this data would be helpful.

    For experiments in Calu-3 cells, is trypsin added to the culture media following infection? If not, what percentage of HA is proteolytically cleaved? I would expect these cells to express activating proteases, but if activation is less efficient, this could favor the filamentous strain (as discussed in ref 49).

    The schematic in Figure 4D illustrates mucins as tethered to the cell surface. This does not reflect the experiments in Figure 4E and F, where secreted mucins are added to the overlay media.

    There are a few small typos. Line 61: "to results in" and Line 111: "neutralizing antibodies against hemagglutinin are more effectively blocking virions with spherical morphology."

    Significance

    A strength of this manuscript is the quantitative rigor of the approaches used, which reveal interesting differences in the spread of filamentous and spherical influenza. These differences are compelling, but are limited somewhat in their significance by the difficulty of evaluating whether or not some of the observations would be preserved in differentiated airway epithelial cells. The authors do not over-generalize their conclusions, but more detailed discussion of these potential limitations is warranted.

  5. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14941493.

    We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: 

    Morphology-dependent entry kinetics and spread of influenza A virus

    Sarah Peterl, Carmen M. Lahr, Carl N. Schneider, Janis Meyer, Xenia Podlipensky, Vera Lechner, Maria Villiou, Larissa Eis, Steffen Klein, Charlotta Funaya, Elisabetta Ada Cavalcanti-Adam, Frederik Graw, Christine Selhuber-Unkel, Karl Rohr, Petr ChlandabioRxiv 2024.08.01.605992; doi: https://doi.org/10.1101/2024.08.01.605992

    We will adhere to the Universal Principled (UP) Review guidelines proposed in: 

    Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029 

    SUMMARY: Influenza A viruses (IAVs) display filamentous and spherical morphologies in nature, but functional differences between these morphologies remain largely understudied, in part because cell culture adaptations are typically accompanied by commitment to a spherical morphology. This report describes new research methods to study the biological significance of IAV spherical and filamentous morphologies in vitroand in vivo. Using reverse genetics, the authors created IAV WSN/33(H1N1) reporter viruses encoding mScarlet, with M1 genes that support either spherical or filamentous morphology, to enable direct comparisons of the biological effects of different virus morphologies on otherwise identical backgrounds. Using a variety of assays, they demonstrate that spherical virions enter cells and spread faster than filamentous virions. By contrast, filamentous virions, bearing more hemagglutinin copies per virion, were better able to evade neutralizing antibodies. These studies provide a starting point for further investigation using this approach.

    OVERALL ASSESSMENT: This report describes functional differences between spherical and filamentous IAV morphologies in controlled settings, building upon previous studies. The central claims of the manuscript are generally well supported by the data. We identified some areas where quantitative analyses and data presentation can be strengthened.

    STRENGTHS: The manuscript is generally well written. Discrete control of virion morphology through M1 mutagenesis was viewed as a strength, and these tools will undoubtedly be used by others to advance the field. For the most part, advanced microscopy methods yielded informative, high-quality data. Phenotypes were confirmed in multiple cell lines, demonstrating a certain extent of generalizability in the findings.

    WEAKNESSES: The mScarlet reporter gene was lost from a significant proportion of viruses, as evidenced by plaque analysis, and mScarlet-deficient plaques were larger than mScarlet expressing plaques. The authors acknowledged this shortcoming but persisted in using these reporter viruses for the study, instead of further reporter gene optimization. As there were several assays that did not rely on the fluorescent reporter, reverting to reporter gene-deficient parental viruses could have been an alternative approach to power most of the study. Statements about numbers of viral particles in endosomes in Figure 3 would have been better supported by quantitative data rather than representative images. While it is interesting to observe filamentous viral particles in one endosome, this single observation does not provide sufficient insight to draw conclusions about the fate of filamentous virions in endosomes. The authors should clearly indicate the basis for estimates of number of HA proteins per virion, which are based on virion size rather than any direct measurements; we discussed the potential for flow virometry approaches for single virion analysis of HA levels. Some figures lacked statistical analysis, and overall, statistical methods could have been better described throughout.

    DETAILED U.P. ASSESSMENT: 

    OBJECTIVE CRITERIA (QUALITY) 

    1.   Quality: Experiments  

    ·     Figure by figure, do experiments, as performed, have the proper controls?

    ·     Overall, these experiments appear to be properly controlled.

    ·     Are specific analyses performed using methods that are consistent with answering the specific question?  

    ·     It is unclear on why specific cell lines were used for specific experiments (e.g. A549 cells in Figure 3 and MDCK cells elsewhere). Rationale for cell line choice should be provided.

    ·     The use of the fluorescent reporter virus, which was unstable and had a plaque formation defect relative to WT parental virus, was not properly justified. Further assay development and refinement could have mitigated these drawbacks for reporter viruses, or they could have been dispensed with entirely, as several assays did not require a reporter gene. 

    ·     Is there appropriate technical expertise in the collection and analysis of data presented?

    ·       Yes

    ·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?  

    ·   There were a number of instances where quantitative methods were not performed, which undermined the authors' conclusions.

    ·   Flow virometry could provide a beneficial alternative approach to study HA content and particle morphology. Here are some relevant studies that demonstrate this approach:

    https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpz1.368

    https://journals.asm.org/doi/10.1128/jvi.01765-17

    https://journals.asm.org/doi/10.1128/jvi.01717-24

    ·     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.  

    ·       Citation of one source to justify inference of HA content based on virion size in Figure 5. This approach could be better supported.

    2.   Quality: Completeness  

    ·     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. 

    ·       In the abstract, the authors state that they show "cellular junction integrity... do[es] not exert morphology-dependent inhibition of IAV cell-to-cell spread" – this would be in reference to Fig 4 B-C where they show that knocking out catenin doesn't change the performance of the filamentous virions relative to the spherical. Is this really a measure of the importance of cellular junction integrity, or is it showing that even when cells are growing farther apart/at a lower density, the spherical virions are still better?

    ·     Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. 

    ·   N/A.

    3. Quality: Reproducibility  

    ·     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

    ·       The observation of single endosome with filamentous particles in it (Fig. 3G) clearly does not adhere to these principles of quantitative analysis. The reader should be made aware that this was a single event and cannot be broadly interpreted as a generalizable phenotype without further validation.

    ·     Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

    ·       Yes

    ·     If a ''discovery' dataset is used, has a ''validation' cohort been assessed and/or has the issue of false discovery been addressed?  

    ·       N/A

    4. Quality: Scholarship  

    ·     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? 

    ·       In this system, a slightly higher concentration of stalk-binding antibody was required to inhibit filamentous viruses compared to spherical viruses, supporting the idea that the elevated levels of HA on filamentous viruses confers an advantage. However, the plaque size phenotype persists in the presence of anti-HA antibodies, whereby the spherical viruses make much larger plaques. Given these findings, a more nuanced discussion of the effects of anti-HA antibodies on the two viruses would be beneficial. Would polyclonal anti-HA antibodies in vivo have a different effect?

    ·     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? 

    ·       We were unable to locate the citation for the previous study that demonstrated that filamentous virions contain more HA than spherical; this is important to justify the HA quantity estimation based on surface area in Figure 5A.

    ·     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section.

    ·       Overall, the Results section would benefit from greater exposition and the strategic use of summaries to emphasize take-home messages.

    ·       We identified several aspects of scholarship for potential improvement, figure-by-figure:

    ·       Fig. 1:

    ·       1C: When referring to this figure in the Results text, the authors refer to five point mutations in M1; these are more properly described as amino acid substitutions, as there are certain to be many synonymous mutations throughout.  

    ·       1D/E: These representative images should have text indicating which virus is used. They are logically placed below cartoons, but since they are separate panels, the reader needs a little more guidance.

    ·       1D/E/G: Scale bars are present, but no values are shown, and reader must refer to figure legend. Clear scale bar values on the figure are better for the reader.

    ·       1F: This data could be more effectively presented, as the current plot of virion diameter vs. length obscures the magnitude of differences. A histogram or kernel density estimation plot to visualize the length distributions would be more appropriate. In the text they discuss the mean length for the reporter viruses, but it would be helpful to show variance and maybe a t-test.

    Fig. 2. Statement in text "Uninfected cells showed numerous filopodia which have a wider and more variable diameter (69 ± 52 nm, n=10) than filamentous viruses" is not well supported by data. Quantitative methods should be clearly described.

    Fig. 3. This data would benefit from more quantitative analysis, i.e. number of virions/endosome. This approach will reinforce details like the extremely rare observation of filamentous virions (in one endosome). Also, observation of filamentous virions in one endosome seems to be a rare event considering 50% of cells were infected. Detailed analysis of 3D segmentations in Figs 3I-L should be more clearly described in the text to help the reader understand significance. In Fig. 3B, even though there are few biological replicates, variance should be reported. An appropriate statistical test would support the conclusion that spherical viruses infected more cells than filamentous viruses.

    Fig 4.

    Fig. 4A. Including an actual picture of the cells here could be helpful in addition to the cartoon, to give the reader a true sense of the cell density. This is particularly important because the quantitative density analysis reveals a large spread of data.  

    Fig 4F. It is unclear from figure and legend if these are mean radial profiles but from method and text discussing this figure leads to believe that these points are means, if so, there should also be a measure of variance included in the figure.

    Fig. 4H is not currently mentioned in the Results text; the figure label should be "focus formation" not "plaque formation".  

    Fig 5. If authors are assuming that the filamentous virions have more HAs based on their size, then they should also maybe assume that there are more NAs. If there are more NAs, then this would have been relevant to mention in the section about the mucin experiments.  

    MORE SUBJECTIVE CRITERIA (IMPACT): 

    Impact: Novelty/Fundamental and Broad Interest  

    How big of an advance would you consider the findings to be if fully supported but not extended? Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2)

    ·       We appreciated this approach to addressing knowledge gaps about the functional differences between spherical and filamentous virions. The authors position this study in context of the field, where in some cases, similar observations have been made in different systems (i.e. filamentous virions are better at escaping neutralization). This undermines the novelty of this study somewhat. Broadening this study to include an in vivo infection model (e.g. mice) would further extend these studies.

    Competing interests

    The authors declare that they have no competing interests.

  6. Version published to 10.1101/2024.08.01.605992v1 on bioRxiv