Caspase cleavage of influenza A virus M2 disrupts M2-LC3 interaction and regulates virion production

  1. Carmen Figueras-Novoa
  2. Masato Akutsu
  3. Daichi Murata
  4. Anne Weston
  5. Ming Jiang
  6. Beatriz Montaner
  7. Christelle Dubois
  8. Avinash Shenoy
  9. Rupert Beale

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Abstract

Influenza A virus (IAV) Matrix 2 protein (M2) is an ion channel, required for efficient viral entry and egress. M2 interacts with the small ubiquitin-like LC3 protein through a cytoplasmic C-terminal LC3-interacting region (LIR). Here, we report that M2 is cleaved by caspases, abolishing the M2–LC3 interaction. A crystal structure of the M2 LIR in complex with LC3 indicates the caspase cleavage tetrapeptide motif ( 82 SAVD 85 ) is an unstructured linear motif that does not overlap with the LIR. IAV mutant expressing a permanently truncated M2, mimicking caspase cleavage, exhibit defects in M2 plasma membrane transport, viral filament formation, and virion production. Our results reveal a dynamic regulation of the M2–LC3 interaction by caspases. This highlights the role of host proteases in regulating IAV exit, relating virion production with host cell state.

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  1. Version published to 10.1038/s44319-025-00388-7
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    Reply to the reviewers

    We thank the reviewers for their positive assessment of our manuscript. We agree that there are some further experiments suggested by the reviewers that would enhance our study. We have highlighted further proposed experimental work in bold for clarity.

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

    1. EVIDENCE, REPRODUCIBILITY AND CLARITY Summary: The Matrix 2 (M2) protein of influenza A virus (IAV) is a single pass transmembrane protein known to act as a tetrameric ion channel that is important for both viral entry and egress. The paper by Figueras-Nova et al. entitled "Caspase cleavage of Influenza A virus M2 disrupts M2-LC3 interaction and regulates virion production" reports on the regulation of IAV virion production through a regulatory interplay between a caspase cleavage site and a LC3 interacting region (LIR) motif in M2. In its C-terminal cytoplasmic tail the IAV M2 protein contains a C-terminal LIR motif interacting with LC3. The authors show that this LIR motif is preceded by a functional caspase cleavage motif cleaved predominantly by caspase-6, with some contribution from caspase-3: The motif 82-SAVD-85 directs cleavage after the aspartate (D) at position 85. The cleavage leads to loss of the remaining C terminal sequence from amino acid 86 to 97. The core LIR motif 91-FVSI-94 LIR motif is then lost from M2 which can no longer bind LC3. As previously described by the same group using point mutations in the LIR motif (Ref 12.), loss of a functional LIR., here by caspase- mediated deletion of the LIR, affects the virion production and inhibits filamentous budding. LC3B lipidation is increased upon treatment with a caspase inhibitor. The authors show for the first time that LC3 is included into IAV virions via binding to M2. Furthermore, they also report a co-crystal structure of the M2 C terminus (aa 70-97), containing the caspase cleavage site and LIR, and LC3B (aa 3-125) adding new insights into this interaction and showing that the caspase cleavage site is in a flexible region N-terminal to the LIR. This work shows how caspase cleavage may modulate LC3B lipidation, trafficking to the plasma membrane, incorporation of LC3B in the virions, filamentous budding and virion production (viral titer).

    Major comments: The findings reported here are very well supported by the data shown. This is a very clearly written paper with well described and nicely visualized results that are accompanied by adequate statistical analyses.

    We thank the reviewer for their assessment of our manuscript.

    The authors report a new way the LC3B binding to the C-terminal tail of the M2 proteins is regulated and suggest that this is an adaptation the virus has made to adjust virion production to host cell status by hijacking the function of host caspases. They show that the caspase cleavage motif is evolutionary conserved and use that as an argument. Perhaps it could be discussed if it also could be an argument that the host protects itself against a too massive virion production as this could be too detrimental to the host? Would it not also be an evolutionary advantage to the virus in the long run by avoiding killing the host?

    This is an interesting point. We agree there could be advantage for the virus not to overproduce virions under certain circumstances. Consistent with this caspase-6 deficient mice had increased mortality in response to IAV PR8 infection, and presented and increase in viral spread in the lungs (Zheng, 2021; doi: 10.1016/j.cell.2020.03.040). This is also relevant for the comments made by Reviewer 2. The manuscript will be updated to include a discussion of this point.

    A question I may raise which is optional as it may be too much work to address as part of this study is if the reported regulation of LC3B binding has any role in regulating the ion channel function of the M2 tetramer?

    It is well established that there is no impact of distal C-terminal truncations on M2 ion channel activity (Cady et al., 2009, doi: 10.1021/bi9008837 Schnell and Chou, doi: 10.1038/nature06531; Nguyen et al., 2008, doi: 10.1021/bi801315m; Tobler et al., 1999, doi: 10.1128/jvi.73.12.9695-9701.1999). This is also consistent with data from our lab (Ulferts et al., 2021, doi: 10.1016/j.celrep.2021.109899, Beale et al., 2014, doi: 10.1016/j.chom.2014.01.006) as well as others (Ren et al., 2015, doi: 10.1128/JVI.00576-15) showing the effects of the LIR motif and the proton channel are distinct. We appreciate the reviewer suggesting further work here as optional, but there is already compelling evidence to show there is no substantial effect of the LIR motif on ion channel activity. (See also Reviewer 2 points 4 and 5).

    Minor comments: Delete "with" in line 145.

    This will be changed in the updated manuscript.

    Line 217: It should be written more specifically how "cells were surface stained with M2"

    The protocol for surface staining of M2 will be explained in more detail in the updated manuscript.

    1. SIGNIFICANCE

    This is a very well performed study with a sound experimental strategy and well performed assays with clear results increasing our insight into the interplay between the Influenza A virus and host cells. Although caspase mediated cleavage of the autophagy receptor and signaling scaffold protein p62 (Ref. 25), removing the LIR and LC3-binding, has been reported before I consider this study as novel in reporting this type of regulation of LC3 binding. The cleavage of p62 deletes a large part of the protein while here it is a "clean" deletion of the LIR sequence representing a conceptual advance of regulation of LC3 binding. The study also reports for the first time on LC3B incorporated into virions. The effects on trafficking to the plasma membrane and viral budding and virion production are similar to those reported before (Ref. 12) using viruses with point mutations crippling the LIR motif. This research will be of interested to all studying virus- host interaction and to the autophagy field both as a non autophagic role of LC3B, and as a regulatory mechanism of LIR-LC3B interactions involving the irreversible caspase cleavage-mediated deletion of the LIR motif.

    We thank the reviewer for this assessment of our manuscript.

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

    The influenza A virus (IAV) M2 protein is small transmembrane protein which plays a role in virus entry and egress. In a previous study, Beale et al. (2014) identified an LC3-interacting region (LIR) in the M2 cytoplasmic domain that was found to recruit the LC3B protein to the plasma membrane. Recombinant IAV harboring mutations in the LIR motif showed reduced particle stability and lost filamentous morphology.

    In the present study, Figueras-Novoa et al. show that the LIR motif is removed in response to activation of cellular caspases. The authors demonstrate that in in IAV-infected THP-1 cells M2 is partially cleaved at the motif (82)SAVD(85)¯A by caspase 6. Caspase inhibitors abolished cleavage, and a mutant virus harboring the D85A substitution was found to be resistant to caspase action. A crystal structure of purified M2 C- terminus and LC3B revealed that the caspase cleavage site lies in a flexible region that is accessible to caspases.

    Mutant virus encoding a truncated M2 protein (M2D86-97) was unable to interact with LC3, in accordance with the absence of the LIR motif. The M2D86-97 mutant showed reduced lipidation of LC3, while enhanced lipidation of LC3 was observed when wild-type virus-infected cells were treated with caspase inhibitors. The authors also observed that cell surface transport of M2D86-97 but not M2-D85A was impaired. However, in purified virus particles a mix of cleaved and uncleaved M2 was detected. The authors also demonstrated that lipidated LC3B was present in purified virions of wild-type virus particles but even more abundant in M2-D85A virions. Finally, M2D86-97 mutants produced significantly less infectious particles compared to wild-type virus while the D85A cleavage mutant replicated to similar titers than wt virus.

    Based on these findings the authors concluded that caspases regulate the interaction of M2 protein with LC3 which impacts virion production. Specifically, they propose that caspase-mediated removal of the LIR motif may enable a switch between filamentous and non-filamentous budding in response to depletion of cellular resources. However, the authors were unable to rescue a filamentous IAV with a truncated M2 protein and therefore could not provide direct proof for their guess.

    While the data are sound and presented well, they do not support the conclusions of the authors.

    1. To the authors opinion, the conserved caspase cleavage site in the M2 protein might provide an evolutionary advantage for the virus. However, the M2-D85A mutation has no effect on viral replication, so the biological significance of why M2 needs to be cleaved at all is unclear. The conclusion that caspase-induced M2 cleavage is a fine-tuning mechanism of IAV has not been supported by experiments.

    We thank the reviewer for the assessment of our data. We think the reviewer is specifically objecting to the phrase “We conclude that this highly conserved interaction and cleavage act as a regulatory mechanism exploited by IAV to fine-tune virion production in different cellular contexts.” This is a reasonable inference from our results, but we accept that it is not proven. We will change the wording to make it clear this has not been definitively demonstrated.

    1. The finding that the permanently truncated IAV M2 mutant virus was substantially attenuated does not necessarily mean that abrogation of M2-LC3 interaction was responsible for this attenuation. As the M2 protein plays a role in virus budding at the plasma membrane (recruitment of M1 protein, induction of membrane curvature, membrane scission), the impaired transport of the truncated M2 protein might already explain that the virus was attenuated and that incorporation of the protein into the viral envelope was reduced.

    We will confirm this further with additional experiments using LIR mutants. Recapitulating the plasma membrane transport defect of truncated M2 with LIR mutants including the newly characterised M2D87A and M2D88A mutants and a more severe mutant with a FVSI_AAAA substitution would strongly imply this truncation mutant phenotype is due to the lack of LIR motif.

    1. It is also not clear whether the loss of the C-terminal 11 amino acids may have affected the interaction of the M2 protein with other proteins such as TRAPPC6A-delta (Zhu et al., 2017).

    This is a reasonable point, however Zhu et al., 2017 (https://doi.org/10.1128/jvi.01757-16) reported that the interaction with TRAPPC6A retains M2 intracellularly. If the phenotype observed with our truncation was due to the loss of interaction with TRAPPC6A, the opposite phenotype would be observed (more M2 in the plasma membrane with the truncated M2∆86-97 mutant). To address this point directly we will attempt to rescue an M2 mutant virus that has disrupted the reported TRAPPC6A binding site and assess M2 plasma membrane localization.

    The authors did not rule out whether the truncation of the M2 protein by 11 amino acids would have an effect on proton channel activity. Proton channel activity, however, might be important to preserve the metastable conformation of HA in the secretory pathway and might be also important for virus uncoating.

    M2D86-97 induced less LC3 lipidation than wild-type M2 or the D85A mutant. The remaining lipidation was attributed to the ion channel activity of the M2 protein. Can the authors rule out that the truncation of the M2 protein led to reduced ion channel activity which in turn led to reduced LC3B lipidation?

    We have addressed points 4 and 5 in response to Reviewer 1.

    The suggested role of caspase cleavage as a regulatory switch between filamentous and spherical virions (lines 304- 313) is highly speculative as long as the authors do not provide any experimental proof for it. The authors indicated that they were unable to rescue filamentous IAV with M2D86-97. However, would it be possible to use caspase inhibitors to test their hypothesis?

    We acknowledge that M2∆86-97 could not be rescued in a filamentous background. The use of caspase inhibitors would only increase the amount of full length M2 present, and does not provide an alternative strategy for increasing the proportion of truncated M2. However, since M2∆86-97 mutant could not be rescued, we will attempt to rescue additional LIR motif mutants to address this point. In particular, D87A and D88A mutants could be generated in a MUd background, as well as the F91S mutant.

    The authors used only the PR8 strain for their studies, a highly cell culture-adapted strain with spherical morphology. Are the findings obtained with this strain are also valid for others IAV strains?

    As we highlight in Figure 2I, both the caspase cleavage motif and LIR motif are highly conserved in human IAV strains. PR8 was used as it is the reverse genetic system in use and approved for use in the lab. We will attempt to address this by testing whether other IAV strains we are able to obtain also undergo caspase mediated cleavage of M2. If possible, we will obtain recent clinical isolates to show cleavage of M2 in a strain that has not adapted to cell culture.

    1. The authors mainly used the THP-1 cells for their studies, a human macrophage-like cell line. However, human IAV mostly replicate in epithelial cells of the respiratory tract and cause only abortive infections of macrophages. Why did the authors choose this cell line? Can the findings obtained with this cell line be translated to epithelial cells of the airways?

    THP-1 cells are widely used for the study of caspase activity. However, we also show M2 cleavage in MDCK cells and HAP1 cells. PR8 infection of A549 cells does not induce significant amounts of cell death in the infection time points used and, as caspase activation is linked to cell death, we did not observe M2 cleavage in this cell type. We will attempt to infect some epithelial cell types to confirm this phenotype.

    1. Minor issues:
    • Fig. 1C: There seem to be quite some differences in the cleavage efficiency of M2 between panels A, B, C, and D? Any explanations?

    Different cell types (THP-1 cells and HAP1 cells) are used for the experiments mentioned above, which accounts for the different amount of M2 cleavage.

    • Fig. 1: Panel E: The labeling of the first amino acids as aa 76 seems to be wrong!

    We thank the reviewer for pointing this out, this will be corrected in the updated manuscript.

    Line 147: ...caspase mediated disruption of the M2-LC3 interaction (Fig 2A-B). Should be Fig. 2A-C.

    This sentence was referring to Figure 2A-B, as it refers to LC3B lipidation and not the coIP. This sentence will be changed in the text to reflect the intended meaning.

    • Growth kinetics of the various mutant viruses are missing?

    __We will provide growth kinetics for the relevant mutants ____(M2D85A and M2∆86-97).______

    • Line 195: The authors speculate that aa85 is important for viral fitness: That should be demonstrated!

    This speculation is based on the very strong conservation of D85 in human IAV strains. The importance of D85 in viral fitness (permitting cleavage of M2) is only likely to be directly demonstrable in transmission models (for example ferrets) which is not feasible or justifiable.

    Reviewer #2 (Significance (Required)):

    Authors concluded that caspases regulate the interaction of M2 protein with LC3 which impacts virion production. Specifically, they propose that caspase-mediated removal of the LIR motif may enable a switch between filamentous and non-filamentous budding in response to depletion of cellular resources. However, the authors were unable to rescue a filamentous IAV with a truncated M2 protein and therefore could not provide direct proof for their guess.

    As stated in the response to the comments above, we will attempt to rescue LIR mutant viruses (____D87A and D88A) in a MUd background which would provide further support for our hypothesis. Our data has significance for the understanding of the cell biology of influenza infection as commented on by Reviewers 1 and 3.

    • Reviewer #3 (Evidence, reproducibility and clarity (Required)): Summary : In this article, the authors identify a caspase cleavage site in the influenza A virus (IAV) Matrix 2 protein (M2) that leads to a truncated form of M2 deleted from its C-term LC3-interacting region (LIR). This cleaved form of M2 is seen and accumulates starting at 12 hours post-infection. IAV expressing M2 delta 86-97 mutant, corresponding to cleaved M2, seems to disrupt LC3B localization to cell plasma membrane upon infection. The authors also show that the IAV M2 delta 86-97 has a reduced viral titer compared to IAV WT. Overall the data are quite exciting where the authors identify the specific caspase responsible for the cleavage and show the residues of M2 necessary for LC3 interaction. However, some of the data showing the consequence of the cleavage for viral replication could be better clarified.

    We thank Reviewer 3 for their kind comments and we propose further experiments to clarify the consequences of cleavage.

    Major comments:

    • In Fig3A-B, the authors seek to demonstrate that the localization of M2 to the plasma membrane requires LIR motif. However, the representative images for cell infected with the delta 86-97 mutant show relatively few cell are expressing M2 raising questions of the infectivity of this mutant virus or if the overall expression of M2 in this assay is less for the delta 86-97 mutant. The authors should consider first quantifying the ratio of M2 cell surface staining over total M2 staining and second re-evaluate the representative images chosen.

    __We will include more examples of permeabilised cells in which comparable numbers of cells are M2 positive between mutants. We will also include high-content microscopy based quantification to support this. __To clarify, we confirm that the quantification of M2 intensity in the plasma membrane is carried out relative to the number of M2 positive cells, as the reviewer agrees is the most accurate way. To avoid confusion, we will update figure legends to describe more accurately the quantification process. A comparison between surface M2 and total M2 cannot be done on an individual cell basis, as once cells are permeabilized (to look for internal M2), robust differentiation between surface and internal M2 is difficult. The above clarification and additional data should provide the necessary support for our conclusions.

    In fig3E, it is unclear what is being quantified in the graph as the legend and text lines 222-223 mention that spot intensity was measured but the y axis indicates LC3 relocalization intensity. Given LC3 is punctated particularly in the cytosol, It is unclear which spots of LC3 they are referring to. Based on the images shown, using a graph with LC3 surface staining as performed for M2 would clarify the data. The authors should clarify the reporting of these data in the results section. Additionally, the images of the control non-infected cells should be added to 3C.

    We agree with the reviewer on this point. The figure will be updated to describe more accurately what is being quantified. Additionally, images for uninfected cells in 3C will be added.

    The data in Fig4 and FigS3 need to be strengthened to be conclusive. The volcano plot in FigS3A indicates that there is more LC3B and IAV proteins in M2 D85A than M2delta86-97. However in Fig4E, both LC3 I and LC3 II are increased in virions M2 delta 86-97 compared to M2 D85A which is opposite to the authors' conclusions in lines 244-245. In other words, the total amount of lipidated LC3 is higher in virions from IAV M2 without LIR motif than M2 with LIR. LC3II/I ratio in fig4F would suggest in virions containing M2 with LIR motif, LC3B II may be preferentially incorporated compared to virions containing M2 without LIR, which incorporates both LC3B I and LC3B II. Since this is a critical point made by the authors, performing a co-immunoprecipitation of M2 D58A and M2delta86-97 in the particles and then assessing for binding of LC3 I or II would bolster their conclusions.

    Figure 4F quantifies the ratio of LC3II to LC3I in infectious particles. Another two repeats used to quantify this ratio will be shown in addition, with a better representation of increased amounts of lipidated LC3II in M2D85A infectious particles, as well as an increased LC3II/LC3I ration in said particles when compared to M2∆86-97. Because of the low yield acquired from the purification of IAV virions, performing an IP would be difficult. Even if this were technically feasible it would not prove that M2 is binding LC3 inside the virion – we do not make this claim in our paper, merely that LC3B can be detected in the purified viral particles. We will clarify this point in the revised manuscript.

    • In Fig4J, even if statistically significant, the PFU difference between M2 D85A and M2 delta86-97 is minimal, performing growth curve assay would help appreciate this difference over time. In Thp1 cells, as the authors show caspase cleavage of M2 at time point 12h 14h 16hpi etc... (fig1), they should also show PFU data at these same time points for M2 mutant D85A compared to WT and M2 delta 86-97.

    We agree with the reviewer and indeed this was a point we attempted to make in our manuscript: Figure 4J shows a statistically significant difference between the titers. However, in the text we state that, even though statistically significant, the difference is much smaller than in other titer quantifications performed. Given the nature of a plaque assay, differences of less than a log fold cannot be considered as definitively indicating biological significance. We will clarify this in a revised manuscript. We will also provide the relevant growth kinetics (as per response to Reviewer 2).

    • The title of Fig4 and FigS3 and in text line 226 should be changed as M2 incorporation into virions is not shown and not described in the text. Plus, in figS3B, the authors show that between the M2 mutants, there is no difference in the abundance of M2 and other viral proteins compared to M1.

    The title of Figures 4 and S3 will be changed to more accurately reflect all of the points made by the figure.

    • In the image shown in Fig4H the number of plaques is higher for M2delta86-97 even though the size in smaller than M2 WT. Could the authors clarify in the text of the results section how they quantify PFU in their plaque assay and if they used a size criterion when quantifying the number of plaques?

    The images of plaques are taken at different dilutions, with the M2∆86-97 image belonging to two dilutions lower than the M2WT image. We will include the calculation used for PFU/mL, which does not take into account plaque size. Furthermore, images of the whole plate, showing plaqued serial dilutions will be shown.

    • In fig3B, the legend indicates 8 hpi but on the graphs it is 9 hpi.

    We thank the reviewer for pointing out this mistake. Both should read 8 hpi, this will be corrected in the new manuscript.

    Reviewer #3 (Significance (Required)):

    The authors demonstrated that IAV M2 binding to LC3 is regulated by caspase cleavage. The authors clearly identify the cleavage site and the caspase involved: caspase 6. The cleaved form of M2 seems relevant to IAV infection as it is accumulating after 12hpi. Using a M2 mutant D85A that cannot be cleaved by caspase 6 and truncated M2 mutant delta86-97 mimicking caspase cleaved M2, the authors are able to elegantly address the role of M2 cleavage. However, the importance of M2 caspase cleavage on IAV infection is not demonstrated. Eventually, addressing the impact of the caspase cleavage of M2 LIR motif on autophagy or CASM would be interesting.

    • Advance: conceptual.
    • Audience: basic research, specialized in virology, specialized in autophagy.
    • Field of expertise: virology, autophagy.

    We agree with the reviewer that we have made a conceptual advance in our understanding of the cell biology of influenza A virus infection. We have also determined the structure of the terminal part of the M2 tail in complex with LC3B. The biological importance of the phenotypes we show are most likely in transmission of the virus between hosts, which for IAV would require animal experiments outside the scope of this study. We have demonstrated regulation of the LIR motif by caspase cleavage in a variety of ways, using cell biological and biochemical methods. IAV is a very significant human and animal pathogen, and we believe we have made an important advance in describing a host-pathogen interaction of relevance for viral egress.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this article, the authors identify a caspase cleavage site in the influenza A virus (IAV) Matrix 2 protein (M2) that leads to a truncated form of M2 deleted from its C-term LC3-interacting region (LIR). This cleaved form of M2 is seen and accumulates starting at 12 hours post-infection. IAV expressing M2 delta 86-97 mutant, corresponding to cleaved M2, seems to disrupt LC3B localization to cell plasma membrane upon infection. The authors also show that the IAV M2 delta 86-97 has a reduced viral titer compared to IAV WT. Overall the data are quite exciting where the authors identify the specific caspase responsible for the cleavage and show the residues of M2 necessary for LC3 interaction. However, some of the data showing the consequence of the cleavage for viral replication could be better clarified.

    Major comments:

    • In Fig3A-B, the authors seek to demonstrate that the localization of M2 to the plasma membrane requires LIR motif. However, the representative images for cell infected with the delta 86-97 mutant show relatively few cell are expressing M2 raising questions of the infectivity of this mutant virus or if the overall expression of M2 in this assay is less for the delta 86-97 mutant. The authors should consider first quantifying the ratio of M2 cell surface staining over total M2 staining and second re-evaluate the representative images chosen.
    • In fig3E, it is unclear what is being quantified in the graph as the legend and text lines 222-223 mention that spot intensity was measured but the y axis indicates LC3 relocalization intensity. Given LC3 is punctated particularly in the cytosol, It is unclear which spots of LC3 they are referring to. Based on the images shown, using a graph with LC3 surface staining as performed for M2 would clarify the data. The authors should clarify the reporting of these data in the results section. Additionally, the images of the control non-infected cells should be added to 3C.
    • The data in Fig4 and FigS3 need to be strengthened to be conclusive. The volcano plot in FigS3A indicates that there is more LC3B and IAV proteins in M2 D85A than M2delta86-97. However in Fig4E, both LC3 I and LC3 II are increased in virions M2 delta 86-97 compared to M2 D85A which is opposite to the authors' conclusions in lines 244-245. In other words, the total amount of lipidated LC3 is higher in virions from IAV M2 without LIR motif than M2 with LIR. LC3II/I ratio in fig4F would suggest in virions containing M2 with LIR motif, LC3B II may be preferentially incorporated compared to virions containing M2 without LIR, which incorporates both LC3B I and LC3B II. Since this is a critical point made by the authors, performing a co-immunoprecipitation of M2 D58A and M2delta86-97 in the particles and then assessing for binding of LC3 I or II would bolster their conclusions.
    • In Fig4J, even if statistically significant, the PFU difference between M2 D85A and M2 delta86-97 is minimal, performing growth curve assay would help appreciate this difference over time. In Thp1 cells, as the authors show caspase cleavage of M2 at time point 12h 14h 16hpi etc... (fig1), they should also show PFU data at these same time points for M2 mutant D85A compared to WT and M2 delta 86-97.

    Minor comments:

    • The title of Fig4 and FigS3 and in text line 226 should be changed as M2 incorporation into virions is not shown and not described in the text. Plus, in figS3B, the authors show that between the M2 mutants, there is no difference in the abundance of M2 and other viral proteins compared to M1.
    • In the image shown in Fig4H the number of plaques is higher for M2delta86-97 even though the size in smaller than M2 WT. Could the authors clarify in the text of the results section how they quantify PFU in their plaque assay and if they used a size criterion when quantifying the number of plaques?
    • In fig3B, the legend indicates 8 hpi but on the graphs it is 9 hpi.

    Significance

    The authors demonstrated that IAV M2 binding to LC3 is regulated by caspase cleavage. The authors clearly identify the cleavage site and the caspase involved: caspase 6. The cleaved form of M2 seems relevant to IAV infection as it is accumulating after 12hpi. Using a M2 mutant D85A that cannot be cleaved by caspase 6 and truncated M2 mutant delta86-97 mimicking caspase cleaved M2, the authors are able to elegantly address the role of M2 cleavage. However, the importance of M2 caspase cleavage on IAV infection is not demonstrated.
    Eventually, addressing the impact of the caspase cleavage of M2 LIR motif on autophagy or CASM would be interesting.

    • Advance: conceptual.
    • Audience: basic research, specialized in virology, specialized in autophagy.
    • Field of expertise: virology, autophagy.
  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The influenza A virus (IAV) M2 protein is small transmembrane protein which plays a role in virus entry and egress. In a previous study, Beale et al. (2014) identified an LC3-interacting region (LIR) in the M2 cytoplasmic domain that was found to recruit the LC3B protein to the plasma membrane. Recombinant IAV harboring mutations in the LIR motif showed reduced particle stability and lost filamentous morphology.

    In the present study, Figueras-Novoa et al. show that the LIR motif is removed in response to activation of cellular caspases. The authors demonstrate that in in IAV-infected THP-1 cells M2 is partially cleaved at the motif (82)SAVD(85)A by caspase 6. Caspase inhibitors abolished cleavage, and a mutant virus harboring the D85A substitution was found to be resistant to caspase action. A crystal structure of purified M2 C- terminus and LC3B revealed that the caspase cleavage site lies in a flexible region that is accessible to caspases.

    Mutant virus encoding a truncated M2 protein (M286-97) was unable to interact with LC3, in accordance with the absence of the LIR motif. The M286-97 mutant showed reduced lipidation of LC3, while enhanced lipidation of LC3 was observed when wild-type virus-infected cells were treated with caspase inhibitors. The authors also observed that cell surface transport of M286-97 but not M2-D85A was impaired. However, in purified virus particles a mix of cleaved and uncleaved M2 was detected. The authors also demonstrated that lipidated LC3B was present in purified virions of wild-type virus particles but even more abundant in M2-D85A virions. Finally, M286-97 mutants produced significantly less infectious particles compared to wild-type virus while the D85A cleavage mutant replicated to similar titers than wt virus.

    Based on these findings the authors concluded that caspases regulate the interaction of M2 protein with LC3 which impacts virion production. Specifically, they propose that caspase-mediated removal of the LIR motif may enable a switch between filamentous and non-filamentous budding in response to depletion of cellular resources. However, the authors were unable to rescue a filamentous IAV with a truncated M2 protein and therefore could not provide direct proof for their guess.

    While the data are sound and presented well, they do not support the conclusions of the authors.

    1. To the authors opinion, the conserved caspase cleavage site in the M2 protein might provide an evolutionary advantage for the virus. However, the M2-D85A mutation has no effect on viral replication, so the biological significance of why M2 needs to be cleaved at all is unclear. The conclusion that caspase-induced M2 cleavage is a fine-tuning mechanism of IAV has not been supported by experiments.
    2.  The finding that the permanently truncated IAV M2 mutant virus was substantially attenuated does not necessarily mean that abrogation of M2-LC3 interaction was responsible for this attenuation. As the M2 protein plays a role in virus budding at the plasma membrane (recruitment of M1 protein, induction of membrane curvature, membrane scission), the impaired transport of the truncated M2 protein might already explain that the virus was attenuated and that incorporation of the protein into the viral envelope was reduced.
    3. It is also not clear whether the loss of the C-terminal 11 amino acids may have affected the interaction of the M2 protein with other proteins such as TRAPPC6A-delta (Zhu et al., 2017).
    4. The authors did not rule out whether the truncation of the M2 protein by 11 amino acids would have an effect on proton channel activity. Proton channel activity, however, might be important to preserve the metastable conformation of HA in the secretory pathway and might be also important for virus uncoating.
    5. M286-97 induced less LC3 lipidation than wild-type M2 or the D85A mutant. The remaining lipidation was attributed to the ion channel activity of the M2 protein. Can the authors rule out that the truncation of the M2 protein led to reduced ion channel activity which in turn led to reduced LC3B lipidation?
    6. The suggested role of caspase cleavage as a regulatory switch between filamentous and spherical virions (lines 304- 313) is highly speculative as long as the authors do not provide any experimental proof for it. The authors indicated that they were unable to rescue filamentous IAV with M286-97. However, would it be possible to use caspase inhibitors to test their hypothesis?
    7. The authors used only the PR8 strain for their studies, a highly cell culture-adapted strain with spherical morphology. Are the findings obtained with this strain are also valid for others IAV strains?
    8. The authors mainly used the THP-1 cells for their studies, a human macrophage-like cell line. However, human IAV mostly replicate in epithelial cells of the respiratory tract and cause only abortive infections of macrophages. Why did the authors choose this cell line? Can the findings obtained with this cell line be translated to epithelial cells of the airways?

    Minor issues:

    • Fig. 1C: There seem to be quite some differences in the cleavage efficiency of M2 between panels A, B, C, and D? Any explanations?
    • Fig. 1: Panel E: The labeling of the first amino acids as aa 76 seems to be wrong!
    • Line 147: ...caspase mediated disruption of the M2-LC3 interaction (Fig 2A-B). Should be Fig. 2A-C.
    • Growth kinetics of the various mutant viruses are missing?
    • Line 195: The authors speculate that aa85 is important for viral fitness: That should be demonstrated!

    Significance

    Authors concluded that caspases regulate the interaction of M2 protein with LC3 which impacts virion production. Specifically, they propose that caspase-mediated removal of the LIR motif may enable a switch between filamentous and non-filamentous budding in response to depletion of cellular resources. However, the authors were unable to rescue a filamentous IAV with a truncated M2 protein and therefore could not provide direct proof for their guess.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The Matrix 2 (M2) protein of influenza A virus (IAV) is a single pass transmembrane protein known to act as a tetrameric ion channel that is important for both viral entry and egress. The paper by Figueras-Nova et al. entitled "Caspase cleavage of Influenza A virus M2 disrupts M2-LC3 interaction and regulates virion production" reports on the regulation of IAV virion production through a regulatory interplay between a caspase cleavage site and a LC3 interacting region (LIR) motif in M2. In its C-terminal cytoplasmic tail the IAV M2 protein contains a C-terminal LIR motif interacting with LC3. The authors show that this LIR motif is preceded by a functional caspase cleavage motif cleaved predominantly by caspase-6, with some contribution from caspase-3: The motif 82-SAVD-85 directs cleavage after the aspartate (D) at position 85. The cleavage leads to loss of the remaining C terminal sequence from amino acid 86 to 97. The core LIR motif 91-FVSI-94 LIR motif is then lost from M2 which can no longer bind LC3. As previously described by the same group using point mutations in the LIR motif (Ref 12.), loss of a functional LIR., here by caspase- mediated deletion of the LIR, affects the virion production and inhibits filamentous budding. LC3B lipidation is increased upon treatment with a caspase inhibitor. The authors show for the first time that LC3 is included into IAV virions via binding to M2. Furthermore, they also report a co-crystal structure of the M2 C terminus (aa 70-97), containing the caspase cleavage site and LIR, and LC3B (aa 3-125) adding new insights into this interaction and showing that the caspase cleavage site is in a flexible region N-terminal to the LIR. This work shows how caspase cleavage may modulate LC3B lipidation, trafficking to the plasma membrane, incorporation of LC3B in the virions, filamentous budding and virion production (viral titer).

    Major comments:

    The findings reported here are very well supported by the data shown. This is a very clearly written paper with well described and nicely visualized results that are accompanied by adequate statistical analyses. The authors report a new way the LC3B binding to the C-terminal tail of the M2 proteins is regulated and suggest that this is an adaptation the virus has made to adjust virion production to host cell status by hijacking the function of host caspases. They show that the caspase cleavage motif is evolutionary conserved and use that as an argument. Perhaps it could be discussed if it also could be an argument that the host protects itself against a too massive virion production as this could be too detrimental to the host? Would it not also be an evolutionary advantage to the virus in the long run by avoiding killing the host? A question I may raise which is optional as it may be too much work to address as part of this study is if the reported regulation of LC3B binding has any role in regulating the ion channel function of the M2 tetramer?

    Minor comments:

    Delete "with" in line 145. Line 217: It should be written more specifically how "cells were surface stained with M2" In the Introduction a description of what filamentous vs "spherical" budding is, could perhaps be included as I missed that reading through, although it comes in the end of the Discussion.

    Significance

    This is a very well performed study with a sound experimental strategy and well performed assays with clear results increasing our insight into the interplay between the Influenza A virus and host cells. Although caspase mediated cleavage of the autophagy receptor and signaling scaffold protein p62 (Ref. 25), removing the LIR and LC3-binding, has been reported before I consider this study as novel in reporting this type of regulation of LC3 binding. The cleavage of p62 deletes a large part of the protein while here it is a "clean" deletion of the LIR sequence representing a conceptual advance of regulation of LC3 binding.

    The study also reports for the first time on LC3B incorporated into virions.

    The effects on trafficking to the plasma membrane and viral budding and virion production are similar to those reported before (Ref. 12) using viruses with point mutations crippling the LIR motif. This research will be of interested to all studying virus- host interaction and to the autophagy field both as a non autophagic role of LC3B, and as a regulatory mechanism of LIR-LC3B interactions involving the irreversible caspase cleavage-mediated deletion of the LIR motif.

  6. Version published to 10.1101/2024.04.04.588074v1 on bioRxiv