Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response

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1. Version published to 10.1128/jvi.00394-25

   Jul 22, 2025
2. 

   Review Commons

   Aug 10, 2024

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   Reviewer #1

   Evidence, reproducibility and clarity

   *Summary: *

   *The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited Alpha and Omicron SARS-CoV-2 variants in …

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   Reply to the reviewers

   Point-to-point answer to reviewers comments

   Reviewer #1

   Evidence, reproducibility and clarity

   *Summary: *

   *The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited Alpha and Omicron SARS-CoV-2 variants in Vero76 and human bronchial epithelial Calu-3 cells at micromolar concentrations. This inhibition restored autophagic flux, degrading newly synthesized viral proteins, and reduced lipid metabolism, affecting viral factories. Additionally, MK-8722 treatment increased the type I interferon (IFN-I) response. Post-infection treatment with MK-8722 efficiently suppressed viral replication and restored the IFN-I response without altering the SARS-CoV-2-specific CD8+ T cell response elicited by Spike vaccination. The authors concluded that, MK-8722 acts as an effective antiviral against SARS-CoV-2 infection, even when applied post-exposure, suggesting potential for preclinical tests to inhibit viral replication and alleviate patient symptoms. *

   __Major comments: __

   • • Are the key conclusions convincing?** Partially. See comments below! *

   *- *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   From my perspective, the title "Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response" is a clear overstatement. In no way can the authors make statements about the autophagic flux, as it simply was not measured. The study would greatly benefit from conducting an autophagy/autophagic flux assay. See specific comments below!

   Answer: As suggested by the author, we have now investigated the autophagic flux by staining the cells for LC3b expression and colocalization with the lysosomal marker LAMP1. Results are shown in the new Fig4.D and E and detailed in the results section to read lines 468-475 page 23-24:

   “To assess directly the impact of MK-8722 on the autophagic flux, Calu3 cells were infected by SARS-CoV-2 without and with MK-8722 (5uM), double labelled with LC3b and LAMP1, and co-localisation of the two marker quantified (Fig.4D). MK-8722 treatment, compared with no treatment, increased LC3b colocalization in the LAMP1 compartment as shown by the increase in MOCs in treated versus non treated infected cells (LAMP1 signal in LC3b signal 0.078±0.014 vs 0.01±0.006, Mann-Whitney pand lines 480-481 page 24:

   “This result indicates that MK-8722 restores the autophagic flux to address viral components to the lysosome, where they are degraded.”

   *- *Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   See below specific comments regarding cell line consistency and autophagy measurements.

   - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   This question depends on various factors such as access to relevant biosafety labs, availability of required reagents, etc. In my estimation, experiments involving WT viruses and autophagy measurements could be conducted within 3-4 months. The proposed experiments with the delta-N-SARS-CoV-2 mutants, of course, depend on access to such viruses. Overall, I believe all experiments could be completed within 6 months. The costs of those assays are not very high.

   - Are the data and the methods presented in such a way that they can be reproduced?

   In the present study, a total of 3 cell lines and human PBMCs were utilized for various experiments. Please indicate why each cellular model was chosen and highlight the differences between these models considering what is known for SARS-CoV-2 infection and autophagy! Furthermore, the study would greatly benefit if the key findings were consistently demonstrated in a single cell line.

   Answer: We agree with the reviewer that three cell lines and PBMC from patients were used but it was necessary for the experimental design of our experiments as detailed below.

   In Figures 1 and 2, experiments use both Vero76 and Calu3 cells for the following reasons. We first used the simian Vero76 cells in order to validate the inhibitory effect of MK-8722 in widely a used cell line in virology, but which lacks the interferon response. This lack of the IFN response renders Vero cells a poor model for the pathophysiology of SARS-CoV-2. We therefore then used the IFN-competent human lung Calu-3 cell line as a more relevant cell model.

   In Figures 3 both cells were again and western blots show a similar pattern of activation downstream AMPK after activation by MK-8722 and similar antiviral activity of MK-8722 in Vero76 and Calu3 cells. In Figure.4, we then mostly focused on deciphering the antiviral mechanism of MK-8722 and therefore focused on Calu3, which is more relevant from a pathophysiological point of view.

   In Figure 5, we then investigated the T-cell response and therefore used primary human, namely PBMC/purified CD8 T-cells from healthy donors. As the reviewer knows, T-cell activation is restricted by HLA-TCR interaction, or in other words, only matched HLA cells can activate CD8 T-cells. As HLA-A2 is the main HLA expressed in human and only a HLA-A2 antibody is available on the market, for these experiments we used only CD8 T cells from HLA-A2 patients and had to find an additional HLA-A2-expressing epithelial cell susceptible to SARS-CoV-2 infection. It is the case of Caco2 cells, which are HLA-A2+ susceptible to infection and competent for IFN responses. We could not use simian Vero cells that are IFN-deficient and do not express human MHC, nor Calu3 cells being HLA-A2 negative. Altogether, in experiments in Figure 5 addressing HLA-A2 restricted antigen presentation, the use of Caco-2 cells was appropriate in contrast to that of Vero or Calu3.

   Furthermore, for sake of clarification, we have now added the following p. 19 lines 390-392:

   “The trends in modification of all markers by MK-8722 treatment by being conserved between cell lines indicates a common antiviral mechanism. We therefore focused our study on Calu-3 cells since they are more relevant to SARS-CoV-2 infection.”

   The authors conclude that selective activation of AMPK has a pro-autophagic effect which in turn leads to a reduced SARS-CoV-2 replication. I would generally agree with this statement, but throughout the entire manuscript, no real autophagy assays are shown. This should definitely be rectified. It is important to demonstrate that (1) MK-8722 is capable of increasing autophagy and particularly autophagic flux in the cell models used, (2) that in the cell models employed, SARS-CoV-2 infection leads to modulation of autophagy, and (3) that SARS-CoV-2 infected cells, when co-treated with MK-8722, lead to a re-established autophagy. The autophagy assays should be performed according to the expert-curated guidelines by Klionsky et al. This is extremely important so that the results can be compared with the now vast number of existing autophagy-SARS-CoV-2 studies.

   We fully agree with the reviewer that it was important for our study to formally address the impact of MK-8722 on the autophagic flux. Following reviewer recommendation and reading of the guidelines for autophagy study, we therefore have evaluated whether MK-8722 affected localization of LC3b, which is essential for autophagosome biogenesis/maturation and also functions as an adaptor protein for selective autophagy, in lysosomal compartment (labelled, by LAMP1). Therefore we labelled cells infected in the presence or absence of MK-8722 for LC3b expression and colocalization with the lysosomal marker LAMP1. Results are shown in the new Fig4.D, E. Please see our above answer (p1) for results description. These results indicate that MK-8722 restored the autophagic flux that had been interrupted by SARS-CoV-2 infection in Calu3 cells.

   - Are the experiments adequately replicated and statistical analysis adequate?

   Minor comments:

   - Specific experimental issues that are easily addressable.

   Typo: Line 288: It should be "MK-8722" instead of "MK-7288".

   In general, a space between value and unit is not consistently used.

   Please indicate always the phosphosite of substrate proteins when phosphorylation is described. E.g. line 288 and throughout the manuscript: regarding ACC phosphorylation.

   Answer: We apologize for these typos, which have now been corrected in the revised MS.

   • Are prior studies referenced appropriately?

   See comment below. Fundmental work that describes the virus-autophagy relationship, such as the work by the Beth Levine lab would be important to add. Also the work bei Konstantin Sparrer and colleagues is important and leads to the current work presented here.

   Answer: Konstantin Sparrer’s work is already cited as by ref 17. However, as suggested by the reviewer, the work by the Beth Livine lab has now been added in the revised MS in p29-30 lines 610-613, to read:

   .“Convergence of Beclin-Atg14 and P62 activation could stimulated the selective clearance of viral components, in a process called virophagy64–66. We thus propose that AMPK pharmacological activation induce virophagy and is responsible, at least partially, for its antiviral effect.”

   - Are the text and figures clear and accurate?

   Introduction:* In general, for some of the statements claimed in the introduction, which is in sum nicely written, informative and well structured, citations are required. For example - line 59 "...autophagy is sequentially activated and inhibited."*

   Answer: Citation has been added, namely: Koepke 2021 Autophagy ref 17

   Lines 59-65. In the introduction, the interaction between autophagy and SARS-CoV-2 is primarily described in a one-sided manner. There are now several studies demonstrating that both inhibition of autophagy and also induction of autophagy in context of coronaviral infection. Both aspects should be illuminated and introduced here.

   Answer: The reviewer points towards a crucial point of autophagy during ß-coronaviruses infection. Indeed, we fully agree that the autophagy is both inhibited and activated, at different levels, by different proteins as exemplified by, Sparrer’s group (ref 17). As we have mentioned in the initial version of our MS: “Throughout the SARS-CoV-2 viral cycle, autophagy is sequentially activated and inhibited 17–20”. As it may have not been clear enough, we have reformulated this paragraph as follows to read line 68-75 p5:

   “Throughout the SARS-CoV-2 viral cycle, autophagy is sequentially activated and inhibited 17–20. Indeed, autophagy is initiated by the early expressed nsp6, resulting in the formation of autophagosomes that are essential for the establishment of viral factories 17,21 and subsequent viral proteins expression 17,21. In turn, viral proteins OFR3a and ORF7a expressed at a latter time post-infection, prevent the fusion between autophagosomes and lysosomes, thereby blocking completion of autophagy, as evidenced by increased LC3-B expression, activation of the ULK1 kinase and increase in the autophagy cargo receptor sequestosome-1/p62. Overall, this disruption of autophagy protects newly formed virus from degradation in the LAMP1+ lysosome 19,22–25”

   Discussion: The selectivity of the compound should be discussed.

   Answer: Manuscript have been revised as follows to discuss both specificity in regards of AMPK and tissue accessibility:

   • lines 557-560, p27: “We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which blocks infection at a µM concentration, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49”

   • Line 576-581, p28: “In contrast, MK-8722, as systemic drug, may reach these tissues with minimal side effects, as a daily treatment in diabetic Non-human primates (NHPs) with MK-8722 (10 mg/kg) for a month induced only a limited and reversible cardiac hypertrophy 36”

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   The presentation of the data is understandable. For reader with a less mechanistic background it would be helpful to present a schematic figure like a graphical abstract.

   Answer: As suggested by the reviewer, we have now introduced a graphical abstract in the revised MS.

   SECTION B – Significance

   ======================== - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   • Even though there is now a plethora of studies on autophagy and SARS-CoV-2, this study is important and of great interest to a broad readership. Not only virologists, immunologists, and autophagy researchers will eagerly anticipate this study, but especially researchers focusing on pharmacology around AMPK and autophagy will recognize the importance of the data presented here.*

   Answer: We thank the reviewer for this positive appreciation of our work.

   - Place the work in the context of the existing literature (provide references, where appropriate).

   The work builds upon a variety of studies on the interplay between coronaviruses and the mechanism of autophagy. Foundational contributions to this research stem from groundbreaking preliminary work conducted in the laboratories of Gassen and Müller (Gassen et al. 2019 and Gassen et al. 2021), as well as general virus-autophagy studies from the laboratory of Beth Levine. Additionally, recent work by Konstantin Sparrer and colleagues would be important to cite, as it underscores the insights gained in this manuscript.

   Answer: As suggested and previously mentioned, these references have now been added to the discussion p27 lines 557-560: “We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which blocks infection at a µM concentration, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49”.

   In addition, Gassen et al. 2019 is already cited as ref 18

   - State what audience might be interested in and influenced by the reported findings.* See comment above!*__ __

   Reviewer #2

   Evidence, reproducibility and clarity

   In the current manuscript, Cottignes-Calamarte et al. have shown tha pharmacological activation of AMPK can be a strategy for overcoming SARS-CoV-2 infection induced reprogramming of host degradation pathways and innate immune response, without hindering the efficacy of spike expression from vaccine agents. Though the suggestion of selective activity of MK-8722 in degrading viral proteins in infection but not the ectopic spike peptides expression is interesting, the evaluation of the mechanism and providing therapeutic index for the drug will overall improve the study.

   • Majorly, I have these suggestions;*

   1. • The manuscript did not clarify the mechanism clearly but correlated the reduction in viral proteins and their association to LAMP1 as the mechanisim of activity of MK-8722. In this way, the authors did not separate whether the reduction in SARS-CoV-2 infection could lead to the potentiation of these effects. There is mentioning of potential mechanism of the drug by inhibiting the activity of SARS-CoV-2 proteins that reduce the autophagic flux without directly showing this. SARS-CoV-2 Orf3a is a known inhibitor of autophagosome maturation and hence the authors should directly probe the activity of MK-8722 in overcoming the suppression of autophagic flux in cells by ectopically expressing Orf3a.* Answer: We fully agree with the reviewer that our work correlated the reduction in viral proteins and their association to LAMP1 to explain the mechanism of MK-8722 activity and did not focus on particular viral protein(s) that could induced autophagic flux.

   Indeed, our approach has been to describe the MK-8722 antiviral activity against full primary SARS-CoV-2 viruses (not ectopically expressed viral proteins or recombinant viruses) in a pathologically relevant cell model including primary CD8+ T cells to mimic at best the initial steps of SARS-CoV-2 infection. Although the ectopical expression of Orf3a is recognized as a potent tool to study the principle of autophagy in the guidelines of autophagy measurement methods, when applied to infection, this ectopical expression of Orf3a will modify the endogenous level of Orf3a (as compared to infection level) and probably affect by itself the autophagy kinetics. To address the question of autophagic flux as suggested by the reviewer, we therefore preferred to use another recommended technique from the guidelines of autophagy measurement methods directly applicable to infected cells. We now evaluated the colocalization of LC3b with LAMP1 during treatment and infection of Calu-3 cells by primary viruses as now described in figure 4.D and E and discussed lines 468-475 pages 23-24 :

   “To assess directly the impact of MK-8722 on the autophagic flux, Calu3 cells were infected by SARS-CoV-2 without and with MK-8722 (5uM), double labelled with LC3b and LAMP1, and co-localisation of the two marker quantified (Fig.4D, E). MK-8722 treatment, compared with no treatment, increased LC3b colocalization in the LAMP1 compartment as shown by the increase in MOCs in treated versus non treated infected cells (LAMP1 signal in LC3b signal 0.078±0.014 vs 0.01±0.006, Mann-Whitney p

   • As the authors propose MK-8722 as a preclinical candidate, they should present therapeutic measures and indexes. All across the data presented, there was no mentioning or measurement of drug toxicity for extended uses up to 36h pi.*

   Answer: Our study aimed to describe the antiviral activity of MK-8722 in a cellular model mimicking only the initial steps of infection up to 3 dpi. Complementary experiments were conducted as required and show that treatment with MK-8722 up to 10mM did not result in an increase in cell death at any time post treatment as shown in the new Fig S2I and corresponding description in line 328-332 p17, which read__: __

   __“__Furthermore, we also investigated MK-8722 toxicity at 4h, 24h and 96h (Fig. S2I) and found that the drug was not toxic up to 10mM. The 50% toxicity dose was calculated to be 57mM at 96h in Calu3 cells. This allows us to determine a therapeutic index of 76 against the SARS-CoV-2 Alpha variant and 36 against the Omicron variant, thus, placing MK-8722 as an attractive antiviral candidate.”

   • Also, it was not clarified if the drug reduced the replication or exclusively worked by restoration of autophagic flux. For the earlier, the authors can consider two assays, i.e., a direct assay of looking into the replicon of SARS-CoV-2 (Bigotti et al 2024; PMID 38387750), or looking at earlier time points of infection (up to 8h pi; Twu et al. 2021; PMID 34788596) with intracellular sgRNA specific RNA probes.In this regards, 24h or 32h (Fig 1F-G) is too long to only measure single-round of infection.*

   Answer: We thank the reviewer for this interesting question. The drug likely acts on both steps. Concerning replication, as shown in figure 1 GH at 24 and 32hrs, there is a block in replication but not in virus entry into the cell, since there is no difference in viral N cellular content after 1hr chase. Concerning the autophagic flux, the new Fig4 D and E shows that MK-8722 restores the autophagic flux in infected cells, which had been interrupted by the infection in the absence of the drug.

   The viral cycle of ß-coronaviruses is highly dependent on hijacking cellular autophagy and lipid biosynthesis, which are both necessary for the assembly of DMV, essential for viral replication. Since MK-8722 treatment reduces cellular lipid content and increases autophagic flux, the use of viral replicons deficient in structural proteins or early post-infection timepoints will most likely show a decrease in viral genome replication under the effect of MK-8722 due to the inability of the replicons to establish such favourable environments (indicated in the corresponding Fig3A, B and C). However, the effect of MK-8722 on the replicon system will not distinguish between whether MK-8722 affects the viral replication complex and whether it is unable to induce viral factory formation.

   • Are the viral components degraded by restored lysosomal activity include replicase or replication organelle components? This needs to be shown with intracellular nsp3/nsp4 levels in infection or ectopic expression.*

   Answer: The reviewer raises a question highly relevant to the biology of coronaviruses, which we have addressed in Figure 4. Indeed, the Replication-Transcription Complex (RTC) is a multifactorial complex, in which N protein bound to the viral RNA is believed to help recruiting RdRp (Cong 2020 J Virol 10.1128/jvi.01925-19, Scherer 2022 Sci Adv DOI: 10.1126/sciadv.abl4895). The increased colocalisation of in N staining in LAMP-1+ compartments observed after MK-8722 treatment indicates that MK-8722 addresses RTC to the lysosomes and therefore our results indicate that replication-transcription organelles could be degraded in lysosomes.

   • Previous studies suggested the decrease in AMPK phospharhorylation in SARS-CoV-2 infection What is inconsistent to previous studies should be commented by the authors. Eg, why is AMPK suppression not see in SARS-CoV- 2 inefction as reported before (Parthasarathy et al 2023; PMID 36417940)*

   Answer: We agree with the reviewer and had already mentioned in the discussion that SARS-CoV-2 infection can have different outcome on AMPK activation as reported in ref 45 and 46 in the original version. Indeed, Gassen and colleagues reports that activation of AMPK and resulting phosphorylation are decreased in cells infected by SARS-CoV-2, as shown in (Ref 18), whereas Parthasarathy reports an increase in AMPK phosphorylation which is clear only at 96 h p.i. (Ref 47). Interestingly in this later study at earlier time point (24hr p.i.), authors report a tendency of AMPK phosphorylation to decrease although not in a statistically significant manner but for only n=3. In our present study, no statistically significant differences were found in AMPK phosphorylation although a small increase tendency is observed. Furthermore, cells used in all these studies differ: Gassen et al. used Vero-FM cells whereas Parthasarathy et al. used intestinal Caco2 cells, and in our study Vero 76 and lung Calu3 cells. Thus the differences reported on the effect of SARS-CoV-2 infection on AMPK activation are likely due to a question of kinetics and/or cellular background. Of note, the level of AMPK phosphorylation observed after SARS-CoV-2 infection is not to compare with the AMPK phosphoryalton induced by drug activation such as MK-8722.

   This heterogenity in AMPK activation during SARS-CoV-2 infection might contribute to the various effect of AMPK activator as antiviral reported in the literature. Indeed, as mentioned p27 lines 560-563 “metformin, a drug approved by FDA since 1994, and the adenosine analogue AICAR that activates AMPK indirectly can inhibit replication of SARS-CoV-2 as well as Flaviviruses, but at a concentrations of 10 and 1 mM, respectively 35,47 ”. The reported discrepancy on the antiviral effect of AMPK activation might rely then on an activation threshold, as 10mM metformin and 1mM AICAR blocks infection, while 25__m__M AICAR does not (ref 18, 47). This later concentration was not evaluated in Gassen et al. on AMPK activation. Furthermore; the time frame evaluated in each study is different and could also be a confusing factor. Finally, as shown by Myers and colleagues (Myers 2017, Science ref 36), MK-8722 activates AMPK more efficiently than AICAR, most probably due to its direct activation of AMPK. We believe that direct AMPK activation by MK-8722 and the higher activation level of AMPK is responsible for the antiviral effect reported in our study.

   As suggested by the reviewer, we have now clarified this section in the revised MS to read p27-28, lines 557-568:

   “We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which, at a µM concentration, blocks infection, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49” .In line, metformin, a drug approved by FDA since 1994, and the adenosine analogue AICAR that activates AMPK indirectly can inhibit replication of SARS-CoV-2 as well as Flaviviruses, but at concentrations of 10 and 1 mM, respectively 35,47. These AMPK-sensitive viruses replicate all in viral factories, disturbing lipid synthesis and escaping autophagy 50,51. AMPK activation by metformin at high concentration (10mM) inhibits SARS-CoV-2 replication in vitro 47. However, AMPK activation with 5-amino-imidazolecarboxamide riboside (AICAR), a non-metabolised analogue of AMP able to activate AMPK, used at 25mM is unable to inhibit SARS-CoV-2 infection 18 while at 1mM has been proven to reduce by 10-fold viral production 47, suggesting that AMPK needs to reach an activation threshold to inhibit viral replication. “

   • Although AMPK activation is shown to be antiviral for SARS-CoV-2 before, e.g., with Metformin (Parthasarathy et al 2023; PMID 36417940), the role of AMPK-related kinases are shown to be pro-viral (e.g., NUAK2; Prasad et al. 2023; PMID 37421942). The authors should discuss these points to provide the reader a context in this sub-field.*

   Answer: As suggested by the reviewer, we have now included in the discussion the following p28 lines 568-571:

   “Conversely, NUAK2, an AMPK-related kinase, was reported to stimulate viral replication in A549 and Calu3 cells50. Altogether our results, in agreement with the literature, indicate that AMPK-dependent antiviral activity is restricted to AMPK-members only, confirming their distance with AMPK-related kinases such as NUAK2 53”

   Minor

   1. • Was there an increase of lipid droplets in SARS-CoV-2 infected cells to non-infected condition? It is not mentioned here.* Answer: Yes indeed, lipid droplets content was increased in Infected non treated cells as indicated in Figure Suppl. 4B (p21 lines 409-411) and as stated in the Result section as follows:

   ” Infection increased the overall Nile Red staining by 40±10% compared to non-infected cells (Fig. S4B, ANOVA: p

   • There are several typos and grammatical errors.*

   Answer: We have now carefully checked the text for typographical and grammatical errors, which have been corrected.

   There is no Fig S2I but is mentioend in the legend.

   Answer: We apologize for this mistake in labelling figure S2 panel and have corrected their labelling as follows:

   __“____G-H: __ACE2 expression and viability were evaluated in Vero76 cells after 24h or in Calu-3 cells after 4days of MK-8722 continuous treatment (1 µM or 5 µM respectively) by flow cytometry. ACE2 expression is expressed as MFI (G) and viability as frequency of cells non-stained by the amine-reactive dye Viobility (H). n=3 independent experiments. “

   • Fig 4A - degradation is not shown, only association.*

   Answer: We agree with the reviewer and have modified the text accordingly to read p37 lines 873-874:

   Figure 4: MK-8722 treatment increases the ____autophagic flux directing viral components ____to ____lysosomes and restores type I interferon response.”

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   • Line 333 - there is no 3d pi post exposure treatment with MK-8722 in Fig 1l.*

   Answer: We meant that treatment initiated at 1dpi lasted until harvest at 3dpi. This is clarified now to read, p18 line 351

   “Post-exposure treatment at 1dpi and until harvest at 3dpi, …”

   Significance

   General assessment*: *

   Strengths and limitations:* The study provides evidence supporting previous results that the activation AMPK can be harnessed as a strategy to combat SARS-CoV2- infection. Whereas the results suggesting that this is targeted in infection by restoration of autopahgic flux and hence is selective is interesting, but the authors did not directly investigated the SARS-CoV-2 protein that is implicated in this effect. The study also does not discuss the context of AMPK and related kinases in discussion.**

   Advance -* The study makes a pre-clinical advance, and has potential to make it fundamentally or conceptually sound. *

   Audience -* Virologists and Clinicians.*

   Expertise -* SARS-CoV-2 infection biology, Cell biology an Molecular virology*__ __

   __Reviewer #3 __

   *Evidence, reproducibility and clarity (Required): *

   • Summary.** The authors address a topic of great importance to the development of antivirals, particularly in light of the possibility of future pandemics due to hitherto understudied viruses: the evaluation of interfering with host pathways (host-directed antivirals), potentially leading to compounds that can be used against a broad spectrum of viruses which require the same host cell functions for their life cycle. The authors studied the effects of activating AMPK with a small molecule (MK-8722) on various aspects of infectivity of SARS-CoV-2. They find that the compound indeed markedly reduced viral infectivity in two cell lines (Vero and Calu3), which correlated with the ability of the compound to activate signaling downstream of AMPK and was associated with increased lysosomal genesis/function and reduced density of "lipid viral factories". *

   General assessment.* The study provides important proof of concept in reductionist cellular models, which may lead to pharmacologically more conclusive in vivo studies later on. It is limited by the use of cell lines, instead of primary human cells, for viral infectivity. The caption "MK-8722 restores the IFN I pathway" is an overstatement, as the observed increase in ISG expression is quite modest.*

   Answer: We agree with the reviewer that we did not use primary cells in our study, which was designed to evaluate the antiviral activity of MK-8722 in a simple epithelial cell model still relevant for the pathophysiology. However, we believe that MK-8722 antiviral effect we observed in the present study will be conserved using primary cells given the breadth of cell lines we used such as Vero, pulmonary Calu-3 and intestinal Caco2 cells. Furthermore, concerning the magnitude of the IFN I response we report, we remind the reviewer that Calu-3 cells are infected with an MOI of 0.05, which does not result in 100% cells infected after 24h. Consequently, the IFN-I response as limited to the sole infected cells in a limited amount in this setup and thus, bulk RT-qPCR of Type I IFN and targeted ISG mRNA is expected to remain modest.

   In future studies, as suggested by the reviewer, we plan to include few ALI culture treated primary cells. We have clarified this limitation of our study in the Discussion p. 31 line 636-638 to read:

   “The antiviral effect of AMPK activation in primary lung reconstruction and preclinical models such as hamster remains to be tested.”

   __ Significance (Required): __

   *Taken as an early proof-of-concept study, the findings are quite important to investigators aiming to develop host-directed antivirals. However, the overall interest and impact of the manuscript would greatly benefit from verifying key findings in a primary cell model. Also, checking at least one other viral species (the authors mention flaviviruses) would be helpful to test how broadly applicable the *current compound (or others to be developed in the future) would be as host-directed antivirals.

   Answer: We thank the reviewer for the positive evaluation of our work. We fully agree with the reviewer in that preparedness is the key to fighting future pandemics. However to our knowledge, the literature about Flaviviruses is already stating that AMPK activation by metformin is an antiviral strategy with regards to its lipid metabolism normalization and autophagy activation (Farfan-Morales 2021 Sci Rep doi: 10.1038/s41598-021-87707-9. Ref 50). Furthermore in a previous review, we discussed the role of AMPK activation on viral infection that could be either pro- or antiviral depending on the viral family and even within a viral family such as HSV-1 which first inhibits AMPK in early infection while activating AMPK in the latter stage (Moreira, D. et al. Curr Drug Targets 17, 942–953 (2016), ref 34). Hence, adding more viruses to our study will not add novelty to the study, even though the molecule is much stronger compared to metformin.

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   Review Commons

   Aug 10, 2024

   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

   Summary. The authors address a topic of great importance to the development of antivirals, particularly in light of the possibility of future pandemics due to hitherto understudied viruses: the evaluation of interfering with host pathways (host-directed antivirals), potentially leading to compounds that can be used against a broad spectrum of viruses which require the same host cell functions for their life cycle. The authors studied the effects of activating AMPK with a small molecule (MK-8722) on various aspects of infectivity of SARS-CoV-2. They find that the compound indeed markedly reduced viral infectivity in two cell lines …

   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

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   Referee #3

   Evidence, reproducibility and clarity

   Summary. The authors address a topic of great importance to the development of antivirals, particularly in light of the possibility of future pandemics due to hitherto understudied viruses: the evaluation of interfering with host pathways (host-directed antivirals), potentially leading to compounds that can be used against a broad spectrum of viruses which require the same host cell functions for their life cycle. The authors studied the effects of activating AMPK with a small molecule (MK-8722) on various aspects of infectivity of SARS-CoV-2. They find that the compound indeed markedly reduced viral infectivity in two cell lines (Vero and Calu3), which correlated with the ability of the compound to activate signaling downstream of AMPK and was associated with increased lysosomal genesis/function and reduced density of "lipid viral factories".

   General assessment. The study provides important proof of concept in reductionist cellular models, which may lead to pharmacologically more conclusive in vivo studies later on. It is limited by the use of cell lines, instead of primary human cells, for viral infectivity. The caption "MK-8722 restores the IFN I pathway" is an overstatement, as the observed increase in ISG expression is quite modest.

   Significance

   Taken as an early proof-of-concept study, the findings are quite important to investigators aiming to develop host-directed antivirals. However, the overall interest and impact of the manuscript would greatly benefit from verifying key findings in a primary cell model. Also, checking at least one other viral species (the authors mention flaviviruses) would be helpful to test how broadly applicable the current compound (or others to be developed in the future) would be as host-directed antivirals.

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   Referee #2

   Evidence, reproducibility and clarity

   In the current manuscript, Cottignes-Calamarte et al. have shown tha pharmacological activation of AMPK can be a strategy for overcoming SARS-CoV-2 infection induced reprogramming of host degradation pathways and innate immune response, without hindering the efficacy of spike expression from vaccine agents. Though the suggestion of selective activity of MK-8722 in degrading viral proteins in infection but not the ectopic spike peptides expression is interesting, the evaluation of the mechanism and providing therapeutic index for the drug will overall improve the study.

   Majorly, I have these suggestions;

   1. The manuscript did not …

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   Referee #2

   Evidence, reproducibility and clarity

   In the current manuscript, Cottignes-Calamarte et al. have shown tha pharmacological activation of AMPK can be a strategy for overcoming SARS-CoV-2 infection induced reprogramming of host degradation pathways and innate immune response, without hindering the efficacy of spike expression from vaccine agents. Though the suggestion of selective activity of MK-8722 in degrading viral proteins in infection but not the ectopic spike peptides expression is interesting, the evaluation of the mechanism and providing therapeutic index for the drug will overall improve the study.

   Majorly, I have these suggestions;

   1. The manuscript did not clarify the mechanism clearly but correlated the reduction in viral proteins and their association to LAMP1 as the mechanisim of activity of MK-8722. In this way, the authors did not separate whether the reduction in SARS-CoV-2 infection could lead to the potentiation of these effects. There is mentioning of potential mechanism of the drug by inhibiting the activity of SARS-CoV-2 proteins that reduce the autophagic flux without directly showing this. SARS-CoV-2 Orf3a is a known inhibitor of autophagosome maturation and hence the authors should directly probe the activity of MK-8722 in overcoming the suppression of autophagic flux in cells by ectopically expressing Orf3a.
   2. As the authors propose MK-8722 as a preclinical candidate, they should present therapeutic measures and indexes. All across the data presented, there was no mentioning or measurement of drug toxicity for extended uses up to 36h pi.
   3. Also, it was not clarified if the drug reduced the replication or exclusively worked by restoration of autophagic flux. For the earlier, the authors can consider two assays, i.e., a direct assay of looking into the replicon of SARS-CoV-2 (Bigotti et al 2024; PMID 38387750), or looking at earlier time points of infection (up to 8h pi; Twu et al. 2021; PMID 34788596) with intracellular sgRNA specific RNA probes.In this regards, 24h or 32h (Fig 1F-G) is too long to only measure single-round of infection.
   4. Are the viral components degraded by restored lysosomal activity include replicase or replication organelle components? This needs to be shown with intracellular nsp3/nsp4 levels in infection or ectopic expression.
   5. Previous studies suggested the decrease in AMPK phospharhorylation in SARS-CoV-2 infection What is inconsistent to previous studies should be commented by the authors. Eg, why is AMPK suppression not see in SARS-CoV- 2 inefction as reported before (Parthasarathy et al 2023; PMID 36417940)
   6. Although AMPK activation is shown to be antiviral for SARS-CoV-2 before, e.g., with Metformin (Parthasarathy et al 2023; PMID 36417940), the role of AMPK-related kinases are shown to be pro-viral (e.g., NUAK2; Prasad et al. 2023; PMID 37421942). The authors should discuss these points to provide the reader a context in this sub-field.

   Minor

   1. Was there an increase of lipid droplets in SARS-CoV-2 infected cells to non-infected condition? It is not mentioned here.
   2. There are several typos and grammatical errors.
   3. There is no Fig S2I but is mentioend in the legend.
   4. Fig 4A - degradation is not shown, only association.
   5. Line 333 - there is no 3d pi post exposure treatment with MK-8722 in Fig 1l.

   Significance

   General assessment

   Strengths and limitations: The study provides evidence supporting previous results that the activation AMPK can be harnessed as a strategy to combat SARS-CoV2- infection. Whereas the results suggesting that this is targeted in infection by restoration of autopahgic flux and hence is selective is interesting, but the authors did not directly investigated the SARS-CoV-2 protein that is implicated in this effect. The study also does not discuss the context of AMPK and related kinases in discussion.

   Advance - The study makes a pre-clinical advance, and has potential to make it fundamentally or conceptually sound.

   Audience - Virologists and Clinicians.

   Expertise - SARS-CoV-2 infection biology, Cell biology an Molecular virology

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

   The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited …

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

   The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited Alpha and Omicron SARS-CoV-2 variants in Vero76 and human bronchial epithelial Calu-3 cells at micromolar concentrations. This inhibition restored autophagic flux, degrading newly synthesized viral proteins, and reduced lipid metabolism, affecting viral factories. Additionally, MK-8722 treatment increased the type I interferon (IFN-I) response. Post-infection treatment with MK-8722 efficiently suppressed viral replication and restored the IFN-I response without altering the SARS-CoV-2-specific CD8+ T cell response elicited by Spike vaccination. The authors concluded that, MK-8722 acts as an effective antiviral against SARS-CoV-2 infection, even when applied post-exposure, suggesting potential for preclinical tests to inhibit viral replication and alleviate patient symptoms.

   Major comments:

   • Are the key conclusions convincing?

   Partially. See comments below!

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   From my perspective, the title "Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response" is a clear overstatement. In no way can the authors make statements about the autophagic flux, as it simply was not measured. The study would greatly benefit from conducting an autophagy/autophagic flux assay. See specific comments below!

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   See below specific comments regarding cell line consistency and autophagy measurements.

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   This question depends on various factors such as access to relevant biosafety labs, availability of required reagents, etc. In my estimation, experiments involving WT viruses and autophagy measurements could be conducted within 3-4 months. The proposed experiments with the delta-N-SARS-CoV-2 mutants, of course, depend on access to such viruses. Overall, I believe all experiments could be completed within 6 months. The costs of those assays are not very high.

   • Are the data and the methods presented in such a way that they can be reproduced?

   In the present study, a total of 3 cell lines and human PBMCs were utilized for various experiments. Please indicate why each cellular model was chosen and highlight the differences between these models considering what is known for SARS-CoV-2 infection and autophagy! Furthermore, the study would greatly benefit if the key findings were consistently demonstrated in a single cell line.

   The authors conclude that selective activation of AMPK has a pro-autophagic effect which in turn leads to a reduced SARS-CoV-2 replication. I would generally agree with this statement, but throughout the entire manuscript, no real autophagy assays are shown. This should definitely be rectified. It is important to demonstrate that (1) MK-8722 is capable of increasing autophagy and particularly autophagic flux in the cell models used, (2) that in the cell models employed, SARS-CoV-2 infection leads to modulation of autophagy, and (3) that SARS-CoV-2 infected cells, when co-treated with MK-8722, lead to a re-established autophagy. The autophagy assays should be performed according to the expert-curated guidelines by Klionsky et al. This is extremely important so that the results can be compared with the now vast number of existing autophagy-SARS-CoV-2 studies.

   • Are the experiments adequately replicated and statistical analysis adequate?

   Minor comments:

   • Specific experimental issues that are easily addressable.

   Typo: Line 288: It should be "MK-8722" instead of "MK-7288". In general, a space between value and unit is not consistently used. Please indicate always the phosphosite of substrate proteins when phosphorylation is described. E.g. line 288 and throughout the manuscript: regarding ACC phosphorylation.

   • Are prior studies referenced appropriately?

   See comment below. Fundmental work that describes the virus-autophagy relationship, such as the work by the Beth Levine lab would be important to add. Also the work bei Konstantin Sparrer and colleagues is important and leads to the current work presented here.

   • Are the text and figures clear and accurate?

   Introduction:

   In general, for some of the statements claimed in the introduction, which is in sum nicely written, informative and well structured, citations are required. For example - line 59 "...autophagy is sequentially activated and inhibited." Lines 59-65. In the introduction, the interaction between autophagy and SARS-CoV-2 is primarily described in a one-sided manner. There are now several studies demonstrating that both inhibition of autophagy and also induction of autophagy in context of coronaviral infection. Both aspects should be illuminated and introduced here.

   Discussion: The selectivity of the compound should be discussed.

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   The presentation of the data is understandable. For reader with a less mechanistic background it would be helpful to present a schematic figure like a graphical abstract.

   Significance

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   Even though there is now a plethora of studies on autophagy and SARS-CoV-2, this study is important and of great interest to a broad readership. Not only virologists, immunologists, and autophagy researchers will eagerly anticipate this study, but especially researchers focusing on pharmacology around AMPK and autophagy will recognize the importance of the data presented here.

   • Place the work in the context of the existing literature (provide references, where appropriate).

   The work builds upon a variety of studies on the interplay between coronaviruses and the mechanism of autophagy. Foundational contributions to this research stem from groundbreaking preliminary work conducted in the laboratories of Gassen and Müller (Gassen et al. 2019 and Gassen et al. 2021), as well as general virus-autophagy studies from the laboratory of Beth Levine. Additionally, recent work by Konstantin Sparrer and colleagues would be important to cite, as it underscores the insights gained in this manuscript.

   • State what audience might be interested in and influenced by the reported findings.

   See comment above!

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

   My expertise is limited to the mechanistic aspects of autophagy, metabolism, and signaling cascades related to autophagy and endosomal-exosomal mechanisms. In some studies, I have been able to investigate the interplay between CoV and autophagy in collaborations with coronavirus experts. I can only provide a superficial assessment of the purely virological (methodological) aspects of the present study.

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6. 

   Version published to 10.1101/2024.02.29.582713 on bioRxiv

   Mar 1, 2024