SIRT2 protects against Japanese encephalitis virus infection in mice

  1. Perumal Arumugam Desingu
  2. Lavanya Dindi
  3. Krishnega Murugasamy
  4. Ankit Kumar Tamta
  5. Venketsubbu Ramasubbu
  6. Sukanya Raghu
  7. Amarjeet Shrama
  8. Raju S Rajmani
  9. Nagalingam R Sundaresan

  • Curated by eLife

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    eLife Assessment

    This important study explores the function of SIRT2 in inhibiting Japanese encephalitis virus infection and disease progression in rodent models. The findings presented are novel as sirtuins are known for their roles in aging, metabolism, and cell survival, but have not been studied in the context of viral infections until recently. The evidence supporting the claims is solid and reveals a mechanism for SIRT2 in modulating acetylated NF-kB transcription factor levels and additional inflammatory response mediators. The work will be of interest to biologists studying viruses, sirtuins, and inflammation.

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Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic RNA virus that causes Japanese encephalitis (JE) and poses a major threat to public health in Southeast Asia and the Western Pacific. Current strategies rely on prophylactic methods to prevent disease, as no effective antiviral therapy exists. Here, we report that SIRT2, an NAD+-dependent deacetylase enzyme, mediates antiviral activity against JEV infection in mice. Interestingly, our study reveals that SIRT2 is downregulated in JEV infection, SIRT2 genetic deficiency/small molecule inhibition increases viral yield in neuronal cells and mice brains thereby reducing the survival rate in the infected mice, whereas SIRT2 gene therapy to the JEV-infected mice by Adeno-associated virus vector reduced the JEV load in mice brains and improved the survival rate. SIRT2 deficiency activates inflammatory cytokines and chemokines response in the JEV-infected mice brains through activating NF-κB transcription factor. Mechanistically, SIRT2 deacetylates NF-κB to reduce the transcriptional factor activity of NF-κB that down-regulates the Beclin-1-mediated autophagy, which is needed for the JEV replication. Overall, the present findings establish SIRT2 as a potential regulator of JEV infection.

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  1. Version published to 10.7554/elife.106778.2 on eLife
  2. Version published to 10.7554/elife.106778 on eLife
  3. eLife

    eLife Assessment

    This important study explores the function of SIRT2 in inhibiting Japanese encephalitis virus infection and disease progression in rodent models. The findings presented are novel as sirtuins are known for their roles in aging, metabolism, and cell survival, but have not been studied in the context of viral infections until recently. The evidence supporting the claims is solid and reveals a mechanism for SIRT2 in modulating acetylated NF-kB transcription factor levels and additional inflammatory response mediators. The work will be of interest to biologists studying viruses, sirtuins, and inflammation.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    Desingu et al. show that JEV infection reduces SIRT2 expression. Upon JEV infection, 10-day old SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival. Conversely SIRT2 overexpression reduced viral titer, clinical outcomes and improved survival. Transcriptional profiling show dysregulation of NF-KB and expression of inflammatory cytokines. Pharmacological NF-KB inhibition reduced viral titer. The authors conclude that SIRT2 is a regulator of JEV infection.

    Strengths:

    This paper is novel because sirtuins have been primarily studied for aging, metabolism, stem cells/regeneration. Their role in infection has not been explored until recently. Indeed, Barthez et al. showed that SIRT2 protects aged mice from SARS-CoV-2 infection (Barthez, Cell Reports 2025). Therefore, this is a timely and novel research topic. Mechanistically, the authors showed that SIRT2 suppresses the NF-KB pathway. Interesting, SIRT2 has also been shown recently to suppress other major inflammatory pathways, such as cGAS-STING (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Together, these findings support the emerging concept that SIRT2 is a master regulator of inflammation.

    Comments on revised version.

    The authors have addressed my comments.

  5. eLife

    Reviewer #2 (Public review):

    The manuscript by Desingu et al., explores the role of SIRT2 in regulating Japanese Encephalitis Virus (JEV) replication and disease progression in rodent models. Using both an in vitro and an in vivo approach, the authors demonstrate that JEV infection leads to decreased SIRT2 expression, which they hypothesize is exploited by JEV for viral replication. To test this hypothesis, the authors utilize SIRT2 inhibition (via AGK2 or genetic knockout) and demonstrate that it leads to increased viral load and worsens clinical outcomes in JEV-infected mice. Conversely, SIRT2 overexpression via an AAV delivery system reduces viral replication and improves survival among infected mice. The study proposes a mechanism in which SIRT2 suppresses JEV-induced autophagy and inflammation by deacetylating NF-κB, thereby reducing Beclin-1 expression (an NF-κB-dependent gene) and autophagy, which the authors consider a pathway that JEV exploits for replication. Transcriptomic analysis further supports that SIRT2 deficiency leads to NF-κB-driven cytokine hyperactivation. Additionally, pharmacological inhibition of NF-κB using Bay 11 (an IKK inhibitor) results in reduced viral load and improved clinical pathology in WT and SIRT2 KO mice. Overall, the findings from Desingu et al. are generally supported by the data and suggest that targeting SIRT2 may serve as a promising therapeutic approach for JEV infection and potentially other RNA viruses that SIRT2 helps control.

    Comments on revised version.

    We believe the authors of Desingu et al. have adequately addressed the major concerns of each reviewer. Thus, we believe the current version of the manuscript is significantly improved, and the data more strongly support the authors' conclusions.

    We would like to point out that the statistics in Figure 8 are difficult to interpret and should be improved to make them useful to the reader and strengthen the paper. Also, line 70 has an extra period that should be removed.

  6. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    Desingu et al. show that JEV infection reduces SIRT2 expression. Upon JEV infection, 10-day-old SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival. Conversely, SIRT2 overexpression reduced viral titer, clinical outcomes, and improved survival. Transcriptional profiling shows dysregulation of NF-KB and expression of inflammatory cytokines. Pharmacological NF-KB inhibition reduced viral titer. The authors conclude that SIRT2 is a regulator of JEV infection.

    This paper is novel because sirtuins have been primarily studied for aging, metabolism, stem cells/regeneration. Their role in infection has not been explored until recently. Indeed, Barthez et al. showed that SIRT2 protects aged mice from SARS-CoV-2 infection (Barthez, Cell Reports 2025). Therefore, this is a timely and novel research topic. Mechanistically, the authors showed that SIRT2 suppresses the NF-KB pathway. Interestingly, SIRT2 has also been shown recently to suppress other major inflammatory pathways, such as cGAS-STING (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Together, these findings support the emerging concept that SIRT2 is a master regulator of inflammation.

    We thank the reviewer for his/her valuable time, clear summary, and critical comments on the work.

    Weaknesses:

    (1) Figures 2 and 3. Although SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival upon JEV infection, the difference is modest because even WT mice exhibited very severe disease at this viral dose. The authors should perform the experiment using a sub-lethal viral dose for WT mice, to allow the assessment of increased clinical outcomes and reduced survival in KO mice.

    We thank the reviewer for the valuable recommendation. We conducted an experiment using a sub-lethal viral dose of 5 × 10^2 PFU in 14-day-old wild-type (WT) and SIRT2 knockout (KO) mice to evaluate clinical outcomes. Our findings showed a significant reduction in survival rates among the SIRT2-KO mice compared to the wild-type mice (Figure 2O).

    (2) Figure 5K-N, the authors examined the expression of inflammatory cytokines in WT and SIRT2 KO cells upon JEV infection, in line with the dysregulation of NF-kB. It has been shown recently that SIRT2 also regulates the cGAS-STING pathway (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Do you also observe increased IFNb, IL1b, and IL18 in SIRT2 KO cells upon JEV infection? This may indicate that SIRT2 regulates systemic inflammatory responses and represents a potent protection upon viral infection. This is particularly important because in Figure 7F, the authors showed that SIRT2 overexpression reduced viral load even when NF-KB is inhibited, suggesting that NF-KB is not the only mediator of SIRT2 to suppress viral infection.

    We agree with the suggestion made by the reviewers. We have quantified the transcript levels of IFNb, IL1b, and IL18 and found that the expression of these genes was increased in JEV-infected SIRT2-KO mice compared to wild-type mice (Figure S5D-S5F). Additionally, we acknowledge the reviewers' comments regarding the role of SIRT2 in regulating systemic inflammatory responses and its significant protective effect against viral infections. Furthermore, NF-κB is not the only mediator involved in SIRT2's suppression of viral infections; other molecular mechanisms may also contribute to this process.

    Reviewer #2 (Public review):

    The manuscript by Desingu et al., explores the role of SIRT2 in regulating Japanese Encephalitis Virus (JEV) replication and disease progression in rodent models. Using both an in vitro and an in vivo approach, the authors demonstrate that JEV infection leads to decreased SIRT2 expression, which they hypothesize is exploited by JEV for viral replication. To test this hypothesis, the authors utilize SIRT2 inhibition (via AGK2 or genetic knockout) and demonstrate that it leads to increased viral load and worsens clinical outcomes in JEV-infected mice. Conversely, SIRT2 overexpression via an AAV delivery system reduces viral replication and improves survival among infected mice. The study proposes a mechanism in which SIRT2 suppresses JEV-induced autophagy and inflammation by deacetylating NF-κB, thereby reducing Beclin-1 expression (an NF-κB-dependent gene) and autophagy, which the authors consider a pathway that JEV exploits for replication. Transcriptomic analysis further supports that SIRT2 deficiency leads to NF-κB-driven cytokine hyperactivation. Additionally, pharmacological inhibition of NF-κB using Bay 11 (an IKK inhibitor) results in reduced viral load and improved clinical pathology in WT and SIRT2 KO mice. Overall, the findings from Desingu et al. are generally supported by the data and suggest that targeting SIRT2 may serve as a promising therapeutic approach for JEV infection and potentially other RNA viruses that SIRT2 helps control. However, the paper does fall short in some areas. Please see below for our comments to help improve the paper.

    We thank the reviewer's valuable recommendation. We measured NF-kB acetylation in JEV-infected Neuro-2a cells treated with Ad-null or Ad-SIRT2. Our findings show that levels of NF-kB acetylation and JEV-NS5 were reduced in the SIRT2-overexpressing cells (Figure S6).

    We conducted an experiment using a sub-lethal viral dose of 5 × 10^2 PFU in 14-day-old wild-type (WT) and SIRT2 knockout (KO) mice to evaluate clinical outcomes. Our findings showed a significant reduction in survival rates among the SIRT2-KO mice compared to the wild-type mice (Figure 2O).

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    In Figures 5C-F, the text is very small.

    Thanks for raising this point. We have increased the text size in the revised Figures 5C-F

    Reviewer #2 (Recommendations for the authors):

    Major concerns:

    (1) Line 71: The introduction does not adequately cite existing work demonstrating the role of SIRT2 in viral infections, including other RNA viruses. It would be more effective to summarize the current literature (similar to lines 162-165) rather than stating that the role of SIRT2 in RNA virus infection is largely unknown.

    We thank the reviewer for the valuable suggestion to improve the manuscript substantially. We have cited existing work demonstrating the role of SIRT2 in viral infections, including other RNA viruses, in the introduction, as suggested.

    (2) Figure 1C: The image should be quantified to state that the observation is statistically significant.

    Thank you for the valuable suggestion. We have quantified the proteins SIRT2, LC3 I, LC3 II, p62, and Beclin in Mock- and JEV-infected Neuro-2a cells, as well as in mouse brain Western blots. These results, along with representative blots (Figure 6I-6J) and n-number-related blots, are presented graphically along with p-values in supplementary figures 7A-7B. Our analysis indicates that these proteins are expressed at statistically significant levels.

    (3) Figure 1I: What tissue is 1I? not stated in the manuscript text or the figure legend.

    Thank you for pointing that out. These samples are from mouse brains. We have included this information in both the text and the figure legend of the revised manuscript.

    (4) Figure 2A: How does Sirt2 expression in WT-infected cells compare to the levels observed in the KD? Are they at comparable levels?

    Thank you for highlighting an important point. We used control siRNA to compare SIRT2 knockdown in SIRT2-siRNA-treated cells in JEV infection. In Figure 2A, “control siRNA” was labelled as “control.” In the revised Figure 2A, we updated this to read “control siRNA.”

    (5) Figure 2L-N: Clinical outcome comparing WT and SIRT2KO mice, JEV-infected mice seems modest. Can statistical analysis be performed to determine whether the results are statistically significant?

    We agree with the suggestion. Clinical signs (Figure 2L) and paralysis (Figure 2M) onset were earlier in SIRT2-KO mice compared to WT mice, and the survival curve (Figure 2N) shows statistical significance. We conducted an experiment using a sub-lethal viral dose of 5 × 10^2 PFU in 14-day-old wild-type (WT) and SIRT2 knockout (KO) mice to evaluate clinical outcomes. Our findings showed a significant reduction in survival rates among the SIRT2-KO mice compared to the wild-type mice (Figure 2O).

    (6) Figure 3: AGK2 can also inhibit other Sirtuins. Thus, to test the contribution of other Sirtuins, the experiment could be repeated using wild-type and Sirt2 KO mice.

    We agree with the suggestion. We repeated the experiment using wild-type and SIRT2-KO mice and found that the differences in survival rates were statistically insignificant between JEV-infected wild-type mice treated with AGK2 and JEV-infected SIRT2-KO mice treated with AGK2 (Figure 3K).

    (7) Figure 5: A-B: Clearly label diagrams and explain what is being compared. G-I: Please annotate genes in the heat maps and volcano plots.

    Thank you for pointing out an important aspect. The figures include several genes that may not be easily visible at readable levels in the main figure. To address this, we have enlarged the figures and labelled them with the gene names in a clearer format, as shown in the supplementary Figures S2-S4. Additionally, we have updated the figure legend for Figure 5 to include this information.

    (8) Line 230-232: "Taken together, these findings reveal that JEV infection negatively regulates SIRT2 levels, resulting in a concomitant increase in acetylated NF-κB mediated virus replication". Data does not support this statement. The experiments presented do not demonstrate that acetylated NF-kB mediates viral replication. In fact, it would be good to measure NF-kB acetylation in AdSIRT2 JEV-infected cells compared to WT-infected cells, to verify that the acetylation of NF-kB is truly linked to SIRT2 expression levels.

    We thank the reviewer for his/her valuable recommendation. We measured NF-kB acetylation in JEV-infected Neuro-2a cells treated with Ad-null or Ad-SIRT2. Our findings show that levels of NF-kB acetylation and JEV-NS5 were reduced in the SIRT2-overexpressing cells (Figure S6).

    (9) Figure 6E: The Graph is hard to interpret. It is also missing a figure legend/key, and also a misplaced A in the image.

    Thank you for highlighting an important point. We have highlighted NF-κB and other colour representations in the signalling network graph (Figure 6E). We revised the figure legend for clarity and removed the misplaced "A" from the figure.

    (10) Line 260-261: "These results indicate that SIRT2 regulates JEV replication through the NF-κB-beclin-autophagy axis.". Data does not support this statement.

    We modified the statement as follows: “These results suggest that SIRT2 regulates JEV replication through the NF-κB-autophagy axis.

    (11) Line 270: Do the authors mean decrease? Bay 11 is an NF-κB inhibitor.

    We agree with the suggestion. This is a typographical error. We have replaced “increase” with “decrease” in the revised manuscript.

    (12) Figure 8: The phenotype seems modest. Have the authors considered that Sirt2 may regulate JEV via other acylation targets?

    We agree with the reviewers' suggestion. NF-κB is not the only mediator involved in SIRT2's suppression of viral infections; other molecular mechanisms may also contribute to this process.

  7. Version published to 10.1101/2025.03.24.645101v2 on bioRxiv
  8. eLife

    eLife Assessment

    This important study explores the role of SIRT2 in regulating Japanese encephalitis virus replication and disease progression in rodent models. The findings presented are novel as sirtuins are known for their roles in aging, metabolism, and cell survival, but have not been studied in the context of viral infections until recently. The evidence supporting the claims is solid, although additional experiments to further characterize the clinical outcomes and directly test the link between acetylated NF-kB and SIRT2 expression would have strengthened the study. The work will be of interest to biologists studying viruses, sirtuins, and inflammation.

  9. eLife

    Reviewer #1 (Public review):

    Summary:

    Desingu et al. show that JEV infection reduces SIRT2 expression. Upon JEV infection, 10-day-old SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival. Conversely, SIRT2 overexpression reduced viral titer, clinical outcomes, and improved survival. Transcriptional profiling shows dysregulation of NF-KB and expression of inflammatory cytokines. Pharmacological NF-KB inhibition reduced viral titer. The authors conclude that SIRT2 is a regulator of JEV infection.

    Strengths:

    This paper is novel because sirtuins have been primarily studied for aging, metabolism, stem cells/regeneration. Their role in infection has not been explored until recently. Indeed, Barthez et al. showed that SIRT2 protects aged mice from SARS-CoV-2 infection (Barthez, Cell Reports 2025). Therefore, this is a timely and novel research topic. Mechanistically, the authors showed that SIRT2 suppresses the NF-KB pathway. Interestingly, SIRT2 has also been shown recently to suppress other major inflammatory pathways, such as cGAS-STING (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Together, these findings support the emerging concept that SIRT2 is a master regulator of inflammation.

    Weaknesses:

    (1) Figures 2 and 3. Although SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival upon JEV infection, the difference is modest because even WT mice exhibited very severe disease at this viral dose. The authors should perform the experiment using a sub-lethal viral dose for WT mice, to allow the assessment of increased clinical outcomes and reduced survival in KO mice.

    (2) Figure 5K-N, the authors examined the expression of inflammatory cytokines in WT and SIRT2 KO cells upon JEV infection, in line with the dysregulation of NF-kB. It has been shown recently that SIRT2 also regulates the cGAS-STING pathway (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Do you also observe increased IFNb, IL1b, and IL18 in SIRT2 KO cells upon JEV infection? This may indicate that SIRT2 regulates systemic inflammatory responses and represents a potent protection upon viral infection. This is particularly important because in Figure 7F, the authors showed that SIRT2 overexpression reduced viral load even when NF-KB is inhibited, suggesting that NF-KB is not the only mediator of SIRT2 to suppress viral infection.

  10. eLife

    Reviewer #2 (Public review):

    The manuscript by Desingu et al., explores the role of SIRT2 in regulating Japanese Encephalitis Virus (JEV) replication and disease progression in rodent models. Using both an in vitro and an in vivo approach, the authors demonstrate that JEV infection leads to decreased SIRT2 expression, which they hypothesize is exploited by JEV for viral replication. To test this hypothesis, the authors utilize SIRT2 inhibition (via AGK2 or genetic knockout) and demonstrate that it leads to increased viral load and worsens clinical outcomes in JEV-infected mice. Conversely, SIRT2 overexpression via an AAV delivery system reduces viral replication and improves survival among infected mice. The study proposes a mechanism in which SIRT2 suppresses JEV-induced autophagy and inflammation by deacetylating NF-κB, thereby reducing Beclin-1 expression (an NF-κB-dependent gene) and autophagy, which the authors consider a pathway that JEV exploits for replication. Transcriptomic analysis further supports that SIRT2 deficiency leads to NF-κB-driven cytokine hyperactivation. Additionally, pharmacological inhibition of NF-κB using Bay 11 (an IKK inhibitor) results in reduced viral load and improved clinical pathology in WT and SIRT2 KO mice. Overall, the findings from Desingu et al. are generally supported by the data and suggest that targeting SIRT2 may serve as a promising therapeutic approach for JEV infection and potentially other RNA viruses that SIRT2 helps control. However, the paper does fall short in some areas. Please see below for our comments to help improve the paper.

  11. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    Desingu et al. show that JEV infection reduces SIRT2 expression. Upon JEV infection, 10-day-old SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival. Conversely, SIRT2 overexpression reduced viral titer, clinical outcomes, and improved survival. Transcriptional profiling shows dysregulation of NF-KB and expression of inflammatory cytokines. Pharmacological NF-KB inhibition reduced viral titer. The authors conclude that SIRT2 is a regulator of JEV infection.

    This paper is novel because sirtuins have been primarily studied for aging, metabolism, stem cells/regeneration. Their role in infection has not been explored until recently. Indeed, Barthez et al. showed that SIRT2 protects aged mice from SARS-CoV-2 infection (Barthez, Cell Reports 2025). Therefore, this is a timely and novel research topic. Mechanistically, the authors showed that SIRT2 suppresses the NF-KB pathway. Interestingly, SIRT2 has also been shown recently to suppress other major inflammatory pathways, such as cGAS-STING (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Together, these findings support the emerging concept that SIRT2 is a master regulator of inflammation.

    Weaknesses:

    (1) Figures 2 and 3. Although SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival upon JEV infection, the difference is modest because even WT mice exhibited very severe disease at this viral dose. The authors should perform the experiment using a sub-lethal viral dose for WT mice, to allow the assessment of increased clinical outcomes and reduced survival in KO mice.

    (2) Figure 5K-N, the authors examined the expression of inflammatory cytokines in WT and SIRT2 KO cells upon JEV infection, in line with the dysregulation of NF-kB. It has been shown recently that SIRT2 also regulates the cGAS-STING pathway (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Do you also observe increased IFNb, IL1b, and IL18 in SIRT2 KO cells upon JEV infection? This may indicate that SIRT2 regulates systemic inflammatory responses and represents a potent protection upon viral infection. This is particularly important because in Figure 7F, the authors showed that SIRT2 overexpression reduced viral load even when NF-KB is inhibited, suggesting that NF-KB is not the only mediator of SIRT2 to suppress viral infection.

    We thank the reviewer for the valuable recommendation. We are willing to conduct an experiment using a sub-lethal viral dose in wild-type (WT) mice to assess increased clinical outcomes and reduced survival in knockout (KO) mice, as recommended.

    Furthermore, we acknowledge reviewers' comments that SIRT2 regulates systemic inflammatory responses and provides potent protection against viral infection. Additionally, NF-κB is not the only mediator of SIRT2's suppression of viral infection; other possible molecular mechanisms are also involved in this process.

    Reviewer #2 (Public review):

    The manuscript by Desingu et al., explores the role of SIRT2 in regulating Japanese Encephalitis Virus (JEV) replication and disease progression in rodent models. Using both an in vitro and an in vivo approach, the authors demonstrate that JEV infection leads to decreased SIRT2 expression, which they hypothesize is exploited by JEV for viral replication. To test this hypothesis, the authors utilize SIRT2 inhibition (via AGK2 or genetic knockout) and demonstrate that it leads to increased viral load and worsens clinical outcomes in JEV-infected mice. Conversely, SIRT2 overexpression via an AAV delivery system reduces viral replication and improves survival among infected mice. The study proposes a mechanism in which SIRT2 suppresses JEV-induced autophagy and inflammation by deacetylating NF-κB, thereby reducing Beclin-1 expression (an NF-κB-dependent gene) and autophagy, which the authors consider a pathway that JEV exploits for replication. Transcriptomic analysis further supports that SIRT2 deficiency leads to NF-κB-driven cytokine hyperactivation. Additionally, pharmacological inhibition of NF-κB using Bay 11 (an IKK inhibitor) results in reduced viral load and improved clinical pathology in WT and SIRT2 KO mice. Overall, the findings from Desingu et al. are generally supported by the data and suggest that targeting SIRT2 may serve as a promising therapeutic approach for JEV infection and potentially other RNA viruses that SIRT2 helps control. However, the paper does fall short in some areas. Please see below for our comments to help improve the paper.

    We thank the reviewer for the valuable recommendation. We are willing to measure NF-kB acetylation in AdSIRT2 JEV-infected cells compared to WT-infected cells, to verify that the acetylation of NF-kB is truly linked to SIRT2 expression levels as per the reviewers' suggestion.

    We are willing to conduct an experiment using a sub-lethal viral dose in wild-type (WT) mice to assess increased clinical outcomes and reduced survival in knockout (KO) mice, as recommended.

    We are accepting the reviewer's suggestion that AGK2 can also inhibit other Sirtuins. Thus, to test the contribution of other Sirtuins, the experiment could be repeated using wild-type and Sirt2 KO mice. We are willing to conduct the AGK2 experiment using JEV-infected wild-type and Sirt2 knockout mice.

  12. Version published to 10.7554/elife.106778.1 on eLife
  13. Version published to 10.1101/2025.03.24.645101v1 on bioRxiv