SIRT2 protects against Japanese encephalitis virus infection in mice

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.

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.

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.