Lactylation of Influenza Virus Polymerase Acidic Protein Promotes Viral Replication and Pathogenicity

Reviewer #1 (Public review):

Summary:

This work characterizes the regulation of lysine lactylation on influenza A virus PA protein, and describes how this post-translational modification at residues K605/K609 facilitates asymmetric polymerase dimerization at the ANP32 interface. The authors identify ATAT1 as the host enzyme mediating PA lactylation and SIRT1 as the enzyme responsible for removing this modification. They present evidence that PA lactylation enhances viral polymerase activity and viral replication, while suppression of lactylation impairs viral growth, polymerase function, and viral pathogenicity in vivo.

Strengths:

Overall, this manuscript explores a virus-host interaction axis illustrating how host metabolic signaling modulates influenza polymerase function. These findings are likely to attract broad interest, including influenza virologists studying polymerase regulation, as well as researchers investigating the functional roles of lactylation. This mechanistic insight may also offer clues for developing host-targeted antiviral strategies.

Weaknesses:

The manuscript lacks direct experimental evidence connecting lactylation to the proposed functional mechanism. While lactylation is detected in virions and overexpression systems, it remains unclear whether lactylation dynamically modulates polymerase function during infection. It is also unknown what proportion of PA undergoes lactylation at distinct infection stages, and whether lactylation specifically takes place within replication-competent asymmetric polymerase dimers. Importantly, the authors have not shown that mutation of K605/K609 abrogates the functional effects induced by lactate supplementation or ATAT1/SIRT1 overexpression in viral replication assays, which would help establish a direct connection between lactylation and viral replication. Therefore, although a correlation exists between these residues and viral replication, a direct mechanistic link between lactylation and the proposed replication model has not been firmly established. At minimum, the authors are encouraged to acknowledge these key limitations and moderate (tone down) their conclusions. For example, the observations are consistent with, but do not definitively prove, a functional role for PA lactylation in viral genome replication.

Major points:

(1) All experiments were performed using PR8, a laboratory-adapted H1N1 strain. Although this strain is commonly used for mechanistic investigations, evidence demonstrating conservation of this mechanism in currently circulating viral strains or other subtypes of influenza viruses would substantially support the conclusion that lactylation promotes viral pathogenicity. In the absence of such data, it remains unclear whether the observed findings apply broadly or are limited to the PR8 strain. In addition, the authors are encouraged to verify these phenotypes in additional cell lines.

(2) While the authors cite published work indicating that ATAT1 possesses lactyltransferase activity, it would be valuable to clarify whether ATAT1 directly catalyzes PA lactylation or functions indirectly as an intermediate. Similar considerations apply to SIRT1 regarding its potential role in removing lactylation from PA. Direct biochemical evidence, such as in vitro modification assays, would help strengthen the proposed mechanism.

(3) The available data cannot rule out the possibility that phenotypic changes induced by K605/K609 mutations stem from structural or charge alterations independent of lactylation. In fact, the results in Figure 4A and 4B support this alternative explanation: the K609R mutant shows reduced lactylation without obvious alterations in polymerase activity. This observation raises the question of whether the functional effects of these residues are driven by modified lactylation status or merely charge alterations.

(4) The proviral effect of ATAT1 appears largely independent of its enzymatic activity (Figure 2H), making it challenging to clarify whether ATAT1 functions by modifying PA to regulate polymerase activity and viral replication. Experiments examining SIRT1 on viral replication encounter similar interpretative limitations.

(5) Several siRNA knockdown results warrant careful interpretation. In Figure 3F and Figure 2E, the knockdown efficiency of SIRT1 and ATAT1 appears limited, especially at 12 and 24 h.p.i. Additional independent experiments with improved silencing efficiency or complementary approaches (such as CRISPR knockout) would help strengthen these observations.

Reviewer #2 (Public review):

This work reveals a role for PA lactylation in influenza virus replication, and the proposed involvement of ATAT1 and SIRT1 is certainly intriguing. These observations open up new avenues for understanding how host metabolism may influence viral infection. Nevertheless, a few mechanistic issues remain to be clarified. Notably, the direct evidence for ATAT1 and SIRT1 acting as the writer and eraser of this modification is still incomplete, and the functional relevance of the identified sites could be further substantiated.

Major Comments:

(1) The direct evidence supporting ATAT1 as a PA lactyltransferase and SIRT1 as a PA delactylase is still lacking. It remains possible that these two molecules affect PA lactylation indirectly. Therefore, in vitro lactylation/delactylation assays to clarify whether ATAT1 and SIRT1 act directly on PA should be performed.

(2) Although Figure 4A shows that K605A/K609A mutations reduce PA lactylation, the use of lactylation-mimetic mutants in functional complementation assays could further strengthen the conclusion.

(3) ATAT1 and SIRT1 are known to regulate multiple substrates. Therefore, whether the viral phenotypes resulting from ATAT1/SIRT1 manipulation truly operate via PA K605/K609 remains to be demonstrated. Complementation experiments would help address this issue.

(4) The mechanistic analysis currently focuses on polymerase dimerization. IP-MS assays comparing the host protein interaction profiles of PA WT versus K605/K609 mutants could reveal whether additional host factors are involved.

(5) The downstream consequences of PA lactylation have not been explored in the context of host antiviral immunity, particularly type I interferon (IFN-I) signaling. We would suggest examining the expression of IFN-β, ISG56, and other ISGs upon infection with WT versus PA K605/K609 mutant viruses.

Author response:

We thank the editors and reviewers for their thoughtful and constructive assessment of our manuscript. We appreciate the reviewers’ insightful comments and suggestions, which will help strengthen the mechanistic rigor of our work. Below, we outline the key revisions we plan to undertake in the revised version.

Response to Reviewer 1

(1) Dynamic regulation of PA lactylation during infection.

We plan to examine PA lactylation levels at multiple time points post-infection to assess whether PA lactylation changes dynamically during the viral replication cycle.

(2) Epistasis experiments linking K605/K609 to lactate- or enzyme-dependent phenotypes.

We acknowledge that multiple viral proteins undergo lactylation and that ATAT1/SIRT1 may mediate lactylation of multiple viral proteins. We will perform viral replication assays in the context of K605/K609 mutant viruses under lactate supplementation or ATAT1/SIRT1 manipulation conditions. These experiments will allow us to assess whether the effects of lactate or ATAT1/SIRT1 manipulation on viral replication are dependent, at least in part, on PA K605/K609.

(3) Conservation across viral strains and cell lines.

We will further examine key phenotypes in additional influenza A virus subtypes (e.g., H1N1 swine influenza and H9N2 avian influenza strains) and in additional cell lines to assess the extent to which the observed mechanism is conserved beyond the PR8 laboratory-adapted strain.

(4) Direct biochemical evidence for ATAT1 and SIRT1 activity on PA.

We plan to perform in vitro lactylation and de-lactylation assays using purified recombinant PA, ATAT1, and SIRT1 proteins to investigate whether ATAT1 and SIRT1 can directly modulate PA lactylation, respectively.

(5) Disentangling lactylation from charge/structural effects.

We acknowledge the reviewer’s point that K609R shows reduced lactylation without obvious changes in polymerase activity. We will include K-to-Q substitution mutants (e.g., K605Q/K609Q) in functional assays to further assess the functional consequences of these substitutions. Although K-to-Q substitutions do not strictly mimic lysine lactylation, these mutants may help distinguish effects related to lysine charge/chemical properties from those specifically attributable to lactylation. We will also temper our conclusions and acknowledge that charge and/or structural effects may contribute independently to the observed phenotypes.

(6) Enzymatic activity dependence of ATAT1 and SIRT1.

We will further investigate the enzymatic activity-dependent versus -independent contributions of ATAT1 and SIRT1 using catalytically inactive mutants, together with the epistasis experiments described above. We will also revise the text to clarify the interpretive limitations of these experiments.

(7) Improved loss-of-function approaches.

We will complement the existing siRNA experiments with CRISPR/Cas9 knockout cell lines for ATAT1 and SIRT1 and, where feasible, repeat key assays in ATAT1- and SIRT1-knockout cells.

In the revised manuscript, we will also add a detailed methodological explanation in the figure legend and Methods section to clarify how the luciferase complementation system distinguishes asymmetric from symmetric polymerase dimers, show individual data points overlaid on bar graphs with error bars, and correct spelling errors throughout the manuscript.

Response to Reviewer 2

(1) Direct in vitro lactylation/de-lactylation assays.

As noted above, we will perform in vitro modification assays with purified proteins to investigate whether ATAT1 and SIRT1 directly modulate PA lactylation and de-lactylation, respectively.

(2) Functional assays with lysine-to-glutamine substitution mutants.

Although we recognize that K-to-Q substitutions do not strictly mimic lysine lactylation, we will generate K-to-Q substitution mutants (e.g., K605Q/K609Q) and evaluate their effects on polymerase activity and viral replication to further assess the functional relevance of these sites.

(3) Complementation experiments linking ATAT1/SIRT1 phenotypes to PA K605/K609.

As noted above, we plan to perform viral replication assays in the context of K605/K609 mutant viruses under ATAT1/SIRT1 manipulation conditions to assess whether PA K605/K609 contributes to the effects associated with ATAT1/SIRT1 manipulation.

(4) IP-MS comparison of PA WT versus mutant host interaction profiles.

Our study focuses on the mechanism by which PA lactylation modulates viral polymerase activity and replication. A comprehensive host interactome analysis via IP-MS represents a broader systematic investigation beyond the scope of this focused work. We will discuss this as an important future research direction in the revised manuscript.

(5) Exploration of host antiviral immunity downstream of PA lactylation.

This work focuses on the direct effects of PA lactylation on viral polymerase activity and replication. As the PA mutants exhibit altered replication capacity, differences in IFN/ISG expression would be largely secondary and difficult to disentangle from the direct effects of altered viral replication. We therefore consider this question beyond the scope of the current study and will add it as a future research direction in the Discussion section.

In the revised manuscript, we will also examine PA lactylation levels under increasing lactate concentrations to assess their relationship with the dose-dependent changes in viral titers. We will revise the text to clarify the interpretive limitations and, where feasible, perform endogenous co-immunoprecipitation experiments to further assess the interactions between PA and ATAT1/SIRT1 under physiological expression conditions.

We believe these revisions will strengthen the mechanistic evidence and help address the core concerns raised by both reviewers.