Influenza virus vRNP subunits are lactylated

(A) A549 cells were infected with PR8 influenza virus at a MOI of 0.01 and subsequently treated with 25, 50 or 100 mM of oxamate. Viral titers were quantified using TCID50 assay at 24 hpi. (B) A549 cells were transfected with non-targeting (NC) or LDHA-targeting siRNA; 24 hours post-transfection, cells were infected with PR8 (MOI=0.01). Viral titers were determined at various h post-infection using TCID50 assay; LDHA knockdown efficiency, and viral NP expression were assessed by immunoblotting. (C) A549 cells were infected with PR8 (MOI=0.01) and treated with 0, 2.5, 5, 10, or 20 mM of lactate. Viral titers were determined with TCID50 assay at 24 hpi; viral protein expression was detected by immunoblotting. (D) HEK293 cells were transfected with plasmids encoding FLAG-tagged viral PB1, PB2, PA, or NP. At 24 hours post-transfection, anti-FLAG immunoprecipitation was performed, and protein lactylation was detected by immunoblotting with a Pan-L-lactyl lysine antibody. (E) A549 cells were infected with PR8 (MOI=0.01); at 24 hpi, anti-Pan-L-lactyl lysine immunoprecipitation was conducted, and enrichment of viral proteins was detected by immunoblotting with viral protein-specific antibodies. (F) A549 cells were infected with PR8 (MOI=1) and treated with 20 mM sodium lactate. At 12 hpi, anti-Pan-L-lactyl lysine immunoprecipitation was performed, and viral protein enrichment was detected using immunoblotting with viral protein-specific antibodies. Band intensities were quantified by ImageJ and normalized to that of Mock-treated group. (G) A549 cells were infected with PR8 (MOI=1) and then treated with 40 μM galloflavin; anti-Pan-L-lactyl lysine immunoprecipitation was carried out at 12 hpi, and viral protein enrichment was detected by immunoblotting with viral protein-specific antibodies. Band intensities were quantified by ImageJ and normalized to that of Mock-treated group. (H-I) Workflow for the identification of influenza virus proteins by mass spectrometry following Pan-L-lactyl lysine antibody enrichment; enriched samples in (H) were subjected to silver staining (I). Schematic diagrams were generated using BioRender.com. Data are presented shown as mean ± SD, and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

ATAT1 promotes influenza virus PA lactylation

(A-B) HEK293T cells were co-transfected with Flag-tagged PA and HA-tagged ATAT1 or empty vector; anti-FLAG (A) or anti-HA (B) co-IP assays were performed at 24 h post-transfection, co-precipitated proteins were detected by immunoblotting. (C) HEK293T cells were co-transfected with FLAG-tagged PA and either HA empty vector or HA-tagged ATAT1. At 24 h post-transfection, FLAG-tagged proteins were enriched by anti-FLAG immunoprecipitation, and PA lactylation was detected by immunoblotting with a Pan-L-lactyl lysine antibody; the band intensities were analyzed using ImageJ and normalized to empty vector-transfected group. (D) A549 cells were transfected with NC or ATAT1-targeting siRNA for 24 h, followed by influenza virus infection (MOI=1) for 12 h; lactylated proteins were enriched by a Pan-L-lactyl lysine antibody, PA enrichment was detected by immunoblotting using viral PA-specific antibodies. Band intensities were analyzed using ImageJ and normalized to NC-transfected group. (E) A549 cells were transfected with NC or ATAT1-targeting siRNA; 24 hours later, cells were infected with influenza virus at a MOI of 0.01. Viral titers were detected using TCID50 assay at various h post-infection; ATAT1 knockdown efficiency, and viral NP expression were assessed by immunoblotting. (F) HEK293T cells were transfected with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter, pRL-TK internal control, and 0, 0.25, 0.5, 1, or 2 μg of HA-tagged ATAT1. At 24 hours post-transfection, Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system, and protein expression was detected by immunoblotting. (G) HEK293T cells were co-transfected with FLAG-tagged PA and HA empty vector, HA-tagged ATAT1, or HA-tagged ATAT1-D157N. At 24 h post-transfection, FLAG-tagged proteins were enriched with an anti-FLAG antibody, and PA lactylation was detected by immunoblotting using a Pan-L-lactyl lysine antibody. Band intensities were quantified by ImageJ and normalized to that of empty vector group. (H) HEK293T cells were transfected with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter, pRL-TK internal control, and either HA-tagged ATAT1 or HA-tagged ATAT1-D157N. At 24 hours post-transfection, Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system, and protein expression was detected by immunoblotting. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

SIRT1 de-lactylates viral PA

(A-B) HEK293T cells were co-transfected with Flag-tagged PA and HA-tagged SIRT1 or empty vector; anti-FLAG (A) or anti-HA (B) co-IP assays were performed at 24 h post-transfection, co-precipitated proteins were detected by immunoblotting. (C) A549 cells were infected with influenza virus (MOI=0.01); anti-SIRT1 co-IP was performed at 24 hpi, precipitation of viral PA protein was detected by immunoblotting with viral PA-specific antibody. (D) HEK293T cells were co-transfected with FLAG-tagged PA and HA empty vector or HA-tagged SIRT1. FLAG-tagged proteins were enriched by anti-FLAG IP at 24 h post-transfection, and PA lactylation was detected by immunoblotting with a Pan-L-lactyl lysine antibody; band intensities were quantified by ImageJ and normalized to that of empty vector group. (E) A549 cells were transfected with NC or SIRT1-targeting siRNA for 24 h, followed by influenza virus infection (MOI=1) for 12 h; lactylated proteins were enriched by a Pan-L-lactyl lysine antibody, viral PA enrichment was detected by immunoblotting using viral PA-specific antibodies; band intensities were analyzed using ImageJ and normalized to NC-transfected group. (F) A549 cells were transfected with NC or SIRT1-targeting siRNA; 24 hours later, cells were infected with influenza virus at a MOI of 0.01. Viral titers were determined by TCID50 assay at various h post-infection; SIRT1 knockdown efficiency, and viral NP expression were detected by immunoblotting. (G) HEK293T cells were transfected with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter, pRL-TK internal control, and increasing concentrations of HA-tagged SIRT1. At 24 h post-transfection, Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system, and protein expression was detected by immunoblotting. (H) Schematic diagram of the SIRT1 catalytic domain and C-terminal regulatory segment. (I) HEK293T cells were co-transfected with FLAG-tagged PA and HA empty vector, HA-tagged SIRT1, or HA-tagged SIRT1-ΔCata. FLAG-tagged proteins were enriched by anti-FLAG immunoprecipitation at 24 h post-transfection, and PA lactylation was detected by immunoblotting with a Pan-L-lactyl lysine antibody. Band intensities were quantified by ImageJ and normalized to that of empty vector group. (J) HEK293T cells were co-transfected with FLAG-tagged PA and HA-tagged SIRT1 or SIRT1-ΔCata. Anti-FLAG co-IP was performed at 24 h post-transfection; co-precipitated proteins were detected by immunoblotting. (K) A549 cells were transfected with HA-tagged SIRT1 or SIRT1-ΔCata; 24 hours later, cells were infected with influenza virus at a MOI of 0.01. Viral titers were determined by TCID50 assay at 24 hpi, protein expression was detected by immunoblotting. (L) HEK293T cells were transfected with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter, pRL-TK internal control, and HA-tagged SIRT1 or SIRT1-ΔCata. Firefly and Renilla luciferase activities were measured with a dual-luciferase reporter assay system at 24 h post-transfection, and plasmid expression was detected by immunoblotting. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

K605/K609 residues of PA are critical for influenza virus replication

(A) HEK293T cells were transfected with FLAG-tagged PA-WT or PA-K356R/K358R/K361R/K362R/K605R/K609R mutants; anti-FLAG IP was performed at 24 h post-transfection, and PA lactylation was detected by immunoblotting with a Pan-L-lactyl lysine antibody. Band intensities were quantified by ImageJ and normalized to that of WT-PA-transfected group. (B) HEK293T cells were transfected with influenza virus PB1/PB2/ NP, pPol I-luc reporter, pRL-TK internal control, and either WT PA or PA carrying alanine- or arginine-substituted mutations at residues 356, 358, 361, 362, 605, or 609. At 24 hours post-transfection, Firefly and Renilla luciferase activities were measured with a dual-luciferase reporter assay system. (C) HEK293T cells were transfected with influenza virus PB1/PB2/NP, pPol I-luc reporter, pRL-TK internal control, PA-K605A/K605R/K609A mutants, and PA-C95A or PA-D108A mutant; Firefly and Renilla luciferase activities were measured at 24 h post-transfection with a dual-luciferase reporter assay system. (D) A549 cells were infected with WT or PA K605A/K605R/K609A mutant PR8 (MOI=0.01); viral titers in the supernatant were determined at 12/24/36 h post-infection by TCID50 assay. (E) A549 cells were infected with WT or PA K605A/K605R/K609A mutant PR8 (MOI=1), treated with DMSO or 100 μg/mL CHX; total cellular RNA was extracted at 2/5/8 h post-infection, and the fold-change of viral m/c/vRNA was detected by qRT-PCR with GAPDH serving as the internal control. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

K605/K609 residues of PA are vital for influenza virus pathogenicity

Six- to eight-week-old female BALB/c mice were intranasally infected with a lethal dose (10 PFU) of WT or PA-K605A/K605R/K609A mutant PR8. Body weight (A) and survival rate (B) were monitored for 14 consecutive days. (C) Viral titers in lung tissues were determined at 3 and 5 dpi by TCID50 assays. (D-E) Lung tissues were collected at 3 and 5 dpi for pathological section and hematoxylin-eosin (H&E) staining (Scale bar, 200 μm). (F) Virus distribution in the lung tissues at 5 dpi was analyzed by immunofluorescence staining (Scale bar, 200 μm). Statistical analyses were performed using unpaired Student’s t-tests for viral titers and Log-rank (Mantel-Cox) test for survival curves; **P<0.01, ***P<0.001.

Lactylation of PA at K605/K609 facilitates ANP32A-mediated polymerase asymmetric dimerization and viral genome replication

(A) H7N9 influenza virus polymerase asymmetric dimer (PDB: 8RMR) in complex with ANP32A (green) showing PA K605 and K609. (B) HEK293T cells were co-transfected with PB2, PB1-Gluc-N, ANP32A-Gluc-C, and WT or PA K605A/K605R/K609A mutants; Gaussia luciferase activity was measured at 24 h post-transfection; protein expression was detected by immunoblotting. (C) HEK293T cells were co-transfected with FLAG-ANP32A, PB1, PB2, and WT or PA K605A/K605R/K609A mutants; anti-FLAG co-IP was performed at 24 h post-transfection; co-precipitated proteins were detected by immunoblotting with targeted antibodies. Band intensities were quantified by ImageJ and normalized to that of WT-PA-transfected group. (D) HEK293T cells were co-transfected with PB2, PB1-Gluc-N, PB1-Gluc-C, and WT or PA K605A/K605R/K609A mutants; Gaussia luciferase activity was measured at 24 h post-transfection; protein expression was detected by immunoblotting. (E) ANP32A/B double-knockout HEK293T cells were co-transfected with PB2, PB1-Gluc-N, PB1-Gluc-C, WT or PA K605A/K605R/K609A mutants; Gaussia luciferase activity was measured at 24 h post-transfection. (F-G) HEK293T cells were transfected with PB1/PB2/NP, Pol I-viral segment 5 vRNA (F) or Pol I- viral segment 5 cRNA (G), WT or PA K605A/K605R/K609A mutants; total RNA was extracted at 18 h post-transfection, and viral cRNA/vRNA levels were quantified by qPCR (GAPDH as internal control). (H-I) HEK293T cells were co-transfected with PB2, PB1-Gluc-N, ANP32A-Gluc-C, PA, and HA-ATAT1 (H) or HA-SIRT1 (I); Gaussia luciferase activity was measured at 24 h post-transfection; protein expression was detected by immunoblotting. (J-K) HEK293T cells were transfected with PA/PB1/PB2/NP, Pol I- viral segment 5 vRNA, and HA-ATAT1 (J) or HA-SIRT1 (K); total RNA was extracted at 18 h post-transfection, and viral cRNA levels were quantified by qPCR (GAPDH as internal control). Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

Schematic model for PA lactylation-driven viral polymerase asymmetric dimerization and influenza virus replication

ATAT1 promotes, whereas SIRT1 inhibits viral PA lactylation. Lactylation of PA at lysine residues K605/K609 facilitates the ANP32-mediated assembly of viral polymerase asymmetric dimers, which promotes viral genome replication and progeny viral production. Schematic diagram was generated using BioRender.com.

Influenza virus vRNP subunits are lactylated.

(A) A549 cells were treated with 0, 50, 100, or 200 mM of oxamate for 24 h; cell viability was measured by CCK-8 assay and normalized to that of non-treated cells. (B) A549 cells were treated with 0, 1, 2,5, 5, 10, 20, 25, or 30 mM of lactate for 24 h; cell viability was measured by CCK-8 assay and normalized to that of non-treated cells. (C-F) HEK293T cells were transfected with Flag-tagged PB2 (C), PB1 (D), PA (E), or NP (F), and treated with 0, 10, or 20 mM of sodium lactate at 6 h post-transfection. Anti-Flag IP was performed at 24 h post-transfection, and immunoprecipitated samples were detected by immunoblotting with a Pan-L-lactyl lysine antibody. Data are shown as mean ± SD and are representatives of three independent assays. Statistical analyses were performed using unpaired Student’s t-tests; **P < 0.01; ***P < 0.001; ****P < 0.0001.

ATAT1 interacts with viral PA and promotes influenza virus replication

(A) HEK293T cells were co-transfected with FLAG-tagged PA and HA-tagged ESCO1, ESCO2, ATAT1, MYST1, AARS1, or HA empty vector. Anti-HA co-IP was performed at 24 h post-transfection; co-precipitated proteins were detected by immunoblotting. (B) HEK293T cells were single- or co-transfected with Flag-tagged PA and HA-tagged ATAT1; indirect immunofluorescence assay (IFA) was performed at 24 h post-transfection using anti-HA and anti-FLAG antibodies (Scale bar, 25 μm). (C) A549 cells were transfected with increasing doses of HA-tagged ATAT1 for 24 h, then infected with PR8 (MOI=0.01); viral titers were determined by TCID50 assay at 24 hpi; and protein expression levels were detected by immunoblotting. (D) HEK293T cells were transfected with NC or ATAT1-targeting siRNA, and 6 h later with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter and pRL-TK internal control. Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a dual-luciferase reporter assay system. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; *P < 0.05.

SIRT1 interacts with viral PA and inhibits influenza virus replication

(A) HEK293T cells were co-transfected with FLAG-tagged PA and HA-tagged SIRT1, SIRT2, SIRT5, SIRT6, SIRT7, HDAC1, HDAC2, HDAC3, or HA empty vector. Anti-HA co-IP was performed at 24 h post-transfection; co-precipitated proteins were detected by immunoblotting. (B) A549 cells were transfected with empty vector or increasing doses of HA-tagged SIRT1 for 24 h, then infected with PR8 (MOI=0.01); viral titers were determined by TCID50 assay at 24 hpi; protein expression levels were detected by immunoblotting. (C) HEK293T cells were transfected with NC or SIRT1-targeting siRNA, and 6 h later with influenza virus PA/PB1/PB2/NP, pPol I-luc reporter and pRL-TK internal control; Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a dual luciferase reporter assay system. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; *P < 0.05, **P < 0.01, ***P < 0.001.

K605/K609 residues of PA are critical for influenza virus replication

(A) PA sequences from human (n = 9940), and avian (n = 12985) influenza A virus isolates were downloaded from the NCBI Influenza Database (as of September 10, 2025) and analyzed for sequence identity. The frequency of indicated lysine residues at these positions in PA is depicted. (B) H17N10 influenza virus vRNA-bound polymerase complex (PDB: 4WSB) showing PA (blue) residues 351, 353, 356, 357, 600, and 604 (orange red), which correspond to residues 356, 358, 361, 362, 605, and 609 in H1N1 PA, respectively. (C) HEK293T cells were transfected with influenza virus PB1, PB2, and NP expression plasmids, along with the pPol I-luc luciferase reporter plasmid, the pRL-TK Renilla luciferase internal control plasmid, and either WT PA, PA-C95A, PA-D108A, or a combination of PA-C95A and PA-D108A. At 24 h post-transfection, firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; **P<0.01, ***P<0.001.

Lactylation of PA at K605/K609 facilitates ANP32B-mediated polymerase asymmetric dimerization

(A) HEK293T cells were co-transfected with FLAG-tagged PB1 and WT or PA-K605A/K605R/K609A mutants; anti-FLAG co-IP was performed at 24 h post-transfection, co-precipitated proteins were assessed by immunoblotting. Band intensities were quantified by ImageJ and normalized to that of WT-PA-transfected group. (B) H5N1 influenza virus polymerase asymmetric dimer (PDB: 8R1J) with ANP32B (green) showing PA K605 and K609. (C) HEK293T cells were co-transfected with PB2, PB1-Gluc-N, ANP32B-Gluc-C, and WT or PA-K605A/K605R/K609A mutants; Gaussia luciferase activity was measured at 24 h post-transfection; protein expression was detected by immunoblotting. (D) HEK293T cells were co-transfected with Flag-ANP32B, PB1, PB2, and WT or PA-K605A/K605R/K609A mutants; anti-Flag co-IP was performed at 24 h post-transfection; co-precipitated proteins were detected by immunoblotting. Band intensities were quantified by ImageJ and normalized to that of WT-PA-transfected group. (E-F) ANP32A/B double-knockout HEK293T cells were co-transfected with PB2, PB1-Gluc-N, ANP32A-Gluc-C (E) or ANP32B-Gluc-C (F), and WT or PA-K605A/K605R/K609A mutants; Gaussia luciferase activity was measured at 24 h post-transfection. (G) H3N2 influenza virus symmetric dimer (PDB: 6QX8) with PA K605 (blue) and K609 (yellow) depicted. (H-I) HEK293T cells were co-transfected with PB2, PB1-Gluc-N, ANP32B-Gluc-C, PA, and HA-ATAT1 (H) or HA-SIRT1 (I); Gaussia luciferase activity was measured at 24 h post-transfection; protein expression was detected by immunoblotting. Data are presented as mean ± SD and are representative of three independent experiments. Statistical analyses were performed using unpaired Student’s t-tests; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

Mutations on K605/K609 residues of PA do not affect PA ubiquitination level

HEK293T cells were transfected with HA-tagged Ub and WT or PA-K605A/K605R/K609A mutants; anti-HA immunoprecipitation was performed at 24 h post-transfection.