TRIM21 inhibits the replication of H5N1 and H3N2 virus rather than H7N9 virus.

(A-D) TRIM21-expressing A549 cells were infected separately with H7N9, H5N1 and H3N2 at an MOI = 1.0 for 12 h, and then the levels of protein (A), mRNA (B), vRNA (C), and the TCID50 (D) were examined. Wild-type A549 cells were used as the control (*, p 0.05; **, p 0.01; ns, p > 0.05). (E-H) TRIM21-KO A549 cells were infected separately with H7N9, H5N1 and H3N2 at an MOI = 1.0 for 12 h, and then the levels of protein (E), mRNA (F), vRNA (G), and the TCID50 (H) were examined. Wild-type A549 cells were used as the control. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD. *, p 0.05; **, p 0.01; ns, p > 0.05. (TIF)</p

TRIM21 directly interacts with the M1 protein of H9N2, H3N2, and H5N1 viruses.

(A) Mass spectrometry identification of TRIM21 as a potential binding partner of H9N2 M1. A549 cells infected with H9N2 virus at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 mAb or mouse IgG, and analyzed by mass spectrometry. The TRIM21 was detected in anti-M1 IP samples. Red indicates matched B ions, blue indicates matched Y ions, green indicates unmatched ions, grey indicates precursor ions. (B) TRIM21 interacts with H9N2 M1. Myc-tagged TRIM21, Flag-GST-tagged H9N2 M1 plasmids were transfected into HEK293T cells individually. Cell lysates were subjected to coimmunoprecipitation and western blotting using the indicated antibodies. (C) Endogenous association of TRIM21 with H9N2 M1. A549 cells infected with H9N2 virus at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 or control IgG, and analyzed by western blotting with the indicated antibodies. (D) TRIM21 interacts directly with H9N2 M1. Purified His-H9-M1 protein mixed with the GST or GST-TRIM21 proteins was pulled down with GST Sepharose, and analyzed by western blotting with the corresponding antibodies. (E) TRIM21 colocalizes with H9N2 M1. HEK293T cells were transfected with Flag-tagged vector or Flag-tagged TRIM21 plasmid for 24 h, infected with H9N2 virus at an MOI = 1.0 for 12 h, and then incubated with the anti-Flag rabbit mAb, anti-Myc mouse mAb, anti-M1 mouse mAb, FITC-Labeled goat anti-rabbit IgG, and Alexa Fluor 546-conjugated donkey anti-mouse IgG. DAPI staining revealed the nuclei. The images were obtained by confocal microscopy. Scale bar = 10 mm. (F) TRIM21 interacts with H3N2 M1 and H5N1 M1. Myc-tagged TRIM21 and Flag-GST-tagged PR8 M1 or H3N2 M1, H5N1 M1, H7N9 M1 plasmids were individually transfected into HEK293T cells. Cell lysates were subjected to coimmunoprecipitation and western blotting using the indicated antibodies. (G) Endogenous association of TRIM21 with H3N2 M1 and H5N1 M1. A549 cells infected with PR8, H3N2, H5N1 and H7N9 viruses at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 or control IgG, and analyzed by western blotting with the indicated antibodies. Each experiment was independently performed with three biological repeats.</p

R⁹⁵ of M1 protein is critical for interacting with the PRY/SPRY domain of TRIM21.

(A) Amino acid sequence alignment of the M1 protein from various IAV, including PR8, H3N2, H5N1, H7N9, H9N2, H5Nx, and H7Nx. (B) The structural prediction of the M1 proteins from PR8, H3N2, H5N1, H7N9, and H9N2 viruses using SWISS MODEL and PyMOL software. (C-E) R95 of M1 is required for the TRIM21 interaction. HEK293T cells were separately transfected with plasmids encoding Flag-GST H3 M1, H5 M1, H9 M1 and its mutants (T37A, R95K, S242N, K242N), Myc-tagged TRIM21 plasmid for 48 h individually, and then cell lysates were incubated as indicated for a coimmunoprecipitation assay and analyzed by western blotting with the indicated antibodies. Each experiment was independently performed with three biological repeats.</p

The primers used for cloning and quantitative real-time PCR.

The primers used for cloning and quantitative real-time PCR.</p

Excel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Fig panels 3A, 4B-4D, 4F-4K, 5A, 5D-5E, 5H-5J, 6A, 6D, 6F-6H, 6J, 6M, 6O-6Q, 7F, S2B-S2D, S2F-S2H, S3B-S3D, S3F-S3H, S4C-S4G, S5A-S5B, S5G-S5H, and S5J-S5L.

Excel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Fig panels 3A, 4B-4D, 4F-4K, 5A, 5D-5E, 5H-5J, 6A, 6D, 6F-6H, 6J, 6M, 6O-6Q, 7F, S2B-S2D, S2F-S2H, S3B-S3D, S3F-S3H, S4C-S4G, S5A-S5B, S5G-S5H, and S5J-S5L.</p

Effect of TRIM21 knockdown and M1 mutation (R⁹⁵K and K²⁴²N) on pathogenicity in mice.

The 8-week-old C57BL/6 mice (13 per group) were intranasally infected with WT H9N2 virus, H9N2 mutant viruses R95K, K242N, or R95K/K242N at a dose of 107.0 TCID50 H9N2 virus, respectively. In another experiment, the 3-week-old C57BL/6 mice (thirteen per group) were intranasally infected with 1011.0 TCID50 of AAV6 with the TRIM21-shRNA (shTRIM21) or scrambled shRNA control (shCtrl). Four weeks after infection, the treated mice were intranasally infected with H9N2 (107.0TCID50). Three mice per group were euthanized on day 6 post-infection to check for lesions and virus replication in the lungs. The remaining mice (10 per group) were monitored until day 14. Mice with a weight loss of more than 30% of their initial body weight were euthanized and recorded as dead. (A and B) Curves of body weight (A) and survival (B) in mice (n = 10 mice) from 0 to 14 days after infection with WT H9N2 virus or H9N2 mutant viruses R95K, K242N and R95K/K242N. (C) Gross and histopathological lesions in the lung at 6 day after infection. (D) The lesion area was measured as a percentage and μm2 of the total lung area in (C). (E-H) The lung of infected mice was harvested to detect the levels of viral proteins (E), mRNA level of M gene (F), viral RNA copies (G), and the TCID50 (H). (I) Validation of shTRIM21 knockdown effect in mice. 3-week-old C57BL/6 mice were intranasally infected with shCtrl or shTRIM21 for four weeks, and TRIM21 expression in mouse lungs was detected by Western blot using the TRIM21 antibody. (J-K) Curves of body weight (J) and survival (K) in TRIM21 knocked down mice from 0 to 14 days after infection with WT H9N2 virus (n = 10 mice). (L) Gross and histopathological lesions in lungs of TRIM21 knocked down mice at 6 days after infection. (M) The lesion area was measured as a percentage and μm2 of total lung area in (L). (N-Q) The lungs of TRIM21 knocked down mice with H9N2 infection were harvested to detect the levels of viral proteins (N), mRNA level of M gene (O), viral RNA copies (P), and the TCID50 (Q). Each experiment was independently performed with three biological repeats. All results are presented as means ± SD.*, p 0.05; **, p 0.01; ns, p > 0.05. The photo by Lulu Lin.</p

Replication ability of H9N2 virus with or without the residue R⁹⁵ of M1.

(A-E) Proportions of the R95K mutation in the M1 protein of H3N2 (A), H5N1 (B), H9N2 (C), H7N9 (D), and H1N1 (E) viruses. M1 sequence data of different IAV subtypes were obtained from the NCBI GenBank Database. The image was created using the website https://app.biorender.com/. (F) A549 cells were co-infected with WT (MOI = 0.1) and R95K mutant H9N2 viruses (MOI = 0.1, 0.5. and 1.0) for 12 h, and then the vRNA was examined. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD. *, p 0.05; **, p 0.01; ns, p > 0.05.</p

R95K and K242N mutations affect budding process.

TRIM21-KO HEK293T cell lines were infected with H9N2-WT virus and mutant viruses (H9N2-R95K and H9N2-K242N) at an MOI of 20 for 10 h, and the cells were analyzed using transmissible electron microscopy. (TIF)</p

TRIM21 ubiquitinates H9N2 M1 via the R⁹⁵ and K²⁴² residues.

(A) TRIM21 degrades H3/H5/H9 M1 rather than H1/H7 M1. Myc-tagged vector or Myc-tagged TRIM21 and Flag-tagged H1/H3/H5/H7/H9 M1 were co-transfected into HEK293T cells, then the cell lysates were detected using the indicated antibodies. The number under the gel band represent the relative protein concentration calculated using Image J software with the gray value of GAPDH as the control standard. (B) TRIM21 mediates H9N2 M1 degradation through the proteasomal pathway. HEK293T cells expressing Flag-tagged H9N2 M1 were transfected with Myc-tagged vector or Myc-tagged TRIM21 for 12 h, followed by treatment with MG132 (6.25μM, 12.5μM, 25μM) or chloroquine phosphate (12.5μM, 25μM, 50μM) for 6 h. Additionally, HEK293T cells expressing Flag-tagged H9N2 M1 R95K were co-transfected with Myc-tagged vector or Myc-tagged TRIM21 for 12 h, followed by treatment with MG132 (25μM) or chloroquine phosphate (50μM) for 6 h. The cell lysates were detected using the indicated antibodies. (C) TRIM21 ubiquitinates the M1 protein of H3N2, H5N1 and H9N2 viruses, but not those of H7N9 and PR8 viruses. Myc-tagged vector or the TRIM21 vector was co-transfected with Flag-GST-tagged H9N2 M1 or H7N9 M1, H5N1 M1, H3N2 M1, PR8 M1 and HA-tagged Ub-WT plasmids into HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation and western blotting using the indicated antibodies. (D) TRIM21 promotes K48-linked polyubiquitination of H9N2 M1. Myc-tagged vector or the TRIM21 vector was co-transfected with Flag-GST-tagged H9N2 M1 and HA-tagged Ub-WT or Ub-K48 or Ub-K63 plasmids into HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation. (E-H) R95 and K242 in H9N2 M1 were necessary for TRIM21 degradation. Myc-tagged vector or Myc-tagged TRIM21 and Flag-tagged H9N2 M1 WT or its mutants (T37A, R95K, S242N, K242N, R95K/K242N) were co-transfected into HEK293T cells, and the cell lysates were detected using the indicated antibodies (E and G). R95 and K242 in H9N2 M1 were required for TRIM21 ubiquitination. Myc-tagged vector and Myc-tagged TRIM21 WT plasmids were co-transfected with Flag-GST-tagged H9N2 M1 WT or its mutants (T37A, R95K, S242N, K242N, R95K/K242N) and HA-tagged Ub-WT plasmids in HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation and western blotting using the indicated antibodies (F and H). (I) E3 ligase activity is indispensable for TRIM21-mediated degradation of H9N2 M1. Myc-tagged vector, Myc-tagged TRIM21 WT, its mutant C16A and TRIM21 deletion (ΔRING, ΔBB, ΔCC and ΔPRY/SPRY) and Flag-tagged H9N2 M1 were co-transfected into HEK293T cells for 12h. Then, the cell lysates were identified using the indicated antibodies. (J) E3 ligase activity was required to catalyze the ubiquitination of H9N2 M1. Myc-tagged vector, Myc-tagged TRIM21 WT, its mutant C16A and TRIM21 deletion (ΔRING, ΔBB, ΔCC and ΔPRY/SPRY) were co-transfected with Flag-gst-tagged H9N2 M1 and HA-tagged Ub-WT in HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. Subsequently, the cell lysates were subjected to immunoprecipitation and western blotting using the indicated antibodies. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD. *, p 0.05; **, p 0.01; ns, p > 0.05.</p

K²⁴² in M1 of H9N2 virus is required for TRIM21-mediated ubiquitination.

(A) All mutants except K242R were degraded by TRIM21. Myc-tagged vector or Myc-tagged TRIM21 and Myc-tagged H9N2 M1 WT or arginine mutants were co-transfected into HEK293T cells, and the proteins in the cell lysates were detected using the indicated antibodies. (B) H9N2 M1 K242R could not be ubiquitinated. Myc-tagged vector, Myc-tagged TRIM21, HA-tagged Ub-WT, and Flag-GST tagged H9N2 M1 WT or arginine mutants were co-transfected into HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h and the cell lysates were then subjected to immunoprecipitation and western blotting using the indicated antibodies. Each experiment was independently performed with three biological repeats. (TIF)</p