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

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

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

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 inhibited IAV H3N2 and H5N1 replication in mice.

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). At four weeks after infection, the treated mice were intranasally infected with H3N2 (106.5 TCID50) and H5N1 (106.7TCID50). On day 6 post-infection, three mice per group were euthanized to check for lung lesions and virus replication, while the remaining ten mice per group were monitored until day 14. Mice with a weight loss exceeding 30% of their initial body weight were euthanized and recorded as dead. (A-D) Curves of body weight (A-B) and survival (C-D) in mice (mean ± SD; n = 10 mice) from day 0 to day 14 post-infection. (E-F) Gross (E) and histopathological (F) lesions in the lungs on day 6 post-infection. H&E staining was performed on lung sections (mean ± SD; n = 3 mice). (G-H) The lesion area was measured as a percentage and μm2 of the total lung area in (E-F). (I-L) The lungs were harvested to detect the levels of viral proteins (I), mRNA (J), vRNA (K), and the TCID50 (L) (mean ± SD; n = 3 mice). 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. (TIF)</p

Effects of TRIM21 on the replication of different subtypes of influenza virus in HEK293T cells.

(A-D) TRIM21 inhibits the replication of H3N2, H5N1 and H9N2 influenza viruses in HEK293T cells, but has no effect on H7N9 and PR8. HEK293T cells were transfected with Myc-tagged vector or Myc-tagged TRIM21 for 24 h, the cells were then infected with H5N1, H7N9, H3N2, H9N2, PR8 at an MOI = 1.0 for 12 h, and the levels of viral proteins (A), M1 mRNA (B), vRNA (C), and the TCID50 (D) were examined. (E-H) TRIM21 increases the replication of H3N2, H5N1 and H9N2 influenza viruses in TRIM21-KO HEK293T cell lines. TRIM21-KO HEK293T cell lines were infected with H5N1, H7N9, H3N2, H9N2, and PR8 at an MOI = 1.0 for 12 h, and the levels of protein (E), M1 mRNA (F), vRNA (G), and TCID50 (H) were 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. (TIF)</p

R⁹⁵K²⁴² of the M1 protein and the TRIM21 are critical for inhibiting IAV replication by ubiquitination degradation.

(A) One-step growth curve of WT and mutant H9N2 viruses. The WT and recombinant viruses were rescued in HEK293T cells and propagated in 9-day-old SPF embryonated chicken eggs. Then, A549 cells were infected with an equal amount of allotonic fluid containing WT and four mutant H9N2 viruses at 0.01 MOI for 24, 36, 48, and 60 h, respectively. The cell supernatant was harvested, and the TCID50 was calculated as described in the Materials and Methods. (B-E) TRIM21 restricted the replication of WT and all mutant H9N2 viruses, except R95K virus and K242N virus, in A549 cells. TRIM21-overexpressing, TRIM21-KO, and control A549 cells were infected with H9N2 WT and mutants H9N2 M1-T37A, H9N2 M1-R95K, H9N2 M1-S224N, and H9N2 M1-K242N at an MOI = 1.0 for 12 h and then the levels of protein (B and C) and the TCID50 (D and E) were examined. (F) the ubiquitination of the H9N2 WT and four mutant viruses except for R95K and K242N virus were markedly promoted by TRIM21. Myc-tagged vector or Myc-tagged TRIM21 and HA-tagged Ub-WT were co-transfected into HEK293T cells. After 24h, the cells were infected with H9N2 WT and mutant IAV (H9N2 M1-T37A, H9N2 M1-R95K, H9N2 M1-S224N, H9N2 M1-K242N) at an MOI = 1.0 for 12h. The cells were then treated with 25μM MG132 for 6 h before harvest, cell lysates were subjected to IP with anti-M1 and the precipitation was analyzed by western blot with the indicated antibodies. (G-J) The RING domain of TRIM21 is critical for inhibiting H9N2 virus replication. HEK293T cells were transfected with Myc-tagged vector or Myc-tagged TRIM21 WT, C16A mutant and ΔRING-deleted TRIM21 plasmids for 24 h, the cells were infected with H9N2 virus at an MOI = 1.0 for 12 h, and then the levels of viral protein (G), mRNA (H), vRNA (I), and the TCID50 (J) were 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

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