Fig 1.
High aggregation potential of H7N9 PB1-F2.
(A) Sequence alignment of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses. Amino acid sequences of PB1-F2 proteins from human WSN, H5N1 and H7N9 viruses were aligned by the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/). Identical, similar and weakly similar residues are indicated by *,: and., respectively. (B) Protein expression. HEK293T and DF-1 cells in 6-well plates were transfected with expression construct (1 μg) for PB1-F2-Flag from WSN (W), H5N1 (H5) and H7N9 (H7) viruses. After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and then subjected to total protein extraction and Western blot analysis with anti-Flag. α-tubulin served as an internal control for protein loading. Slight differences in the actual molecular masses of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses on the 12% polyacrylamide gel might be explained at least partially by the differences in their calculated molecular masses. (C) Bioinformatic prediction of cross-β sheet structure. Amino acid sequences of PB1-F2 from WSN, H5N1 and H7N9 viruses were analyzed for putative cross-β sheet structure by PASTA 2.0 (http://protein.bio.unipd.it/pasta2/). The top panels were the free-energy profile plotting free energy of cross β sheet pairing against residue number k. The bottom panels represented probability matrix that showed probability of pairing of residues m and k of two self-aligned PB1-F2 amino acid sequences. Probability calculation was as described [29]. Dot intensity was proportional to probability of pairing. (D and E) Aggregation and solubility assays. HEK293T cells in 6-well plates were transfected with PB1-F2-Flag expression construct (1 μg). After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and subjected to SDD-AGE and SDS-PAGE analysis (D) as well as protein solubility assay with RIPA lysis buffer (E). GAPDH or β-actin served as internal control for protein loading. Similar results were obtained from three independent experiments.
Fig 2.
Influence of H7N9 PB1-F2 expression on mitochondrial morphology and function.
(A) Mitotracker Red CMXRos staining. HEK293T cells over coverslips in 6-well plates were transfected with 1μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde. PB1-F2 was probed with anti-Flag and nuclei were stained with DAPI. The stained cells were analyzed by confocal microscopy. Arrows indicated distinct mitochondrial clusters colocalized with PB1-F2. vec: vector control. Bars, 20 μm. (B) JC-1 staining. HEK293T cells in 12-well plates were transfected with 0.5 μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with JC-1 dye. For CCCP control, cells were co-treated with 50 μM CCCP and JC-1 dye. JC-1 dye-stained cells were analyzed through fluorescence microplate reader for red and green signals, which respectively indicated healthy and depolarized mitochondria. The data represent mean values ± SD of red-to-green ratio of JC-1 staining of three independent experiments. Unpaired Student’s t-test was used to assess the statistical significance of the difference between vector control (vec) and sample. ***: P < 0.001. **: P < 0.01. *: P < 0.05. n.s.: P > 0.05. (C) Mitochondrial clustering. Cells with clustered mitochondria colocalized with PB1-F2 as indicated by arrows in (A) were calculated by number of cells with clustered mitochondria over total number of cells. For each experiment, at least 90 total cells were counted. The data represent mean values ± SD of cells (%) from three independent experiments. (D) COXIV staining. HEK293T cells over coverslips in 6-well plates were transfected with 1 μg of expression construct for Flag-tagged H7N9 PB1-F2. After 48 hours, cells were fixed with 1:1 methanol: acetone solution and probed with anti-Flag for PB1-F2, anti-COXIV for mitochondria and DAPI for nuclei. The stained cells were analyzed by confocal microscopy. Cells with clustered mitochondria colocalized with PB1-F2 were calculated as in (C).
Fig 3.
Suppression of RIG-I-MAVS antiviral signaling by H7N9 PB1-F2.
(A and B) HEK293T cells in 12-well plates were transfected with 400 ng PB1-F2 expression constructs, 200 ng p125-Luc and 10 ng pRL-TK. After 24 hours, cells were treated with 100 hemagglutinating units/ml of Sendai virus (A) or 1 μg/mL of poly (I:C) (B) for a further 16 hours. Then, cells were harvested for dual-luciferase reporter assay. (C-G) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100ng p125-Luc, 10ng pRL-TK and 50ng expression constructs for type I IFN stimulants including RIG-IN (C), MyD88 (D), MAVS in pEF-Bos vector (E), TBK1 (F) or IRF3-5D (G). After 48 hours, cells were harvested for dual-luciferase reporter assay. (H and I) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100 ng kB-FLuc (H) or IRF3-FLuc (I), 10 ng pRL-TK with or without 50 ng MAVS expression construct for 48 hours before harvested for dual-luciferase reporter assay. (J) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 50 ng MAVS expression construct, 10 ng pRL-TK plus 100ng ISRE-FLuc. After 24 hours, cells were mock-treated or treated with 1000 U/mL human recombinant IFNβ protein for 24 hours before harvested for dual-luciferase reporter assay. (K) Same as in (A), except the inclusion of a group for H7N9 PB1-F2 S66 (S). (L) Same as in (E), except that 50 ng H7N9 PB1-F2 N66 (N) or S66 (S) was used. (M) HEK293T cells in 6-well plates were transfected with 1 μg expression construct for H7N9 PB1-F2 with N66 (N) or S66 (S) for 48 hours before SDS-PAGE and Western blot analysis. Anti-Flag recognized PB1-F2 protein. GAPDH served as internal loading control. All bars denote means ± SD of triplicate experiments. Unpaired Student’s t-test was performed to evaluate the statistical significance of the difference between vector control (vec) and the indicated sample. ***: P < 0.001. **: P < 0.01. *: P < 0.05. n.s.: P > 0.05.
Fig 4.
Suppression of type I IFN response by H7N9 PB1-F2 in cells infected with recombinant influenza A virus.
(A) Schematic representation of the genotypes of H7-2 WT and H7-2 ΔF viruses. (B and C) THP-1 (B) and A549 (C) cells were infected with H7-2 WT and H7-2ΔF at MOI = 1. At 6, 12, and 24 hpi, infected cells were harvested for RT-qPCR assay for IFNβ mRNA, TNFα mRNA, vRNA segment 4 (S4) and segment 6 (S6). The levels of mRNA or vRNA relative to GAPDH mRNA were analyzed by using comparative Ct method. Bars represent means ± SD of triplicate experiments. Unpaired Student’s t-test was performed to judge the statistical significance of the difference between H7-2 WT and H7-2ΔF groups. ***: P < 0.001. **: P < 0.01. *: P < 0.05. n.s.: P > 0.05. (D) WSN PB1-F2 does not suppress type I IFN response during viral infection. THP-1 cells were infected with WSN WT or WSN ΔF at MOI = 1. At 6, 12, and 24 hpi, infected THP-1 cells were harvested for RT-qPCR assay for IFNβ mRNA, TNFα mRNA, vRNA segment 4 (S4) and segment 6 (S6). The levels of mRNA or vRNA relative to GAPDH mRNA were analyzed by using comparative Ct method. Bars represent means ± SD of triplicate experiments.
Fig 5.
Facilitation of MAVS degradation by H7N9 PB1-F2 in immune-stimulated cells.
(A and B) Impact of H7N9 PB1-F2 on MAVS protein level. THP-1 cells were infected with H7-2 WT and H7-2ΔF viruses at MOI = 1. At 6, 12, and 24 hpi, infected cells were harvested for total protein extraction, SDS-PAGE and Western blot analysis with anti-MAVS and anti-H7N9 PB1-F2 (A). PA and GAPDH served as normalization controls for viral infection and loading. Harvested cells were also subjected to RT-qPCR analysis of MAVS mRNA level relative to that of GAPDH mRNA using comparative Ct method (B). (C and D) Impact of WSN PB1-F2 on MAVS protein level. THP-1 cells were infected with WSN WT and WSN ΔF viruses at MOI = 1. MAVS protein and mRNA were analyzed as above. (E) HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg PB1-F2 expression constructs. After 24 hours, 100 μg/mL cycloheximide (CHX) was added. Cells were harvested for total protein extraction at 0, 6, 12 and 24 hours after drug treatment for SDS-PAGE followed by Western blot analysis with anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. (F) HEK293T cells were transfected with 0.5 μg pEF-Bos-Flag-MAVS together with increasing dosage of expression construct for PB1-F2-Flag of WSN and H7N9 (0, 0.25, 0.5, 1, 1.5 and 2 μg). After 48 hours, cells were harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-Flag for detection of both MAVS (70 kDa) and PB1-F2 (15–20 kDa) normalized to GAPDH. (G and H) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constructs. After 24 hours, cells were either mock-transfected or transfected with 1 μg/mL poly (I:C) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (G) or for RT-qPCR analysis of MAVS mRNA (H). (I and J) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constructs. After 24 hours, cells were either mock-infected or infected with Sendai virus (100 hemagglutinating units/ml) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (I) or for RT-qPCR analysis of MAVS mRNA relative to HPRT mRNA by comparative Ct method (J). Bars represent means ± SD of triplicate experiments.
Fig 6.
H7N9 PB1-F2-induced destabilization of MAVS protein aggregate for proteasomal and lysosomal degradation.
(A) Treatment with proteasome and lysosome inhibitors. HEK293T cells in 6-well plates were co-transfected with 1 μg PB1-F2-Flag expression construct and 1 μg pEF-Bos-Flag-MAVS. After 24 hours, 20 μM MG132, 100 nM bafilomycin A1 (BaA1) or their combination was added for 6 or 16 hours before cells were harvested for total protein extraction and SDS-PAGE Western blot analysis against anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the right panel. (B) Analysis of aggregation-defective mutants of MAVS. HEK293T cells were co-transfected with 0.5 μg PB1-F2 expression construct and 0.5 μg pEF-Bos-Flag-MAVS WT, E26A, W56R or R64, 65A. After 48 hours, total protein was extracted from the cells and subjected to SDS-PAGE followed by Western blot analysis with anti-Flag for detection of MAVS at 70 kDa and PB1-F2 at 15-20kDa. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. Three independent experiments were performed with similar results.
Fig 7.
Suppression of MAVS aggregation by H7N9 PB1-F2.
(A) SDD-AGE analysis of MAVS aggregation. HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg expression construct for PB1-F2 of WSN, H5N1 or H7N9 virus. After 48 hours, cells were harvested for SDD-AGE or SDS-PAGE followed by Western blot analysis with anti-MAVS. (B) Co-immunoprecipitation assay for PB1-F2-MAVS association. HEK293T cells in 60mm dishes were co-transfected with 1 μg pCAGEN-myc-MAVS and 0.5 μg PB1-F2-Flag expression construct. After 48 hours, cells were harvested for co-immunoprecipitation with anti-Flag (IP: Flag). Both input and immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti-Myc for MAVS, anti-Flag for PB1-F2 and anti-GAPDH for normalization. (C) Distribution of unaggregated MAVS on fissioned mitochondria in cells expressing H7N9 PB1-F2. HEK293T cells in 6-well plates were co-transfected with 0.25 μg of CAGEN-V5-MAVS and 0.25 μg of PB1-F2-Flag expression constructs. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde and probed with anti-Flag for PB1-F2, anti-MAVS and DAPI. The stained cells were analyzed by confocal microscopy. Fractions of cells with unaggregated and aggregated MAVS were calculated by counting 100 cells per sample. Aggregated MAVS or PB1-F2 was visually defined as concentrated dots or structures with intense MAVS or PB1-F2 signal. Bars, 20 μm. Similar results were obtained from three independent experiments.
Fig 8.
Interaction of MAVS with TRAF3 but not TRAF6-TBK1-IKKε signalosome in the presence of H7N9 PB1-F2.
HEK293T cells in 60mm dishes were co-transfected with 0.5 μg pCAGEN-V5-MAVS, 0.25 μg PB1-F2 expression construct and 0.5 μg expression construct for Flag-TRAF3 (A), Flag-TRAF6 (B), Flag-TBK1 (C) or Flag-IKKε (D). After 48 hours, cells were harvested for co-immunoprecipitation with anti-V5 (IP: V5). Both input and immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti-V5 for MAVS, anti-Flag for TRAF3 (A), TRAF6 (B), TBK1 (C) and IKKε (D). GAPDH served as loading control. Arrowheads point to the target TBK1-Flag and IKKε-Flag bands. IgH: immunoglobulin heavy chain. Three independent experiments were carried out and similar results were obtained.
Fig 9.
Suppression of TRIM31-mediated K63 polyubiquitination of MAVS by H7N9 PB1-F2.
(A) HEK293T cells in 60mm dishes were transfected with 0.5 μg expression construct for Myc-Ub-WT, 0.5 μg pCAGEN-V5-MAVS and 0.25 μg PB1-F2 expression construct. After 48 hours, cells were harvested for immunoprecipitation with anti-V5 (IP: V5). Immunoprecipitates were analyzed with SDS-PAGE and Western blot analysis with anti-Myc for ubiquitin chain and anti-V5 for MAVS. (B) HEK293T cells in 60mm dishes were transfected with 2 μg expression construct for HA-Ub-mutants (K48, K63 or K27), 0.5 μg pCAGEN-myc-MAVS and 0.25 μg PB1-F2 expression construct. Immunoprecipitation and SDS-PAGE were performed as in (A). Western blot analysis was carried out with anti-HA for Ub mutant chains and anti-V5 for MAVS. (C) Same as in (B) except inclusion of WSN PB1-F2. (D-F) HEK293T cells in 60mm dishes were transfected with 2 μg expression construct for myc-TRIM31, 0.2 μg pCAGEN-myc-MAVS and 0.1 μg PB1-F2 expression construct. After 48 hours, cells were harvested for co-immunoprecipitation with anti-V5 (D and F) or anti-Myc (E) as indicated (IP: V5 or IP: Myc). Both input and immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti-V5 for MAVS, anti-Myc for TRIM31 and anti-GAPDH for normalization. Arrows point to the target Myc-TRIM31 and V5-MAVS bands. Two exposures (long and short) of the same blot are presented in (D). IgH: immunoglobulin heavy chain. IgL: immunoglobulin light chain. Results were representative of three independent experiments.