Fig 1.
PIAS1 interacts with multiple proteins in the RNP complex of IAV.
(A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E. coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively.
Fig 2.
PIAS1 restricts IAV replication in vitro.
(A, B) Establishment of a lentiviral-mediated PIAS1-overexpressing A549 cell line. The stable expression of PIAS1 was confirmed by quantitative reverse transcription PCR (RT-qPCR) (A) and western blotting with a rabbit anti-PIAS1 pAb (B). ***, P < 0.001. (C) The PIAS1-overexpressing or control A549 cells were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. **, P < 0.01. (D, E) siRNA knockdown of PIAS1 in A549 cells. A549 cells were transfected with siRNA targeting PIAS1 or with scrambled siRNA for 48 h. Cell lysates were subjected to RT-qPCR (D) or western blotting with a rabbit anti-PIAS1 pAb (E) to confirm the downregulation of PIAS1. ***, P < 0.001. (F) Cell viability of siRNA-treated A549 cells as in (D, E) was determined by using a CellTiter-Glo assay. (G to J) Virus replication in siRNA-treated A549 cells as in (D, E). PIAS1 siRNA- or scrambled siRNA-transfected A549 cells were infected with WSN (H1N1) (MOI = 0.01) (G), AH05 (H5N1) (MOI = 0.1) (H), AH13 (H7N9) (MOI = 0.1) (I), or SH13 (H9N2) (MOI = 0.1) (J) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. *, P < 0.05, ***, P < 0.001. (K) Knockout of PIAS1 in PIAS1_KO A549 cells was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (L) Cell viability of PIAS1_KO A549 cells was determined by using the CellTiter-Glo assay. (M) Virus replication in PIAS1_KO A549 cells. PIAS1_KO or control A549 cells were infected with WSN (H1N1) (MOI = 0.01) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. **, P < 0.01, ***, P < 0.001.
Fig 3.
PIAS1 regulates the gross SUMOylation induced by IAV infection.
A549 cells treated with specific siRNA targeting PIAS1 or scrambled siRNA were infected with WSN (H1N1) virus at an MOI of 0.01. The overall cellular SUMOylation level was assessed at 0, 12, and 24 h p.i. by western blotting with an anti-SUMO1 (A) or anti-SUMO2/3 (B) pAb.
Fig 4.
PIAS1 relies on its SUMO E3 ligase activity to suppress IAV transcription and replication.
(A) siRNA knockdown of PIAS1 in HEK293T cells. HEK293T cells were transfected with PIAS1 siRNA or scrambled siRNA for 48 h. Knockdown of PIAS1 expression was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (B) Minigenome assay in siRNA-treated HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. HEK293T cells treated with siRNA as in (A) were transfected with plasmids for the expression of four viral RNP proteins (WSNPB2, WSNPB1, WSNPA, and WSNNP), together with pHH21-SC09NS F-Luc and pRL-TK. Thirty-six hours later, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the internal control, Renilla luciferase activity. ***, P < 0.001. (C) Establishment of a PIAS1_KO HEK293T cell line. The knockout of PIAS1 was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (D) Minigenome assay in PIAS1_KO HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (B). ***, P < 0.001. (E) RT-qPCR analysis to determine the effect of PIAS1 knockdown on the transcription and replication of viral RNAs. A549 cells were transfected with siRNA targeting PIAS1 or with scrambled siRNA for 48 h, followed by infection with WSN (H1N1) virus (MOI = 5). Total RNA was extracted at 6 h p.i. by using TRIzol reagent, and the levels of NP-specific vRNA, cRNA, and mRNA were analyzed by RT-qPCR and then normalized to GAPDH mRNA. The values shown are standardized to the corresponding RNA expression level in the scrambled siRNA-treated A549 cells (100%). **, P < 0.01, ***, P < 0.001. (F-I) Minigenome assay in HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. HEK293T cells were transfected with plasmids for the expression of four viral RNP proteins and increasing amounts of Myc-PIAS1 or Myc-PIAS1 mutant, together with pHH21-SC09NS F-Luc and pRL-TK. At 36 h post-transfection, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the Renilla luciferase activity. **, P < 0.01, ***, P < 0.001. (J-M) Minigenome assay in PIAS1_KO HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (F-I). *, P < 0.05, **, P < 0.01, ***, P < 0.001.
Fig 5.
IAV PB2, PB1, and NP are differentially SUMOylated in HEK293T cells expressing exogenous SUMOylation components.
(A-I) HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB2 (A-C), WSNPB1 (D-F), or WSNNP (G-I), along with or without Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1 or its mutant. HEK293T cells transfected to express only Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a mouse anti-HA mAb (A-C) or a rabbit anti-HA pAb (D-I), and were then subjected to western blotting with a rabbit anti-PB2 pAb (A-C), a mouse anti-PB1 mAb (D-F), a rabbit anti-NP pAb (G-I), and a mouse anti-Flag mAb (A-I) for the detection of PB2, PB1, NP, and SUMO1/2/3, respectively. Red triangle indicates the corresponding SUMOylated viral protein.
Fig 6.
PIAS1 mediates robust SUMOylation of IAV PB2 and has no effect on IAV PB1 SUMOylation.
(A-C) PIAS1 catalyzes SUMOylation of IAV PB2 with SUMO1 (A), SUMO2 (B), and SUMO3 (C). HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB2, along with or without Flag-SUMO1 (A), Flag-SUMO2 (B), Flag-SUMO3 (C), Ubc9-V5, Myc-PIAS1, and Myc-PIAS1 mutants. HEK293T cells transfected to express only Flag-SUMO1/2/3 and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a mouse anti-HA mAb, and were then subjected to western blotting with a rabbit anti-PB2 pAb and a mouse anti-Flag mAb for the detection of PB2 and SUMO1 (A), SUMO2 (B), and SUMO3 (C), respectively. (D) PIAS1 has no effect on the SUMOylation of IAV PB1. HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB1, along with or without Flag-SUMO1, Ubc9-V5, Myc-PIAS1, and Myc-PIAS1 mutants. HEK293T cells transfected to express only Flag-SUMO1 and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a rabbit anti-HA pAb, and were then subjected to western blotting with a mouse anti-PB1 mAb and a mouse anti-Flag mAb for the detection of PB1 and SUMO1, respectively. Red triangle indicates the corresponding SUMOylated viral protein.
Fig 7.
IAV PB2, PB1, and NP exhibit diverse stability in the presence of exogenously expressed SUMOylation components.
(A-I) Stability of PB2, PB1 and NP in the presence of exogenous SUMOylation components. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Flag-SUMO1/2/3, Ubc9-V5, along with/without Myc-PIAS1 (A-C), or transfected with plasmids for the expression of WSNPB1 (D-F) or WSNNP (G-I) in combination with Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1. At 36 h post-transfection, the cells were treated with CHX. At the indicated timepoints, cell lysates were subjected to western blotting. Data are representative of three independent experiments (A-I). The band intensities of PB2, PB1, and NP, quantified by using ImageJ software, were normalized to GAPDH and are expressed as relative ratios compared with untreated cells at 0 h.
Fig 8.
PIAS1-mediated SUMOylation leads to degradation of PB2 through the ubiquitin-proteasome pathway.
(A-C) Stability of PB2 in cells overexpressing SUMO1/2/3, Ubc9, and PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Ubc9-V5, Myc-PIAS1, and Flag-SUMO1 (A), Flag-SUMO2 (B), or Flag-SUMO3 (C). At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (D-F) Stability of PB2 in cells overexpressing SUMO1/2/3 and PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Myc-PIAS1, and Flag-SUMO1 (D), Flag-SUMO2 (E), or Flag-SUMO3 (F). At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (G) Stability of PB2 in cells overexpressing PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2 and Myc-PIAS1. At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (H, I) Effect of the PIAS1 mutant on the stability of PB2. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Myc-PIAS1, and Myc-PIAS1 W372A (H) or Myc-PIAS1 S90A (I). At 36 h post-transfection, the cells were treated with CHX. At the indicated timepoints, cell lysates were subjected to western blotting. Data are representative of three independent experiments (A-I). The band intensities of PB2, quantified by using ImageJ software, were normalized to GAPDH and are expressed as relative ratios compared with untreated cells at 0 h at the bottom of each panel.
Fig 9.
Pias1 inhibits IAV replication and virulence in mice.
(A) Schematic illustration of the strategy used to generate Pias1+/- mice. Two sgRNAs were designed to delete exon 2 of the Pias1 gene. (B) The genotype of Pias1+/- mice was verified by western blotting with a rabbit anti-PIAS1 pAb. (C) Survival of WT and Pias1+/- mice (n = 6 per group) after intranasal infection with WSN (H1N1) virus (1.2×104 PFU/mouse). (D) Body weight changes of WT and Pias1+/- mice (n = 6 per group) after intranasal infection with WSN (H1N1) virus. (E) Titers of WSN (H1N1) virus in the lungs of WT and Pias1+/- mice (n = 5 per group) on days 2 and 6 p.i.
Fig 10.
Schematic model of PIAS1-mediated SUMOylation and degradation of IAV PB2.
Upon initial binding with sialic acid receptors on the cell surface, IAV is endocytosed into host cells. Following low-pH mediated fusion of viral and endosomal membranes, the vRNPs are released into the cytoplasm, and translocate into the nucleus where they catalyze the transcription and replication of viral genome. PIAS1 mediates robust poly-SUMOylation of PB2 from incoming vRNPs or newly synthesized PB2 by SUMO1/2/3, and also catalyzes minimal SUMOylation of NP by SUMO1. PIAS1-mediated SUMOylation of PB2 most likely recruits SUMO-targeted ubiquitin ligases (STUbLs) to the SUMOylated PB2, resulting in ubiquitination-dependent proteasomal degradation of PB2. SF: stimulating factors.