Research ArticleInnate Immunity

HIPK2 is necessary for type I interferon–mediated antiviral immunity

See allHide authors and affiliations

Science Signaling  19 Mar 2019:
Vol. 12, Issue 573, eaau4604
DOI: 10.1126/scisignal.aau4604
  • Fig. 1 HIPK2 is critical for antiviral immunity in vivo.

    (A) Survival analysis of age- and sex-matched wild-type (WT) and Hipk2+/− mice inoculated with VSV. Data are from a total of eight mice per group from three experiments. d, days. (B to E) qRT-PCR analysis of (B) Ifn-β, (C) Isg15, (D) VSV glycoprotein (VSV-G), and (E) Ifng mRNA expression in blood cells from mice at the indicated times after infection with VSV. Data with means ± SEM of six mice per group from three experiments. NS, not significant. (F to H) qRT-PCR analysis of (F) Ifn-β, (G) Isg15, or (H) VSV-G mRNA expression in wild-type, Hipk2+/−, or Hipk2−/− peritoneal macrophages infected with VSV for 24 hours. MOI, multiplicity of infection. (I) Survival analysis of age- and sex-matched wild-type and Hipk2+/− mice treated with antibody against IFNAR and infected with VSV. Data are from a total of 10 mice per group from three experiments. IgG, immunoglobulin G. (J) Viral load in the blood after wild-type and Hipk2+/− mice were treated with antibody against IFNAR and infected with VSV. Data with means ± SEM are from three experiments. *P < 0.05 and **P < 0.01 by log-rank Mantel-Cox test (A and I) or an unpaired two-tailed nonparametric Mann-Whitney U test (B to E and J).

  • Fig. 2 HIPK2 is an ISG.

    (A) qRT-PCR analysis of HIPK2 mRNA expression in 2fTGH cells treated with increasing amounts of human IFN-β for 24 hours. Data with means ± SEM are from three experiments. (B) qRT-PCR analysis of HIPK2 mRNA in whole blood from wild-type or Ifnαr−/− mice at the indicated times after infection with 200 plaque-forming units (PFU) of WNV. Data with means ± SEM are from three experiments. (C and D) qRT-PCR analysis of HIPK2 mRNA expression in 2fTGH cells (C) and iBMDMs (D) infected with VSV for indicated times. Data with means ± SEM are from three experiments. h, hours. (E) Western blot analysis of HIPK2 abundance in lysates of 2fTGH cells after infection with VSV for the indicated times. Blots (top) are representative of three experiments. Quantified band intensity values (bottom) with means ± SEM are from all experiments. (F) Luciferase assay analysis of HIPK2 promoter activity in HT1080 cells treated with increasing amounts of human IFN-β for 24 hours. Data with means ± SEM of three experiments. *P < 0.05 and **P < 0.01 by Student’s t test.

  • Fig. 3 HIPK2 promotes the expression of type I IFNs.

    (A and B) qRT-PCR analysis of Ifnαs (A) and Ifn-β (B) mRNA expression 24 hours after VSV infection of wild-type and Hipk2−/− iBMDMs. Data with means ± SEM are from three experiments. (C) qRT-PCR analysis of Il10, Ccl2, Ccl5, and Cxcl2 mRNA expression in wild-type or Hipk2−/− iBMDMs after VSV infection for 6 hours. Data with means ± SEM are from three experiments. (D) qRT-PCR analysis of VSV-G mRNA expression in HEK293 cells transfected with scrambled control or HIPK2 small interfering RNA (siRNA) and infected with VSV for 24 hours. Data with means ± SEM are from three experiments. (E) Plaque assay analysis of viral production by wild-type or Hipk2−/− iBMDMs infected with VSV for indicated times. Data with means ± SEM are from three experiments. (F) Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with the indicated expression plasmids and then infected with VSV for 24 hours. Data (top) with means ± SEM are from three experiments. Blots confirming expression of the indicated proteins (lower) are representative of all experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by Student’s t test.

  • Fig. 4 HIPK2 is involved in antiviral immune signaling.

    (A and B) Luciferase assay analysis of IFN-β (A) or IRF-mediated ISRE (B) promoter activity in HEK293 cells transfected with scrambled control or HIPK2 siRNA and the indicated expression plasmids for 24 hours. Data (top) with means ± SEM are from three experiments. Blots confirming expression of the indicated proteins (lower) are representative of all experiments. (C) Bioassay of type I IFN production by wild-type, Irf3−/−, or Irf7−/− iBMDMs transfected with Vec control or HIPK2 expression plasmids and infected with SeV for 24 hours. Data with means ± SEM are from three experiments. (D) Enzyme-linked immunosorbent assay (ELISA) analysis of type I IFNs secreted from wild-type, Irf3−/−, Irf7−/−, and p65−/− iBMDMs transfected with Vec control or HIPK2 expression plasmids and infected with SeV for 6 hours. Data with means ± SEM are from three experiments. (E and F) Bioassay of IFN production by wild-type or p65−/− iBMDMs transfected with Vec or HIPK2 expression plasmids and infected with VSV (E) or SeV (F) for 6 hours. Data with means ± SEM are from three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 5 HIPK2 activates the transcription of IFNs by interacting with ELF4.

    (A) Luciferase assay analysis of wild-type and mutant IFN-β promoter activity in HEK293 cells transfected with the indicated expression plasmids for 24 hours. Data with means ± SEM are from three experiments. (B) Coimmunoprecipitation analysis of ELF4 interaction with HIPK2 in lysates of HEK293 cells infected with VSV for 6 hours. Blots (left) are representative of three experiments. Quantified HIPK2 band intensity values (right) normalized to whole-cell lysate (WCL) are pooled from all experiments. IP, immunoprecipitation. (C) Coimmunoprecipitation analysis of HIPK2 interaction with ELF4 in lysates of wild-type and Hipk2−/− iBMDMs infected with VSV for 6 hours. Blots (left) are representative of 3 experiments. Quantified HIPK2 band intensity values (right) normalized to WCL are pooled from all experiments. (D and E) Luciferase assay analysis of IFN-β (D) or IFN-α2–luc and IFN-α4–luc (E) promoter activity in wild-type or ELF4−/− HEK293 cells transfected with Vec control or HIPK2 expression plasmids and infected with VSV for 24 hours. Data with means ± SEM are from three experiments. **P < 0.01 and ***P < 0.001 by Student’s t test.

  • Fig. 6 HIPK2 translocates to nucleus and is cleaved after virus infection.

    (A) Western blot analysis of HIPK2 cleavage in lysates of HEK293 cells pretreated with dimethyl sulfoxide (DMSO) or caspase inhibitor Z-VAD for 2 hours and infected with VSV for 2 hours. Blots (left) are representative of three experiments. Quantified band intensity values (right) are pooled from all experiments. IB, immunoblotting. (B) Western blot analysis of HIPK2 cleavage in lysates of HEK293 cells transfected with wild-type or D923A mutant HIPK2 and infected with VSV for 2 hours. Blots are representative of three experiments. (C and D) Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with the indicated expression plasmids 24 hours after infection with VSV (C) or transfection with ELF4 (D). Data with means ± SEM are from three experiments. (E) Western blot analysis of the indicated proteins in nuclear and cytoplasmic fractionations of lysates from MEF cells infected with VSV for 2 hours. Blots (top) are representative of three experiments. Quantified HIPK2 band intensity values (right) are pooled from all experiments. (F) Confocal microscopy analysis of HIPK2 localization in Hela cells transfected with Flag-HIPK2 and infected with VSV for 4 hours. Images are representative of three experiments. DAPI, 4′,6-diamidino-2-phenylindole. (G) qRT-PCR analysis of IFN-β mRNA expression in wild-type or Hipk2−/− MEFs reconstituted with full-length HIPK2, HIPK2-D923A, or HIPK2△NLS (deletion of nuclear localization sequence) and infected with VSV for 24 hours. Data with means ± SEM are from three experiments. *P < 0.05 and **P < 0.01 by Student’s t test.

  • Fig. 7 HIPK2 phosphorylates ELF4 at Ser369and contributes to its binding to type I IFN promoters.

    (A) Coimmunoprecipitation (top) and SDS-PAGE (bottom) analysis of lysates from HEK293 cells transfected with Flag-ELF4 and HA-HIPK2 plasmid. ELF4 bands were analyzed for protein modification by mass spectrometry (right). Data are representative of three experiments. aa, amino acid. (B) Alignment of ELF4 proteins from human and nonhuman species. (C) Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with IFN-β luc and the indicated expression plasmids after 24 hours. Data (top) with means ± SEM are from three experiments. Blots confirming expression of the indicated proteins (lower) are representative of all experiments. (D) ChIP analysis of IFN-β or IFN-α2 promoter DNA associated with ELF4 in wild-type or Hipk2−/− peritoneal macrophages infected with VSV for 6 hours. Data with means ± SEM are from three experiments. (E) ChIP analysis of IFN-β promoter DNA associated with Flag-HIPK2 in HEK293 cells transfected with indicated plasmids and infected with VSV for 6 hours. Data (top) with means ± SEM are from three experiments. Blots confirming expression of the indicated proteins (lower) are representative of all experiments. (F) ChIP analysis of IFN-β or IFN-α4 promoter DNA associated with Flag-HIPK2 in ELF4−/− HEK293 cells transfected with indicated plasmids and infected with VSV for 6 hours. Data with means ± SEM are from three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/573/eaau4604/DC1

    Fig. S1. HIPK2 is involved in antiviral immunity.

    Fig. S2. HIPK2 is an ISG.

    Fig. S3. HIPK2 facilitates the induction of type I IFN.

    Fig. S4. HIPK2 is involved in antiviral immune signaling.

    Fig. S5. HIPK2 translocates to nucleus and is cleaved after virus infection.

    Fig. S6. HIPK2 phosphorylates ELF4 at Ser369 and contributes to its binding to type I IFN promoters.

    Fig. S7. HIPK2 promotes IFN-αβ transcription, and type I IFNs augment HIPK2 expression.

    Table S1. Guide RNA used in this study.

    Table S2. Primers used in this study.

  • This PDF file includes:

    • Fig. S1. HIPK2 is involved in antiviral immunity.
    • Fig. S2. HIPK2 is an ISG.
    • Fig. S3. HIPK2 facilitates the induction of type I IFN.
    • Fig. S4. HIPK2 is involved in antiviral immune signaling.
    • Fig. S5. HIPK2 translocates to nucleus and is cleaved after virus infection.
    • Fig. S6. HIPK2 phosphorylates ELF4 at Ser369 and contributes to its binding to type I IFN promoters.
    • Fig. S7. HIPK2 promotes IFN-αβ transcription, and type I IFNs augment HIPK2 expression.
    • Table S1. Guide RNA used in this study.
    • Table S2. Primers used in this study.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article