Fig 1.
LSD1 catalyzes lysine demethylation of IFITM3 at K88.
(A) HEK293T cells grown in six-well plates were transfected with HA-IFITM3 and FLAG-LSD1. Forty-eight hours later, cells were treated with IFNα (200U/ml) for the indicated time periods. IFITM3 was immunoprecipitation (IP) by anti-HA antibodies and LSD1 was probed with indicated antibody. The expression levels of HA-IFITM3, FLAG-LSD1 and β-Actin were shown as loading control (Input). (B) HEK293T cells grown in six-well plates were treated with IFNα (200U/ml) for the indicated time periods and were then collected to perform immunoprecipitation (IP) of endogenous IFITM3. The levels of LSD1 and IFITM3 were detected by immunoblotting with anti-LSD1 and anti-IFITM3 antibodies. Proteins levels in whole cell lysates were shown as Input. (C) The interaction of LSD1 and IFITM3 is direct. A pull down assay was performed with recombinant MBP-LSD1, MBP, and GST-IFITM3 as indicated. Immunoblotting (IB) was used to show protein levels. (D) LSD1 down-regulates monomethylation of IFITM3 at K88 in a dose dependent manner. HEK293T cells were transfected with pcDNA3.1-IFITM3 and increasing amounts of FLAG-LSD1 plasmids (0.6μg, 1.5μg or 2.4μg). Forty-eight hours later, cells were collected and levels of IFITM3-K88me1 were determined by western blotting. (E) The demethylase activity of LSD1 is antagonized with SET7. FLAG-tagged SET7, Myc-tagged LSD1 and HA-tagged IFITM3 were co-transfected into HEK293T cells as indicated. After 48h, the cells were collected and protein levels were detected by western blotting.
Fig 2.
LSD1 enhances the antiviral activity of IFITM3 against VSV infection.
(A) Overexpression of LSD1 promotes the antiviral activity of IFITM3. HEK293T cells were transfected with pcDNA3.1-IFITM3 and increasing amounts of FLAG-LSD1 plasmids (0.6μg, 1.5μg or 2.4μg). Forty-eight hours later, cells were infected with VSV at MOI = 0.02. The cells were then collected at 12h post-infection and tested by qPCR to detect of the vRNA levels of viral L gene. Experimental procedure is shown in B. (C) Knockdown efficiency of shRNAs against SET7 and LSD1. HEK293T cells were infected by lentivirus coding shRNA against either SET7 (shSET7) or LSD1 (shLSD1) followed by qPCR analyses of the mRNA levels of IFITM3, SET7 or LSD1 respectively. (D and E) Knockdown of LSD1 promotes the methylation of IFITM3 at K88, whereas, knockdown of SET7 reduces IFITM3-K88me1. Lentivirus-packaged shRNAs against either SET7 (shSET7) or LSD1 (shLSD1) were transduced into HEK293T cells. None-transduced 293T cells are included as control (Mock).The media was changed to fresh DMEM media 24h later. After another 24h, equal number of 5×105 cells were transferred to twelve-well plates and then treated with IFNα (100U/ml) to induce the expression of IFITM3. Mock cells were treated without IFNα. Twenty-four hours after IFNα treatment. the cells were infected with VSV at MOI = 0.02 and were then collected at 12h post-infection for western blot to detect the levels of IFITM3 and IFITM3-K88me1 (D) or for qPCR to detect the vRNA levels of viral L gene (E). (F) The effects of LSD1 and SET7 are dependent on IFITM3 expression. Experimental procedure is same as procedure shown in B except that lentivirus-packaged shRNAs against either SET7 (shSET7) and IFITM3 (shIFITM3) or LSD1 (shLSD1) and IFITM3 (shIFITM3) were transduced into HEK293T cells on the Day 1. Cells were collected at 12h post-infection for qPCR to detect the vRNA levels of viral L gene. All data are representative of more than three independent experiments, and are shown by the mean value with +s.d. ns, p >0.05; *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 3.
LSD1 enhances the antiviral activity of IFITM3 against IAV infection.
Experimental procedure is shown in A. (B) Knockdown efficiency of shRNAs against SET7 and LSD1. HEK293T cells were infected by lentivirus coding shRNA against either SET7 (shSET7) or LSD1 (shLSD1) followed qPCR analyses of the mRNA levels of IFITM3, SET7 or LSD1 respectively. (C and D) Knockdown of LSD1 promotes the methylation of IFITM3 at K88, whereas, knockdown of SET7 reduces IFITM3-K88me1. Lentivirus-packaged shRNAs against either SET7 (shSET7) or LSD1 (shLSD1) were transduced into A549 cells. None-transduced A549 cells are included as control (Mock). The media was changed to fresh DMEM media 24h later. After another 24h, equal number of 5×105 cells were transferred to twelve-well plates and then treated with IFNα (200U/ml) to induce the expression of IFITM3. Mock cells were treated without IFNα. Twenty-four hours after IFNα treatment, the cells were infected with WSN at MOI = 5 and were then collected at 8h post-infection for western blotting to detect the levels of IFITM3 and IFITM3-K88me1 (C) or for qPCR to detect the vRNA, cRNA and mRNA levels of viral gene (D). (E) The effects of LSD1 and SET7 are dependent of IFITM3 expression. Experimental procedure is same as procedure shown in A except that lentivirus-packaged shRNAs against either SET7 (shSET7) and IFITM3 (shIFITM3) or LSD1 (shLSD1) and IFITM3 (shIFITM3) were transduced into A549 cells on the Day 1. Cells were then collected at 8h post-infection for qPCR to detect the vRNA, cRNA and mRNA levels of viral gene. All data are representative of more than three independent experiments, and are shown by the mean value with +s.d. ns, p >0.05; *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 4.
TCP treatment induces severe disease in IAV-infected mice.
The mice (n = 4) were pretreated with PBS (100μl/kg) or TCP (5mg/kg) through intraperitoneally injection on day 0 (D0). One hour later, mice were infected with either 10000 pfu of WSN virus or 300 pfu of SC09 virus in 50μl PBS or 50μl PBS (mock) intranasally. All mice were injected intrapertoneally with PBS (100μl/kg) or TCP (5mg/kg) once a day. The body weights of mice were monitored throughout the infection time course from Day 0 to Day 14 (A and C). The survival curve of mice was shown in B and D. For mice infected with WSN virus, the differences between TCP-treatment and PBS-treatment groups were significant (p<0.05) from Day 2 to Day 6. For mice infected with SC09 virus, the differences between TCP-treatment and PBS-treatment groups were significant (p<0.05) from Day 3 to Day 6.
Fig 5.
TCP treatment results in more severe lung injuries in IAV-infected mice.
The PBS or TCP were admitted to Mock-infected or WSN-infected mice as above, and mice were sacrificed at Day 9 post-infection. Lung tissues from each group were collected and a representative result were shown in A. (B) The lungs were further sectioned, and stained with hematoxylin and eosin (H&E) for the histopathological analysis. Representative results were shown in B. Scale bar was 100μm in 10-time and 20-time enlargement.
Fig 6.
TCP treatment induces increased levels of IFITM3-K88me1 in IAV-infected mice.
Mice (n = 3) in the indicated group were sacrificed at Day 1, Day 3, Day 6 and Day 9 respectively. Lung tissues were collected and homogenized for western blots to detect the levels of indicated proteins. Representative results are shown as above, data were repeated at least three times.
Fig 7.
The disassociation of LSD1 from IFITM3 by IAV and VSV infections can counteract IFITM3.
(A)Virus infection reduces the interaction between LSD1 and IFITM3. HEK293T cells were transfected with HA-IFITM3 and FLAG-LSD1. Forty-eight hours later, cells were infected with WSN virus at MOI = 2. Cells were then collected at the indicated time-point post-infection and were immunoprecipitated with anti-HA antibodies plus protein A/G beads. HA-IFITM3 and FLAG-LSD1 were detected as above, and levels of viral NP protein and β-Actin were shown in whole cell lysates (Input). (B) HEK293T cells grown in six-well plates were transfected with 0.5μg HA-IFITM3 and 1μg FLAG-LSD1. Forty-eight hours later, cells were infected with VSV at MOI = 0.02. Cells were then collected at the indicated time-point post-infection and were immunoprecipitated with 1μg anti-IFITM3 antibody plus protein A/G beads. Protein blots were probed with the antibodies as indicated, data were repeated at least three times.
Fig 8.
Zika virus infection triggers demethylation of IFITM3.
(A) Zika virus infection increases the monomethylation of IFITM3. Human neural progenitor cells (hNPC) grown in six-well plates were infected with Zika virus at MOI = 1. Cells were then collected at the indicated time-point post-infection for western blot to detect the levels of IFITM3 and IFITM3-K88me1 or for qPCR to detect the NS5 levels of Zika virus. The levels of IFITM3, IFITM3-K88me1, LSD1, SET7 and β-Actin were shown in whole cell lysates. Representative results are shown as above, data were repeated at least three times. The virus growth pattern is shown by Zika virus RNA copies on the right. (B) The levels of IFITM3-associated LSD1 keeps stable under Zika virus infection. Huh7 cells were transfected with HA-IFITM3 and FLAG-LSD1. Forty-eight hours later, cells were infected with Zika virus at MOI = 1. Cells were then collected at the indicated time-point post-infection and were immunoprecipitated with anti-HA antibodies plus protein A/G beads. HA-IFITM3 and FLAG-LSD1 were detected as above, and levels of β-Actin were shown in whole cell lysates (Input). Data were repeated at least three times. (C) Knockdown of LSD1 promotes multiplication of Zika virus. Lentivirus-packaged shRNAs against LSD1 (shLSD1) were transduced into Huh7 cells. None-transduced Huh7 cells are included as control (Mock). The media was changed to fresh DMEM media 24h later. After another 24h, part of the cells was collected to check the knockdown efficiency of shRNAs (left and middle panel), the same time, equal number of 1×106 cells were transferred to six-well plates and then treated with IFNα (200U/ml) to induce the expression of IFITM3. Twenty-four hours after IFNα treatment. The cells were infected with Zika virus at MOI = 1 and were then collected at 72h post-infection for qPCR to detect the NS5 levels of Zika virus (right pannel). All data are representative of more than three independent experiments, and are shown by the mean value with +s.d. ns, p >0.05; *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 9.
Reciprocal regulation of IFITM3 monomethylation by LSD1 and SET7.
In the normal circumstances, there is a balance between the lysine methylation and demethylation of IFITM3 at K88. Under virus infection, the methylation of IFITM3 is increasing. On the contrary, type I IFN signaling will recruit LSD1 to demethylate K88me1 on IFITM3 to promote its antiviral activity during RNA virus infection. Our findings suggest that targeting the enzymatic activities of IFITM3 methylation modification enzymes can be useful to fight RNA virus infections.