Negative regulation of interferon-induced transmembrane protein 3 by SET7-mediated lysine monomethylation

Abstract

Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases.

Keywords: Antiviral Agents; Antiviral Host Restriction Factors; Host Defense; Host-pathogen Interactions; IFITM3; Lysine Methylation; Post-translational Modification; Protein Methylation; SET7.

FIGURE 1.

Monomethylation of IFITM3 at Lys-88 is regulated by SET7. A, the strategy of tandem affinity purification. B, alignment of amino acid sequences of SET protein substrates: IFITM3 (amino acids 79–93) against histones H3 and H4. The red arrow indicates similarly conserved SET protein methylation sites. C, identification of the interaction between IFITM3 and SET7. 1 μg of FLAG-tagged SET7, SET8 or SUV39H1 were co-transfected into HEK-293T cells grown in six-well plates with 0.5 μg of HA-tagged IFITM3. Co-immunoprecipitation was performed with 1 μg of anti-FLAG antibody plus protein A/G beads. Protein blots were probed with the antibodies as indicated. D, reciprocal immunoprecipitation of IFITM3 and SET7. 1 μg of FLAG-tagged SET7 and 0.5 μg of HA-tagged IFITM3 were co-transfected into HEK-293T cells grown in six-well plates. Immunoprecipitation was performed with 1 μg of anti-HA or anti-FLAG antibodies plus protein A/G beads. Protein blots were probed with the antibodies as indicated. E, IFITM3 interacts with SET7 in vitro. A pulldown assay was performed with recombinant His-SET7, GST, and GST-IFITM3 as indicated. Immunoblotting (IB) and Coomassie Brilliant Blue staining (CBB) were used to show protein levels. F, IFITM3 is monomethylated by SET7 at Lys-88. Different combinations of 0.5 μg of HA-IFITM3, 0.5 μg of HA-IFITM3-K88A, 1 μg of FLAG-SET7 and 1 μg of FLAG-SET7-DN (H297A catalytically inactive mutant) were co-transfected into HEK-293T cells grown in six-well plates. Protein blots were probed with the antibodies as indicated. G, SET7 methylates IFITM3 in a cell-free system. In vitro methylation was performed with S-adenosylmethionine (SAM), recombinant His-SET7, His-SET7-DN, and GST-IFITM3 as indicated. Immunoblotting and Coomassie Brilliant Blue staining (CBB) were used to show methylation and protein levels, respectively. All figures are representative of >3 independent experiments. CBP, calmodulin binding protein; DN, dominant negative.

FIGURE 2.

Monomethylation of IFITM3 at Lys-88 is promoted by viral infection but reduced by IFN-α treatment. A and B, monomethylation of IFITM3 at Lys-88 is induced by VSV infection but reduced by IFN-α treatment. HEK-293T cells overexpressing IFITM3 grown in 12-well plates were infected with VSV at a MOI of 0.02 and treated with IFN-α (100 units/ml). Cells were collected at the indicated time points post-infection or treatment for immunoblotting with the indicated antibodies (A) or qPCR for detection of VSV-L (B). C, WSN33 infection increases monomethylation of IFITM3 at Lys-88. A549 cell lines grown in 12-well plates are infected with WSN33 at a MOI of 2 and collected at the indicated time points post-infection for immunoblotting (IB) analysis with the indicated antibodies. Numbers below the IFITM3-K88me1 and IFITM3 bands (in A and C) represent their relative density normalized to 0 h. D, virus infection increases the interaction between SET7 and IFITM3. HEK-293T cells grown in six-well plates were transfected with 0.5 μg of HA-IFITM3 and 1 μg of FLAG-SET7. 48 h later, cells were infected with VSV at a MOI of 0.02. Cells were then collected at the indicated time-points post-infection and were immunoprecipitated with 1 μg of anti-IFITM3 antibody plus protein A/G beads. Protein blots were probed with the antibodies as indicated. E, IFN-α reduces the interaction between SET7 and IFITM3. The experiment was performed as described in D but treated with IFN-α (100 units/ml) without virus for the indicated time periods. F, endogenous interaction between IFITM3 and SET7 is up-regulated by VSV infection but down-regulated by IFN-α treatment. MEF cells grown in six-well plates were infected with VSV (tested 6 h post-infection) or treated with IFN-α (6 h) as indicated and then collected for endogenous co-immunoprecipitation (co-IP). WCL, whole cell lysate; NP, nucleoprotein. G, VSV infection increases IFITM3-K88me1 directly. 2 × 10⁶ MEF cells were pretreated with IFN-α (100 units/ml) for 24 h and then infected with GFP-VSV at a MOI of 1 followed by downstream experiments 8 h post-infection. GFP-VSV negative and positive cells were sorted (labeled “−” and “+”, respectively) and analyzed for IFITM3-K88me1 expression by Western blotting. Numbers below the IFITM3-K88me1 and IFITM3 bands represent their relative density normalized to the controls (Ctrl). Error bars represent the S.D. from the mean (n = 3). All figures are representative of >3 independent experiments. Note that the HA tag epitope is derived from an H3 influenza virus strain and is not present in the WSN33 strain of H1N1 influenza virus.

FIGURE 3.

Knockdown of SET7 promotes the antiviral activity of IFITM3. A–D, knockdown of SET7 affects VSV infection only in the presence of IFITM3 expression. Lentivirus packaged with shRNA was transduced into HEK-293T (A) or HEK-293T cells overexpressing IFITM3 (B) grown in 12-well plates. 72 h later, cells were infected with VSV at a MOI of 0.02. Cells were collected at the indicated time points post-infection for Western blot analysis with the indicated antibodies (A and B) or qPCR (C and D). E, VSV titers in the supernatants of the infected cells were determined by a plaque assay with Vero cells. Error bars represent the S.D. from the mean (n = 3). All figures are representative of >3 independent experiments. IB, immunoblot; NT, no treatment.

FIGURE 4.

Overexpression of SET7 reduces the antiviral activity of IFITM3. A, overexpression of SET7 reduces IFITM3 antiviral function. HEK-293T cells grown in 12-well plates were transfected with 1 μg of pcDNA3.1-IFITM3 and 1 μg of FLAG-SET7 plasmids as indicated. 48 h later, cells were infected with VSV at a MOI of 0.02 and analyzed 12 h post-infection. Cells were then collected and tested by Western blotting with the indicated antibodies (bottom) or subjected to qPCR analysis for VSV-P and VSV-L expression (top). B, mutation of Lys-88 of IFITM3 removes SET7-mediated modulation of its antiviral function. HEK-293T cells grown in 12-well plates were transfected with 0.5 μg of pcDNA3.1-IFITM3 or 0.5 μg of pcDNA3.1-IFITM3-K88R/K88A mutants and increasing amounts of FLAG-SET7 plasmids (0.3 μg, 0.9 μg, or 1.5 μg). 48 h later, cells were infected with VSV at a MOI of 0.02 and collected for analyses 12 h post-infection. Cells were collected and tested by Western blotting with the indicated antibodies (bottom) or subjected to qPCR analysis for VSV-P and VSV-L expression (top). C, VSV titers in the supernatants of the indicated infected cells were determined by a plaque assay with Vero cells using 1.5 μg of FLAG-SET7 and 0.5 μg of pcDNA3.1-IFITM3 WT or K88A plasmids. Error bars represent the S.D. from the mean (n = 3). All figures are representative of >3 independent experiments.

FIGURE 5.

SET7 inhibits anti-VSV potency of ISGs in an IFITM-dependent manner. A, experimental procedure. B, IFITM1 and IFITM2 can interact with SET7 and are sensitive to SET7-mediated monomethylation. KD, knockdown. 1 μg of FLAG-tagged SET7 was co-transfected into HEK-293T cells grown in six-well plates with 0.5 μg of HA-tagged IFITM1, -2, or -3. Co-immunoprecipitation was performed with 1 μg of anti-HA antibody plus protein A/G beads. Protein blots were probed with the antibodies as indicated. C, IFITM shRNA targets IFITM1, -2, and -3. MRC5 cells were tested for knockdown of IFITM1, -2, and -3 by lentivirus containing shIFITM followed by analysis via qPCR. D, knockdown of SET7 promotes antiviral activity of ISGs in an IFITM-dependent manner. Lentivirus-packaged shRNAs were transduced into MRC5 cells grown in six-well plates on day 1. After lentivirus infection for 12 h, the medium was changed to fresh DMEM (day 2). 24 h later (day 3), 5 × 10⁵ cells were counted and transferred to 12-well plates and then treated with IFN-α (100 units (U)/ml) to induce the expression of ISGs. After IFN-α treatment for 24 h, cells were infected with VSV at a MOI of 0.02 and then collected at 16 h post-infection for quantitative PCR (top) or Western blot analysis with the indicated antibodies (bottom). E, VSV titers in the supernatants of the indicated infected cells were determined by a plaque assay with Vero cells. Error bars represent the S.D. from the mean (n = 3). All figures are representative of >3 independent experiments. IB, immunoblot.

FIGURE 6.

SET7 inhibits anti-IAV potency of ISGs in an IFITM-dependent manner. A, experimental procedure. B, knockdown (KD) efficiency of IFITM1–3 examined by qPCR. A549 cells were tested for knockdown of IFITM1, -2, and -3 by lentivirus containing shIFITM followed by analysis via qPCR. C, knockdown of SET7 promotes antiviral activity of ISGs in an IFITM-dependent manner. Lentivirus-packaged shRNAs were transduced into A549 cells grown in six-well plates on day 1. After lentivirus infection for 12 h, the medium was changed to fresh DMEM (day 2). 24 h later (day 3), 5 × 10⁵ cells were counted and transferred to 12-well plates and then treated with IFN-α (100 units (U)/ml) to induce the expression of ISGs. After IFN-α treatment for 24 h, infected cells with WSN33 at a MOI of 2 and collected 24 h post-infection for quantitative PCR (top) or Western blot analysis with the indicated antibodies (bottom). D, WSN33 titers in the supernatants of the indicated infected cells were determined by a plaque assay with MDCK cells. Error bars represent the S.D. from the mean (n = 3). All figures are representative of >3 independent experiments. IB, immunoblot; NP, nucleoprotein gene.