The IFITM proteins inhibit HIV-1 infection

Abstract

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.

FIG. 1.

(A to C) Effect of ISG knockdown on HIV-1 production in the presence of IFN-α2b. Each ISG was targeted with 3 to 5 shRNA clones. The shRNA-transduced SupT1 cells were treated with IFN-α2b for 16 h before being infected with a subtype B HIV-1 (BH10). Production of infectious HIV-1 particles was determined by infecting TZM-bl indicator cells. The fold change for each shRNA was calculated by dividing the amount of infectious HIV-1 made from the shRNA SupT1 cells by that from the control shRNA SupT1 cells. The results are summarized in three graphs. The candidate genes are highlighted by arrows. (D) Western blots of SupT1 cells that were stably transduced with either the control shRNA vector or the shIFITM1 clones. Cells were treated with IFN-α2b (500 U/ml) for 20 h prior to Western blotting. The fold change in HIV-1 production for each shIFITM1 clone is shown. The results shown are the averages of two independent infection experiments.

FIG. 2.

IFITM proteins inhibit HIV-1 replication in SupT1 cells. (A) Illustration of the domain structures of IFITM proteins. The transmembrane domains are highlighted in boxes. A Flag tag was attached to the N termini of IFITM proteins. The conserved amino acid residues are highlighted in red letters. (B) Doxycycline-induced expression of IFITM proteins in SupT1 cells. Stably transduced SupT1 cells were exposed to different amounts of doxycycline (0, 50, and 500 ng/ml) for 16 h before the cell lysates were harvested and subjected to Western blotting using anti-Flag antibodies to detect the expression of IFITM proteins. Control represents a cell line that was stably transduced with the empty retroviral vector. (C) HIV-1 replication in IFITM-expressing SupT1 cells. The cells were treated with doxycycline (Dox) (500 ng/ml) for 16 h before they were exposed to wild-type HIV-1. Virus production over different time intervals was monitored by measuring viral reverse transcriptase activity in the culture supernatants. The results shown represent four independent infection experiments.

FIG. 3.

IFITM proteins inhibit an early step of HIV-1 replication. (A) IFITM2 and IFITM3 diminished the number of HIV-1-infected cells. Following doxycycline treatment (0 and 500 ng/ml) for 16 h, SupT1 cells were infected with the NLEY1-IRES virus, which expresses YFP. Forty hours after infection, the number of YFP-positive cells was scored by flow cytometry. (B) IFITM proteins suppressed production of infectious HIV-1 particles. The amounts of infectious virus particles were determined by infecting TZM-bl indicator cells. The fold change was calculated by dividing the values from doxycycline-treated cells (+Dox) by the values from untreated cells (−Dox). The results shown are the averages of three independent infection experiments shown in panel A. The error bars indicate standard deviations. (C) SupT1 cells were infected with the NLEY1-IRES virus for 2 h before exposure to doxycycline (500 ng/ml) treatment. The effects of IFITM proteins on the number of infected cells and the amounts of infectious HIV-1 are summarized in the bar graph. (D) The IFITM SupT1 cell lines were first treated with Dox (500 ng/ml) for 16 h and then infected with NLEY1-ES-IRES viruses that were pseudotyped with VSV G protein. The number of YFP-positive cells was scored 40 h after infection. The results of three independent infections are summarized in the bar graph.

FIG. 4.

Effect of IFITM proteins on HIV-1 entry. (A) T20 blocks the infection of SupT1 cells by the BH10/BlaM-Vpr viruses. SupT1 cells were infected with the BH10/BlaM-Vpr or the NLEY1-ES-IRES/VSV G/BlaM-Vpr viruses in the absence or presence of the entry inhibitor T20 (1 μg/ml) for 2 h before cells were harvested to detect BlaM activity. The numbers of cells with successful HIV-1 entries are scored in windows Q2. (B and C) IFITM SupT1 cell lines were treated with Dox (500 ng/ml) for 16 h before being infected with BH10/BlaM-Vpr viruses. The results of three independent HIV-1/BlaM-Vpr fusion experiments are summarized in the bar graph. The error bars indicate standard deviations. (D) Illustration of IFITM3 mutations. The N-terminal sequences of IFITM3 were deleted to create mutants Δ(1-6), Δ(1-11), and Δ(1-16). Their expression in the Dox-inducible SupT1 cell lines was examined in Western blots. (E) The Δ(1-11) and Δ(1-16) mutants are less effective in inhibiting HIV-1 entry than the wild-type IFITM3. The results of three independent HIV-1 fusion experiments are shown in the bar graph.

FIG. 5.

Association of IFITM2 and IFITM3 with endocytosis. 293 cells were transiently transfected with vector DNA that expressed Flag-tagged IFITM1, IFITM2, or IFITM3. Forty hours after transfection, the cells were fed with Alexa Fluor 555-conjugated transferrin (red) at 37°C for 10 min before being fixed with 4% paraformaldehyde (in 1× phosphate-buffered saline). IFITM proteins (green) were detected by immunostaining them with anti-Flag antibody.

FIG. 6.

Effects of IFITM proteins on HIV-1 cDNA synthesis and viral Gag expression. (A) IFITM-inducible SupT1 cells were infected with HIV-1 (BH10). Real-time PCR was performed using primers that amplify either the early or the late viral cDNA products. The levels of integrated viral DNA were determined by Alu-gag PCR. The results shown are the averages of three independent infections. The error bars indicate standard deviations. (B) Western blots of HIV-1 Gag protein expression in IFITM-inducible SupT1 cells that were infected with HIV-1 (BH10). The results shown represent three independent infection experiments.

FIG. 7.

The anti-HIV-1 activity of IFITM1 does not depend on its C-terminal sequence. (A) Illustration of IFITM1 deletion mutants. Sequences from the IFITM1 C terminus were deleted. (B) Doxycycline-induced expression of IFITM1 mutants in SupT1 cells. (C) HIV-1 replication in SupT1 cells that express IFITM1 mutants. SupT1 cells were cultured in media with or without doxycycline (500 ng/ml). The results shown represent three independent spreading-infection experiments. (D) Infection of SupT1 cells with the NLEY1-IRES virus. For details, refer to the legend to Fig. 3B. (E) Comparison of doxycycline-induced expression of IFITM1 and Δ(108-125) to that of IFN-α2b-induced endogenous IFITM1. Cell lines were exposed to doxycycline (500 ng/ml), IFN-α2b (500 U/ml), or both for 16 h before the levels of IFITM1 and Δ(108-125) were assessed in Western blots using anti-IFITM1 antibody. Tubulin was probed as the internal control.

FIG. 8.

Roles of the N-terminal sequence and the loop sequence of IFITM1 in suppressing HIV-1 infection. (A) Illustration of IFITM1 mutants. All mutants lack the C-terminal sequence from amino acids 108 to 125. Red letters indicate the mutated amino acids. The mutated peptide sequence for each location is underlined. The transmembrane domains are shown in boxes. (B) Expression of the IFITM1 mutants in stably transduced SupT1 cell lines upon treatment with doxycycline (500 ng/ml). (C) Replication of HIV-1 in SupT1 cell lines that express IFITM1 mutants. The results shown represent three independent spreading-infection experiments. (D) Effects of IFITM1 mutants on HIV-1 infection in one-cycle infection assays. For details, see the legend to Fig. 3B.

FIG. 9.

Knockdown of IFITM1, IFITM2, and IFITM3 increases HIV-1 infection of TZM-bl cells. (A) Expression of endogenous IFITM1, IFITM2, and IFITM3 in different cell lines. Western blots were probed with anti-IFITM1, -IFITM2, or -IFITM3 antibody. (B) Western blots to assess the knockdown of IFITM1, IFITM2, and IFITM3 in TZM-bl cells that were transfected with mixtures of siRNA oligonucleotides. (C) Following transfection with siRNA oligonucleotides, TZM-bl cells were exposed to HIV-1 infection. The levels of luciferase activity in the TZM-bl cell lysates were measured 40 h after infection. The values (relative luciferase units [RLU]) represent the susceptibility of TZM-bl cells to HIV-1 infection under different siRNA treatments. The results shown are the averages of three independent experiments.