Fig 2: IFI44 protein impairs antiviral responses. HAP-1 WT and IFI44 KO cells were transfected with poly(I·C) or treated with IFN-a for 16 h. (A) Total cellular RNA was purified, and the levels of IFIT2 (A), IFI27 (B), and IFN-?1 (C) mRNAs were evaluated by RT-qPCR. Bars represent SDs determined using duplicate wells. Three different experiments were performed with similar results. (D) Cells that had been subjected to mock treatment, transfected with poly(I·C), or treated with IFN-a were infected with rVSV-GFP (MOI of 0.1). Virus production was analyzed at 24 hpi. Bars represent SDs determined using duplicate wells. Three different experiments were performed, with similar results. *, P < 0.05 (for comparisons between HAP-1 WT and IFI44 KO cells).

Fig 3: IFI44 decreases the kinase activity of IKKß and IKKe. Human 293T cells were silenced for IFI44, or for FKBP5, and were transfected with plasmids expressing His-IKKe (A) or MYC-IKKß (B), together with IFI44-HA, and FKBP5-FLAG expression plasmids. At 24 hpt, IKKe (A) and IKKß (B) complexes were purified with anti-His and anti-MYC antibodies, respectively, and these complexes were assayed in kinase assays using IRF-3 (for the IKKe complexes shown in panel A) and IkBa (for the IKKß complexes shown in panel B) as substrates. The levels of phosphorylated and unphosphorylated forms of IRF-3 (panel A, bottom blot) and IkBa (panel B, third and fourth blots) were analyzed by Western blotting using specific antibodies. Levels of IKKe were analyzed using an anti-His-specific antibody (A, first blot) and anti-pIKKe (A, second blot), and levels of IKKß were analyzed using an anti-MYC-specific antibody (B, first blot) and anti-pIKKß (B, second blot). Western blots were quantified by densitometry using ImageJ software (v1.46). Protein expression levels in cells expressing IKKe (A) and IKKß (B) alone were assigned a value of 100% for comparisons with the levels of expression in cells expressing the different combinations of IKKe/IFI44/FKBP5 (A) or IKKß/IFI44/FKBP5 (B) (numbers are indicated below each plot). pIRF-3 and IRF-3 levels (observed in the same bottom blot in panel A) and pIkBa and IkBa (third and bottom blot in panel B) are represented with numbers below each blot. Levels of pIRF-3 and pIkBa normalized to the levels of IKKe and IKKß are represented in the bottom graphs in panels A and B, respectively. Molecular weight markers are indicated (in kilodaltons) on the right.

Fig 4: IFI44 interacts with FKBP5 and does not inhibit binding of FKBP5 to IKKß or IKKe. (A to C) Human 293T cells were transiently cotransfected with the pCAGGS plasmid encoding IFI44-HA and FKBP5-FLAG, or with empty plasmids, as internal controls. IB, immunoblot. (A and B) Coimmunoprecipitation experiments using anti-HA to pull down IFI44 (A) and anti-FLAG to pull down FKBP5 (B) using affinity columns were performed. Western blotting using antibodies specific for the HA tag (to detect IFI44) or the FLAG tag (to detect FKBP5 protein) was performed to detect protein in the cellular lysates (Input) and after the Co-IP. Molecular weight markers are indicated (in kilodaltons) on the right. Three different experiments were performed, with similar results. (C) At 24 hpi, cells were fixed with paraformaldehyde, FKBP5-FLAG and IFI44-HA were labeled with antibodies specific for the tags (in green and red, respectively), and nuclei were stained with DAPI (in blue). Areas of colocalization of both proteins appear in yellow in the third picture and in white in the fourth picture. Scale bar, 10 µm. (D) Human A549 cells were transiently cotransfected with the pCAGGS plasmid encoding IFI44-HA and FKBP5-FLAG, or with empty plasmids, as internal controls. At 24 hpt, the cells were subjected to mock treatment, transfected with poly(I·C), treated with IFN-a, or infected with IAV for 24 h. Western blotting using antibodies specific for the HA tag (to detect IFI44), the FLAG tag (to detect FKBP5), anti-actin, and IAV anti-NP proteins was performed. Molecular weight markers are indicated (in kilodaltons) on the right. Two different experiments were performed, with similar results. (E and F) Human 293T cells were transiently cotransfected with different combinations of pCAGGS plasmids encoding IFI44-HA, FKBP5-FLAG, and MYC-IKKß (E) or IFI44-HA, FKBP5-FLAG, and MYC-IKKe (F). Co-IP experiments using an anti-FLAG affinity column (to pull down FKBP5) were performed. Western blotting using antibodies specific for the MYC tag (to detect IKKß or IKKe), the HA tag (to detect IFI44), and the FLAG tag (to detect FKBP5) was performed to detect protein in the cellular lysates (Input) and after the Co-IP. Molecular weight markers are indicated (in kilodaltons) on the right. Three different experiments were performed, with similar results.

Fig 5: IFI44 negatively affects IKKß and IKKe activation. (A) IFI44-silenced human 293T cells were transiently cotransfected with a plasmid expressing IKKß, a plasmid expressing the Fluc reporter gene under the control of NF-?B (pNF-?B-Fluc), and a plasmid constitutively expressing Rluc. (B) IFI44-silenced human 293T cells were transiently cotransfected with a plasmid expressing IKKe together with a plasmid expressing the Fluc reporter gene under the control of IFN-ß promoter (pIFNß-Fluc) and a plasmid constitutively expressing Rluc. (A and B) At 24 hpt, levels of Fluc were determined and normalized to the levels of Rluc. Data represents means and SDs of results from triplicate wells. Experiments were repeated three times with similar results. (C) Cellular lysates from cells analyzed as described for panel B were collected, and protein levels of pIRF-3 and actin were evaluated by Western blotting. Western blots were quantified by densitometry using ImageJ software (v1.46), and the amounts of pIRF-3 were normalized to the amounts of actin (numbers below the pIRF-3 blot). Molecular weight markers are indicated (in kilodaltons) at the right. (D and E) Tissue culture supernatants from cells analyzed as described for panel B were collected and used to treat fresh A549 cells. After 24 h of incubation, cells were infected (MOI of 0.1) with rVSV-GFP. At 24 hpi, GFP expression was quantified in a microplate reader (D) and GFP-infected cells were analyzed by visualizing GFP expression under a fluorescence microscope (E). Experiments were repeated three times with similar results. *, P < 0.05 (using Student’s t test [panels A, B, and D]). Representative images from a microscope using a 20× objective are shown in panel E. Scale bars, 50 µm.