Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: Vero (A), 293 (B), and HeLa (C) cells were incubated with the indicated concentrations of IFN-α for 24 h and then transfected with AiV replicon RNA. At the indicated time points after transfection, cell lysates were harvested, and luciferase activity was measured. Each right panel represents the protein levels of IFITM1, IFITM2, or IFITM3 by immunoblotting for cell lysates harvested from experiments shown in the left panels. Data are the means ±SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Luciferase, Activity Assay, Western Blot

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Luciferase, Activity Assay, Western Blot, Control, Knockdown, Glo Assay

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: Vero (A), 293 (B), and HeLa (C) cells were incubated with the indicated concentrations of IFN-α for 24 h and then transfected with AiV replicon RNA. At the indicated time points after transfection, cell lysates were harvested, and luciferase activity was measured. Each right panel represents the protein levels of IFITM1, IFITM2, or IFITM3 by immunoblotting for cell lysates harvested from experiments shown in the left panels. Data are the means ±SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Luciferase, Activity Assay, Western Blot

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Luciferase, Activity Assay, Western Blot, Control, Knockdown, Glo Assay

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Two Hybrid Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Western Blot, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid analysis was carried out to examine interactions between IFITM1 and ACBD3, PI4KB, OSBP, or CERT, and the results are shown as described in . Data are the mean ± SD of at least three independent experiments. (B) HA-tagged IFITM1 and FLAG-tagged ACBD3, PI4KB, OSBP, or CERT were cotransfected into 293T cells, and the cell lysates were subjected to immunoprecipitation with anti-HA antibody or control IgG. The resulting immunocomplexes and whole-cell lysates were detected by anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h, the cells were labeled with anti-FLAG and anti-ACBD3 (top), anti-PI4KB (middle), or anti-OSBP (bottom) antibodies. (D) Vero IFITM1 cells were incubated with Tet (−) or Tet (+) for 72 h, and the cells were fixed and double stained with the indicated antibodies. (E) Vero cells were transfected with HA-IFITM1. At 24 h, the cells were labeled using anti-HA, anti-OSBP and anti-EEA1, or anti-CD63 antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation (C-E).

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Immunoprecipitation, Control, Transfection, Labeling, Incubation, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–F) Vero cells were pretreated with DMSO (as the control) or the indicated concentrations of 25-HC (A), U18666A (B), or BF738735 (C) for 24 h and then transfected with AiV replicon RNA. At 10 h after transfection, the cells were analyzed for luciferase activity. Data were normalized to the DMSO treatment, and cell viability was scored. (D–F) Vero IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (–) or 1 μM of 25-HC (D), 1.5 μM of U18666A (E), or 150 nM of BF738735 (F). After 24 h, the cells were transfected with AiV replicon RNA, and luciferase activity was determined at 10 h after transfection. (G) Cell viability was measured in parallel. The maximum value obtained for Tet (–) drug-untreated cells was taken as 100%. Data are the mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Control, Transfection, Luciferase, Activity Assay, Incubation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–E) Vero-IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (A and D), 1 μM 25-HC (B), 1.5 μM U18666A (C), or 150 nM BF738735 (E), followed by pCMV-polyprotein transfection. At 24 h after transfection, the cells were fixed and stained with filipin III (A–C) or anti-PI4P (D and E), anti-IFITM1 and anti-2B antibodies. Bars, 4 μm. Pearson’s correlation coefficient was measured for individual cells.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Staining

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A and B) Vero-IFITM1 cells were incubated with or without Tet for 48 h, and then treated with 3 μM AY9944. After 24 h, the cells were fixed and stained with filipin III and the indicated antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Staining, Standard Deviation

Journal: mBio

Article Title: IFITM3 Reduces Retroviral Envelope Abundance and Function and Is Counteracted by glycoGag

doi: 10.1128/mBio.03088-19

Figure Lengend Snippet: IFITM2 and IFITM3 inhibit MLV infectivity and decrease Env levels in virus-producing cells. (A) 293T were cotransfected with env -deficient MLV (2.5 μg), pBabeLuc (0.6 μg), ecotropic Env (0.5 μg), and empty pCMV6 or pCMV6-IFITM3 (0.03, 0.09, 0.27, or 0.81 μg). (B) Same as in panel A, except that xenotropic Env was used. (C) 293T were cotransfected with env -deficient MLV (2.5 μg), pBabeLuc (0.6 μg), ecotropic Env (0.5 or 0.1 μg), and empty pCMV6 or pCMV6-IFITM3 (0.27 μg). (D) Same as in panel C, except that xenotropic Env (0.5 or 0.1 μg) was used. (E) 293T cells were cotransfected with env -deficient MLV, ecotropic Env (0.1 μg), and empty pCMV6, pCMV6-IFITM1, pCMV6-IFITM2, pCMV6-IFITM3, or pQCXIP-FLAG-mIfitm3 (0.81 μg). (F) Same as in panel E, except xenotropic Env (0.1 μg) was used. The specific infectivities of viruses produced by transfected cells were measured as described in Materials and Methods; the specific infectivity of virus produced with empty vector was set to 100%. Numbers next to blots indicate molecular weight standards in kilodaltons or specific proteins, when indicated. Data represent averages of three to five independent experiments; for each experiment, infectivities were measured in triplicate and averaged. Statistical analysis for panels A, B, E, and F was performed using one-way ANOVA. Statistical analysis for panels C and D was performed using the Student t test. An asterisk marks a statistically significant difference from empty vector or from the paired condition indicated. Error bars represent standard deviations. * , P < 0.05; * * , P < 0.0005.

Article Snippet: MEFs and IfitmDel MEFs were kindly provided by Michael S. Diamond (Washington University) ( , ). pCMV6-XL5 encoding untagged human IFITM1, IFITM2, or IFITM3 was purchased from Origene (SC117830, SC124192, and SC112616). pQCXIP encoding FLAG-Ifitm3 was obtained from Chen Liang (McGill University).

Techniques: Infection, Virus, Produced, Transfection, Plasmid Preparation, Molecular Weight