Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhances PEDV infection in LLC-PK1 cells. ( A ) Western blot analysis of porcine IFITM1 and IFITM2/3 overexpression in LLC-PK1 cells transduced with lentiviral vectors expressing porcine HA-tagged porcine IFITM1, porcine IFITM2/3, or an empty vector as a negative control. Porcine IFITM1- or IFITM2/3-overexpressing LLC-PK1 cells were infected with the PEDV-HM strain. ( B ) Viral RNA copies in the supernatant were quantified by RT-qPCR and are presented as copy numbers. The data are presented as the means ± s.d. from three technical repeats. ( C ) Viral titers in the supernatant were measured via the TCID 50 assay. The data are presented as the means ± s.d. from three independent experiments. ( D ) LLC-PK1 cells overexpressing porcine IFITM1 or IFITM2/3 were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, while background cells were visualized via white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Western Blot, Over Expression, Transduction, Expressing, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Interaction and colocalization of the PEDV S protein with IFITM proteins. ( A ) Co-IP assays were performed to assess the interaction between the PEDV S1 protein and human or porcine IFITM proteins. HEK293T cells were cotransfected with plasmids encoding PEDV S1-Fc and various HA-tagged IFITM proteins. At 24 h post-transfection, the cells were harvested. Immunoprecipitation was conducted using an Fc tag antibody, and the presence of IFITM proteins was detected by Western blotting (IB: Anti-HA). Whole-cell lysates (WCLs) were analyzed to confirm expression levels (IB: Anti-Fc, Anti-HA, Anti-β-Tubulin). The Co-IP was repeated three times, yielding similar results. ( B ) Confocal microscopy images showing the intracellular localization of porcine IFITM1 in LLC-PK1 cells are presented. IFITM1 (red) colocalizes with clathrin (green), EEA1 (green), Rab7 (green), and LAMP7 (green), as indicated in the merged images. DAPI (blue) was used to stain the nuclei. ( C ) Confocal microscopy images demonstrating the colocalization of the PEDV S protein (red) with HA-IFITM1 (green) in LLC-PK1 cells are shown. The merged image shows the nuclei stained with DAPI (blue).

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Staining

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhanced PEDV infection in porcine small intestinal organoids. ( A ) Porcine small intestinal crypts were isolated from neonatal piglets and cultured in vitro . A representative image depicting the morphology of porcine small intestinal organoids is shown. Scale bar: 200 µm. ( B ) Immunofluorescence confirmation of HA-tagged porcine IFITM1 overexpression in PSIOs. Representative images displaying IFITM1 (HA, green) and nuclei (DAPI, blue) are presented. ( C ) The expression of IFITM1 was verified via immunofluorescence in a 2D organoid culture system. Representative images showing HA (green) and nuclei (DAPI, blue) staining are presented. ( D ) Analysis of viral infection in IFITM1-overexpressing and control 2D organoids following PEDV infection is shown. IFITM1-overexpressing or control 2D organoids were infected with PEDV-HM (0.1 MOI). At 24 hpi, the virus titer in the supernatant was quantified according to the TCID 50 . The data are presented as the means ± s.d. from three independent experiments. **, P < 0.01. ( E ) IFITM1-overexpressing or control 2D organoids were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, and the background cells were visualized under white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Isolation, Cell Culture, In Vitro, Immunofluorescence, Over Expression, Expressing, Staining, Control, Virus, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Monoclonal antibody against human IFITM1 (catalog number 60047-1), rabbit polyclonal antibody against human IFITM3 (catalog number 11714-1-AP), which also efficiently recognizes IFITM2 and weakly cross-reacts with IFITM1, were purchased from Proteintech Group, Inc.

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 7. HIV Nef can help overcome IFITM-mediated restriction of protein synthesis. (A) Level of virus production in HEK293T cells transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with a deletion (ΔNef) was measured by p24 ELISA 48 hours post-transfection and the data normalized, fold change of virus production compared to vector control is indicated. Differences were assessed with Student’s t tests. (B) Intracellular level of p55/p24 and IFITMs was measured by immunoblotting. (C) SupT1 cells were infected with the indicated dilutions of wild type or Nef-deleted (ΔNef) HIV-1 NL4-3 inoculum and then treated with 1 μg/ml doxycycline to induce IFITM expression post-entry and 5 μM AMD3100 to limit infections to a single round. Level of virus production was measured by p24 ELISA 72 hours post-infection. Differences were assessed with Two-way ANOVA and Bonferroni post-tests. (D) C8166 cells constitutively expressing either vector control or IFITM1 were infected with either wild type or Nef-deleted (ΔNef) HIV-1 NL4-3. Levels of virus production were measured by p24 ELISA at the indicated time-points post-infection and were normalized to the levels of virus produced from vector controls. Differences were assessed with Two-way ANOVA. (E) HEK293T cells were transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with either wildtype NL4-3 nef or the indicated lentiviral nef alleles. Virus production was measured by p24 ELISA 48 hours post-transfection and the data normalized. Differences were assessed with Student’s t tests. (F) HEK293T cells were transfected with ΔNef HIV-1 NL4-3 proviral DNA, expression vectors for IFITMs and an increasing proportion of HIV-1 Nef-encoding vector versus empty vector in a fixed total quantity of 1 μg. Level of virus production was measured by p24 ELISA 48 hours post- transfection, while levels of viral proteins and IFITM-FLAG expression was analyzed by (G) immunoblotting. Differences were assessed by Student’s t-test. All data show mean + SEM from 3 independent experiments and *denotes p < 0.05.

Article Snippet: The following antibodies were used to detect IFITMs: human IFITM1 (clone 5B5E2, Proteintech), IFITM2 (clone 3D5F7, Proteintech) and human IFITM3 (clone EPR5242, Novus Biologicals).

Techniques: Virus, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Western Blot, Infection, Produced

Journal: Cell reports

Article Title: Tuberculosis exacerbates HIV-1 infection through IL-10/STAT3-dependent tunneling nanotube formation in macrophages

doi: 10.1016/j.celrep.2019.02.091

Figure Lengend Snippet: (A) Vertical scatter plots showing the Median Fluorescence Intensity (MFI) of cell-surface receptors involved in HIV-1 entry (CD4, CCR5, CXCR4) on monocytes differentiated for 3 days under the presence of CmCTR and CmMTB. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. (B) Histogram showing the percentage of HIV-1 fusion with CmCTR- (white) or CmMTB (black)-pre-treated cells, as determined using the Blam-Vpr assay in the presence of entry inhibitor Maraviroc (dashed bars). (C) Left: Representative images of Western Blot analysis illustrating the expression of IFITM1/2/3 and Actin as loading control. Right: Quantification of IFITM1/2/3 expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR (white) and CmMTB (black). n = 6 donors. (D) Representative images of Western Blot analysis illustrating the expression of SAMHD1 and its phosphorylated version (pSAMHD1), and Actin as loading control (left). Quantification of SAMHD1 (center) pSAMHD1 (right) expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 11 donors. (E) Representative images of Western Blot analysis illustrating the expression of C/EBP-β (LAP), C/EBP-β (LIP), and Actin as loading control (left). Quantification of C/EBP-β (LAP, center left) and C/EBP-β (LIP, center right) expressed as a ratio related to actin, and LAP expressed as a ratio related to LIP (right), of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. LAP is an activator of HIV-1-LTR whereas LIP is a repressor of HIV-1-LTR. (F) Representative images of Western Blot analysis illustrating the expression of CUGBP1 and Actin as loading control (left). Quantification of CUGBP1 expressed as a ratio related to actin (right) of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. (G) Quantification of LC3-II expression as a ratio to actin of monocytes differentiated for 3 days under the presence of CmCTR and CmMTB at the indicated time points after 2h treatment with Bafilomycin A1 (BafA1) or DMSO as control, as measured by western blot analysis. Uninfected cells at day 3 of the experiment (left, n = 6 donors), and HIV-infected cells at 1 (day 4, center, n = 4 donors) and 3 (day 6, right, n = 6 donors) days post-infection. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. Data in histograms are represented as mean ± SD. * p≤0.05; ** p≤0.005; *** p≤0.0005; **** p≤0.0001.

Article Snippet: Anti-human IFITM1/2/3 , Santa Cruz Biotechnology , Clone FL-125.

Techniques: Fluorescence, Western Blot, Expressing, Infection

Journal: Cell reports

Article Title: Tuberculosis exacerbates HIV-1 infection through IL-10/STAT3-dependent tunneling nanotube formation in macrophages

doi: 10.1016/j.celrep.2019.02.091

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-human IFITM1/2/3 , Santa Cruz Biotechnology , Clone FL-125.

Techniques: Purification, Recombinant, Luciferase, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Software, Imaging