Journal: PLoS ONE
Article Title: IFITM Proteins Restrict Antibody-Dependent Enhancement of Dengue Virus Infection
doi: 10.1371/journal.pone.0034508
Figure Lengend Snippet: (A) K562 cells were treated with the indicated concentrations of human interferon-α or mock treated, and lysed for Western blot analysis 48 h later, using an antibody that recognizes IFITM1 alone, or one that recognizes both IFITM2 and IFITM3. (B) K562 cells were stably transduced to express human IFITM1, 2, or 3 or with the control vector pQCXIP. IFITM protein expression was measured by Western blot using an anti-c-myc antibody. (C) Stably transduced K562 cells characterized in panel B were infected with an MLV-based retrovirus expressing EGFP and pseudotyped with MACV or IAV H5 entry proteins, as previously described . Infection was determined by flow cytometry, and normalized to K562 cells transduced with vector alone. (D) K562 cells characterized in panel B were infected with infectious DENV2 NGC strain (labeled “DENV2” for virus only) at the indicated MOI, or the same amount of infectious DENV2 pre-opsonized with enhancing titers of antibodies against DENV structural proteins prM or E (“+2H2” and “+4G2” respectively) , . Cells were washed after 1.5 h and incubated for ∼24 h. Intracellular staining of DENV antigen was performed with a DyLight-649-conjugated antibody against prM and infection was determined by flow cytometry. Experiment is representative of three with similar results. (E) An experiment similar to that shown in panel D was performed except that infection was assayed by measuring viral loads in the supernatant by plaque assays using BHK cells. (F) An experiment similar to that in panel D was performed, except that J774A.1 murine macrophage cells were stably transduced to express murine orthologs of IFITM1, 2, or 3 or with the control vector pQCXIP. Stably transduced cells were infected with infectious DENV2 NGC strain at ∼MOI 5 and incubated for ∼2 days before harvesting for flow cytometry. Error bars indicate standard error. Single and double asterisks indicate statistically significant ( P <0.05 and P <0.005, respectively) differences between IFITM protein expressing and control cells for corresponding infection conditions.
Article Snippet: Endogenous IFITM protein expression was detected by polyclonal rabbit anti-IFITM1 (FL-125, Santa Cruz Biotechnology) or rabbit anti-IFITM2 (12769-1-AP, Proteintech Group, cross reacts with IFITM3 protein).
Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Expressing, Infection, Flow Cytometry, Transduction, Labeling, Incubation, Staining