IFITM proteins restrict both direct and ADE-mediated DENV infection of K562 cells.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Huang1" target="_blank">[9]</a>. 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Brass1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Diamond1" target="_blank">[21]</a>. 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 (<i>P</i><0.05 and <i>P</i><0.005, respectively) differences between IFITM protein expressing and control cells for corresponding infection conditions.