The coverslips were dried and mounted in anti-fade fluorescence installation medium (ab104135; abcam). bars represent the standard error of the mean of results of four impartial experiments for A549 cells and standard deviation of results of three impartial experiments for HUVEC, each performed in triplicate. Statistical significance was determined by ordinary two-way ANOVA; values were corrected for multiple comparisons by Dunnetts method (ns, values were corrected for multiple comparisons by Dunnetts method (ns, values were corrected for multiple comparisons by Dunnetts method (ns, values were corrected for multiple comparisons by Dunnetts method (ns, (32, 33). Both viruses encode a set of glycoproteins (g) that mediate entry and are conserved among herpesviruses. Of these, gH, gL, and gB are the most extensively studied (reviewed in reference 34). KSHV and RRV enter many cell types through the conversation of the gH/gL complex with members of the ephrin receptor tyrosine kinase family (Ephs) (35,C37) and, in the case of RRV, also with members of the plexin domain-containing protein family (38). KSHV also interacts with heparan sulfate and integrins (39,C41). Entry of both viruses occurs mainly via endocytotic routes (33, 42,C44). Following internalization, the viral membrane fuses with the host membrane. Several reports implicate the gH/gL complex together with gB as the minimal set of glycoproteins required for membrane fusion (38, 45, 46). One study reported an enhancing ENPP3 role of IFITMs in the infection of the BJAB B cell line and human dermal microvascular endothelial cells (HMVEC-D) cells by KSHV, EBV, and herpes simplex virus 2 (HSV-2) (21). However, given the considerable differences between KSHV and RRV entry into B cells and different adherent cells (33, 36, 37), in particular since KSHV contamination of B cell lines is usually, with a few exceptions, efficient only through cell-to-cell transfer (47,C49), we hypothesized that IFITM-mediated restriction may be dependent on the nature of the target cell. Another question that Pitolisant hydrochloride we sought to address is usually whether IFITMs restrict RRV in human cells. RESULTS KSHV induces IFITM expression in A549 cells. We first validated the specificity of the antibodies used in this study for Western blot analysis after directed expression (Fig.?1A). Next, we examined expression of IFITM proteins at baseline levels and after stimuli such as virus contamination in the human lung epithelial cell line A549, which has been well characterized with regard to IFN signaling and IFITM expression (50,C53). We infected the cells with KSHV BAC16 recombinant virus carrying a green fluorescent protein (GFP) reporter gene and RRV-YFP carrying a yellow fluorescent protein (YFP) reporter gene (Fig.?1B). Treatment with H2O and IFN- served as negative and positive controls for IFITM induction, respectively. IFITM2 and IFITM3 were detected at low levels without IFN treatment, while IFITM1 and human Pitolisant hydrochloride myxovirus resistance protein 1 (MxA), another IFN-induced protein, were not detectable without stimulation (Fig.?1C). At the 1-h time point, neither treatment induced IFITM or MxA expression relative to the background. At the 24-h time point, induction over background levels of Pitolisant hydrochloride IFITM1, IFITM2, IFITM3, and MxA was observed in IFN–treated or KSHV-infected cells but not in RRV-infected cells. At 48 h, IFITM3 was also slightly induced by RRV, and IFITM2 induction relative to H2O treatment was barely discernible anymore. Basal IFITM expression also increased slightly over time after plating. In summary, KSHV-containing inoculum and IFN- induced IFITM expression. Open in a separate window FIG?1 KSHV induces IFITM1, IFITM2, and IFITM3 expression in A549 cells. (A) Western blot of 293T cells transduced with pQCXIP constructs to express IFITM1 to -3 or pQCXIP (empty vector). IFITMs were detected using the respective IFITM antibody, and GAPDH served as a loading control. (B and C) Fluorescence microscopy images (scale bar, 200?m) (B) and Western blot analysis (C) of A549 cells infected with KSHV-GFP or RRV-YFP or treated with H2O or IFN- (5,000 U/ml) for the indicated time and harvested using SDS sample buffer. IFITM expression was detected with antibodies shown in panel A. MxA served as control for IFN-stimulated gene induction; GAPDH served as a loading control. Triple knockout of IFITM1/2/3 enhances KSHV and RRV contamination of A549 cells and human foreskin fibroblasts (HFF). Overexpression of IFITMs alters their subcellular localization (6; our observations), IFITMs are usually induced together, and recent studies report that IFITMs form homo- and hetero-oligomers (54,C56) and might thus take action synergistically. We therefore used CRISPR/Cas9 to generate triple IFITM1/2/3 knockout cells to study.