Biol

Biol. and inhibited PY-STAT1 interaction with karyopherins 1, 5, and 6. Consistent with their ability to inhibit the karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN–induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with karyopherins 1, 5, or 6 by binding within the PY-STAT1 binding region of the karyopherins and that this function is conserved among the VP24 proteins of different Ebola virus species. Ebola virus (EBOV) and Marburg virus comprise the family luciferase reporter plasmid. Twenty-four hours posttransfection, cells were washed and maintained in DMEM containing 0.3% bovine serum albumin, with or without (mock-treated control) 1,000 U/ml of type I interferon (PBL Biomedical Laboratories, Piscataway, NJ). Sixteen hours post-IFN treatment, cells were GSK189254A harvested using reporter lysis buffer (Promega, Madison, WI) and analyzed for CAT and luciferase activities. The CAT activity was quantified by using GSK189254A a PhosphorImager and normalized to the luciferase activity. Alternatively, dual luciferase assays (Promega) were performed, and firefly luciferase activity was normalized to luciferase activity. Open in a separate window FIG. 6. EBOV VP24 proteins inhibit reporter gene expression in human and murine cells. 293T (A), HuH7 (B), NIH-3T3 (C), or MEF (D) cells were transfected with the ISG54-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and either empty vector (vector) or the indicated plasmids expressing various concentrations (a, 500, 100, 20, and 4 ng; b, 500, 100, 20, and 4 ng; c, 500, 100, and 20 ng; and d, 500, 100, and 20 ng) of ZEBOV VP24 (Z-VP24), MA ZEBOV VP24 (MA-VP24), or REBOV VP24 (R-VP24). At 1 day posttransfection the cells were either mock treated or treated with 1,000 U/ml of universal type I IFN or human IFN- (for 293T and HuH7 cells) or murine IFN- (for NIH-3T3 cells and MEFs). At 16 h posttreatment the cells were harvested and assayed for reporter activities. RESULTS VP24 interacts with endogenous karyopherin 1. To determine if VP24 is able to interact with endogenous karyopherin 1, cells were transfected with a FLAG-tagged VP24 or VP35. One day posttransfection, immunoprecipitations were performed with an anti-FLAG monoclonal antibody, and the precipitated material was analyzed by Western blotting. Karyopherin 1 was coprecipitated with FLAG-VP24 but not with FLAG-VP35 (Fig. ?(Fig.1).1). This result indicates that EBOV VP24 is indeed able to interact with endogenous karyopherin 1. Open in a separate window FIG. 1. HA-VP24 interacts with endogenous karyopherin 1. 293T cells were transfected with either FLAG-VP24 or FLAG-VP35, and 1 day posttransfection immunoprecipitation was performed with the anti-FLAG monoclonal antibody M2 bound to agarose beads. The immunoprecipitated material (FLAG-IP) and whole-cell extract (WCE) were then subjected to Western blot analysis with a monoclonal antibody against karyopherin 1 (K1) and an anti-FLAG monoclonal antibody. VP24 and PY-STAT1 bind to overlapping regions of karyopherin 1. We previously demonstrated that EBOV VP24 binds karyopherin 1 and in doing so disrupts the interaction of an activated STAT1-green fluorescent Rabbit Polyclonal to YOD1 protein (GFP) fusion protein with karyopherin 1 (45). PY-STAT1 interacts with the C-terminal armadillo (arm) repeats of karyopherin 1 for transport into the nucleus (39, 48); it was therefore of interest to determine if VP24 binds to a similar region of the karyopherin. Several karyopherin 1 truncation mutants were GSK189254A constructed (Fig. ?(Fig.2A)2A) and tested for their ability to interact with PY-STAT1 and an HA-tagged VP24. Full-length FLAG-tagged karyopherin 1 (FLAG-K1) and the truncation mutants were transfected alone (Fig. ?(Fig.2B)2B) or along with HA-VP24 (Fig. ?(Fig.2C).2C). At 1 day posttransfection, immunoprecipitations were performed with an anti-FLAG monoclonal antibody, and the precipitated material was analyzed by Western blotting. In the absence of IFN- treatment, STAT1 did not coprecipitate with FLAG-K1 (data not shown); however, upon IFN- treatment STAT1.