For cell surface staining of -DG with mAb IIH6, cells were detached with enzyme-free cell dissociation solution (Sigma), resuspended in fluorescence-activated cell sorter (FACS) buffer (1% [vol/vol] FBS, 0
For cell surface staining of -DG with mAb IIH6, cells were detached with enzyme-free cell dissociation solution (Sigma), resuspended in fluorescence-activated cell sorter (FACS) buffer (1% [vol/vol] FBS, 0.1% [wt/vol] sodium azide, PBS), and plated in conical Rabbit Polyclonal to NR1I3 96-well trays. previously shown Tazemetostat hydrobromide for LCMV, we found that protein O mannosylation of -DG is crucial for the binding of arenaviruses of Tazemetostat hydrobromide unique phylogenetic origins, including LFV, Mobala computer virus, and clade C New World arenaviruses. In contrast to the highly conserved requirement for O mannosylation, more generic O glycans present on -DG are dispensable for arenavirus binding. Despite the crucial role of agglutinin, and lectin II were obtained from Vector laboratories (Burlingame, CA). The Steady-Glo and Bright-Glo luciferase assay systems were obtained from Promega (Madison WI). Heparan sulfate (HS) and heparin were purchased from Sigma. Cell lines. Vero-E6 cells, HEK293 cells, A549 cells (ATCC CCL-185), and human hepatoma Huh7 cells (36) were cultured in Dulbecco’s altered Eagle’s medium (DMEM), 10% (vol/vol) fetal bovine serum (FBS) supplemented with glutamine, and penicillin-streptomycin. Human umbilical cord vascular endothelial cells (HUVEC) purchased from Cambrex (Walkersville, MD) were cultured according to the company’s recommendations. The human B-cell lines WIL-2 NS and Daudi, the human T-cell collection Jurkat, and immortalized human T cells (kindly provided by Philippe Galley, Scripps Research Institute) were cultured as explained previously (41). The O-mannosylation-deficient cell lines Lec15.2 and Lec35 were maintained as described previously (24), and the mutant collection psg-745 was maintained as outlined previously (41). DG+/? and DG?/? embryonic stem (ES) cells were maintained as explained previously (22). Computer virus strains, purification, and quantification. Recombinant adenoviral vector (AdV)-Ad5-LARGE-enhanced green fluorescent protein (GFP) (EGFP) and Ad5-EGFP have been explained previously (4), Tazemetostat hydrobromide as have LCMV clone 13 (cl-13) and WE2.2 (1, 47). Purified LCMV stocks were produced and titers were determined as explained previously (15). UV inactivation of LCMV was carried out as explained previously (30). LFV (Josiah), Mobala computer virus, Oliveros computer virus, Parana computer virus (PAR), and Amapari computer virus (AMA) were produced in Vero-E6 cells in a biosafety level 4 (BSL-4) facility, polyethylene glycol precipitated, and -inactivated at the Center for Disease Control and Prevention in Atlanta, GA, according to a method explained previously (16). Inactivation was verified by contamination assay. Work with infectious viruses was carried out at BSL-3, with the exception of LFV, which was dealt with in the BSL-4 laboratories at the Special Pathogens Branch, and LCMV, which was dealt with at BSL-2 at The Scripps Research Institute. Production of retroviral pseudotypes. Recombinant Moloney murine leukemia computer virus (MLV) pseudotypes were produced as explained previously (41). The package cell collection GP2-293 (BD Biosciences) was transfected with the packable MLV genome pLZRs-Luc-gfp, which contains a luciferase reporter gene and a GFP reporter (52), provided by Gary Nabel. The GPs of AMA, LCMV cl-13, LFV, and VSV were provided in by cotransfection with expression plasmids made up of their full-length cDNAs. Briefly, 1.2 107 GP2-293 cells were plated in poly-l-lysine-coated T175 tissue culture flasks. After 16 h, cells were cotransfected with 20 g each of pLZRs-Luc-gfp and the GP expression plasmid using calcium phosphate. Forty hours after transfection, cell supernatants were harvested and cleared by centrifugation for 15 min at 3,000 rpm. Retroviral pseudotypes were then concentrated by ultracentrifugation at 25,000 rpm at 4C for 2 h using an SW28 rotor. Supernatants were discarded after centrifugation, and pellets were resuspended for 16 h in DMEM-20 mM HEPES (pH 7.5) at 4C as described above. For determinations of titers, monolayers of Vero-E6 cells were infected with serial dilutions of pseudotypes. After 48 h, cells were fixed for 10 min in 2% (wt/vol) paraformaldehyde-phosphate-buffered saline (PBS). GFP was detected by using a rabbit anti-GFP polyclonal antibody (3080; Chemicon) diluted 1:100 in PBS-1% (vol/vol) FBS-0.1% (wt/vol) saponin that was incubated overnight at 4C. After several washes, bound main antibody was detected with an anti-rabbit IgG-FITC (Fab)2 (1:50 in PBS-1% [vol/vol] FBS-0.1% [wt/vol] saponin) applied for 45 min in the dark. Specimens were examined under a fluorescence microscope, clusters of GFP-positive cells were counted, and titers were calculated. Detection of arenavirus GP incorporated into retroviral pseudotypes. For the detection of GP in the retroviral pseudotypes, three impartial preparations of concentrated viruses.