FIG. 4.

Expression of the α4 integrin subunit enhances cell permissivity to Py at a postattachment step. (A) Addition of α4 function-blocking Abs prior to or after virus adsorption blocks the increased infectivity of Py in α4-BALB/c 3T3 cells. Mock-transfected and α4-BALB/c 3T3 cells were either pretreated or not pretreated for 1 h at 4°C with 30 μg of anti-α4 or its isotype Ab per ml before infection with Py for 1 h at 4°C (left panel) or after infection with Py for 1 h at 4°C (right panel). After the virus particles were removed, cells were further incubated for 20 h at 37°C before fixation and immunostaining for LT. Results are reported with the value obtained when mock-transfected cells were preincubated with DMEM without competitors (control) considered to be 100% virus infectivity (average of 1% LT positive cells). Results of a representative experiment performed in duplicate are reported as described for Fig. 3B. (B) Characterization of the purity and labeling efficiency of biotinylated Py viral particles. Left panel, SDS-PAGE of purified viral particles (500 ng) stained with GelCode Blue staining reagent. Right panel, Western blot analysis of biotinylated viral particles with HRP-conjugated streptavidin. (C) Function-blocking MAb directed to α4 does not affect the binding of biotinylated Py particles to α4-BALB/c 3T3 cells. α4-BALB/c 3T3 cells were pretreated for 30 min on ice with a 30-μg/ml concentration of Ab directed to α4 integrin (right panel) or its isotype (left panel) before treatment with 500 ng of biotinylated viral particles for 1 h at 4°C. Cells incubated with the binding buffer alone are shown as negative control (shaded area). After two washes, cells were incubated for 30 min with PE-conjugated streptavidin. Fixed cells were analyzed by FACS. The histograms display relative cell numbers as a function of relative fluorescence intensities. (D) SA-containing receptors are involved at the attachment level. One microgram of biotinylated viral particles was incubated for 1 h at 4°C with or without NANA and then added to α4-BALB/c 3T3 cells pretreated with 200 mU of neuraminidase (Neu) and left for 1 h at 4°C. Binding of virus on untreated cells is also shown. Cells incubated with the binding buffer alone as a negative control are shown (shaded area). After two washes, cells were incubated for 30 min with PE-conjugated streptavidin. Fixed cells were analyzed by FACS. The histograms display relative cell numbers as a function of relative fluorescence intensities.