FIG. 3.

CVB3 allows trafficking of nascent MHC class I molecules early in infection and then mediates their rapid disappearance. All experiments were repeated at least three times, and the results shown are for a single representative experiment. (A) To ascertain the kinetics of nascent MHC class I trafficking to the cell surface, a citrate-phosphate low-pH wash strategy was employed (34, 37). Before the wash (left panel), flow cytometric analysis indicated that approximately 98% of the cell population stained positive for β₂M (upper right quadrant). Following a citrate-phosphate wash (pH 3.0) for 1 min, preexisting β₂M associated with the MHC class I heavy chain was rendered virtually undetectable within the cell population (right panel, upper left quadrant). (B) HeLa (RW) cells were mock infected or infected with CVB3 at an MOI of 10. Following adsorption, cells were washed with citrate-phosphate buffer as described above. At the indicated time points, the percentage of cells expressing nascent β₂M at the cell surface was measured by flow cytometry and plotted. One sample contained 2 mM guanidine hydrochloride, a known inhibitor of picornavirus RNA synthesis. Two additional uninfected cell populations contained either 2 μg/ml BrA (to halt anterograde secretory transport) or 100 μg/ml cycloheximide (to stop protein synthesis). (C) To analyze anterograde trafficking kinetics at later times postinfection, three acid-washed cell populations were treated exactly as described for panel B (uninfected [black diamonds], infected with CVB3 [yellow triangles], or left uninfected and treated with BrA [red squares]). Three additional populations were CVB3 infected at time zero and washed at 3, 4, or 5 h postinfection (orange, blue, and green plots, respectively).