HeLa cells were grown in complete medium to 70-80% confluence, and then infected at a multiplicity of infection (MOI) of 10 with CVB3 or sham-infected with phosphate-buffered saline (PBS) for 1 h in serum-free DMEM. Cells were then washed with PBS and cultured in serum-free medium. For inhibition experiments, HeLa cells were infected with CVB3 for 1 h, washed with PBS, and then incubated with DMEM containing various concentrations of inhibitors. Immunoprecipitation and immunoblot analysis Cell lysates were prepared using lysis buffer (50 mM pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 mM Na 3 VO 4 , 10 mM HEPES (pH 7.4), 0.1% Triton X-100, and the protease inhibitor cocktail) as described previously [2] . For immunoblot analysis, equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked for 1 h with nonfat dry milk solution (5% in PBS) containing 0.1% Tween 20. Blots were then incubated for 1 h with the primary antibody followed by incubation for 1 h with the secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). When protein ubiquitination was examined, membrane was heat-activated by autoclaving at 121uC for 35 min prior to blocking with nonfat dry milk solution to enhance antigenic site recognition.