As alluded to earlier, two successive steps are involved in protein degradation: (1) covalent attachment of ubiquitins to the target protein substrate, and (2) degradation of the polyubiquitinated protein by the 26S proteasome with the release of ubiquitin for recycling. To dissect out the role of ubiquitination and degradation in CVB3 infection, we next decided to investigate the protein ubiquitination after CVB3 infection. As shown in Fig. 2A , protein ubiquitination was gradually increased along the time-course of CVB3 infection, which was accompanied by a decrease of free ubiquitin levels. Densitometric analysis further demonstrated that the increases in protein ubiquitination at 3 h, 5 h, and 7 h post-infection were statistically significant as compared to sham infection (Fig. 2B) . We have previously demonstrated that 26S proteasome activities were unchanged during CVB3 infection [9] . Thus, the finding of increased accumulation of ubiquitinated proteins is likely due to enhanced protein ubiquitination as opposed to reduced proteasome activity. Decreased levels of free ubiquitin could be a direct consequence of the increased protein ubiquitination. These results suggest that enhanced ubiquitin conjugation may be a prerequisite for efficient synthesis of CVB3 viral RNA and continuation of its lifecycle.