In the present study, we have provided further evidence that CVB3 manipulates the UPS for its infection. CVB3 infection results in increased protein polyubiquitination and a subsequent decrease in free ubiquitin levels. Knockdown of ubiquitin and ubiquitinmediated protein modification and/or degradation by siRNA It is increasingly apparent that viruses can evolve various strategies to utilize the host UPS for their own benefits. The UPS has been suggested to play a critical role in the different steps of viral lifecycle, including viral entry, viral replication, maturation, viral progeny release, and latent virus reactivation [32] [33] [34] . The mechanisms that the UPS regulates viral infection involve degrading intracellular proteins or excessive viral proteins that are against efficient viral replication and modulating viral protein function through ubiquitin-mediated modification or by directly encoding ubiquitin-related enzymes [35] . The finding in this study that CVB3 infection stimulates protein ubiquitination without inhibition of the core 20S proteasome activity highlights the possibility that CVB3 manipulates the UPS to destabilize or modulate the host and viral proteins. Polyubiquitination and degradation of host antiviral proteins has been suggested to be a mechanism of HIV-1 replication [36] . We have previously identified several proteins, such as cyclin D1, p53 and b-catenin, which are downregulated through the UPS after CVB3 infection [7, 9] . Destabilization of these short-lived host proteins is likely required for CVB3 viral RNA and protein synthesis in its lifecycle. Moreover, it is speculated that nonstructural viral proteins of CVB3 could also be potential targets of the UPS for degradation. Previous studies on picornavirus have shown that several viral proteins, such as encephalomyocarditis virus (EMCV) 3C protease and hepatitis A virus (HAV) 3C protease, are ubiquitinated and present in low concentrations in infected cells [37] [38] [39] . Several E3 ubiquitin ligases, such as human E3a ubiquitin ligase, have been shown to catalyze the ubiquitination of these viral proteins [38, 39] . Although the exact role of ubiquitination and subsequent degradation of nonstructural viral proteins of EMCV and HAV in infected cells remains elusive, such rapid turnover may be required for efficient viral RNA replication, viral protein synthesis and virus maturation.