In addition to blocking proteasome proteolytic activities, proteasome inhibitors are known to reduce free ubiquitin levels in treated cells [27] . It has been suggested that proteasome inhibition negatively affects the budding of retroviruses through reducing free ubiquitin level and subsequently interfering with ubiquitination of viral Gag proteins [22, 24, 25] . Ubiquitin is generated in the cell by proteolysis of polyubiquitinated proteins or ubiquitin fused to carboxyl extension proteins (CEPs) [28] . To investigate whether protein ubiquitination is beneficial to CVB3 replication in HeLa cells, we used the ubiquitin-specific siRNA to gene-silence the expression of human ubiquitin-CEP Uba80, which codes for ubiquitin fused to ribosomal protein S27a [29] . As shown in Fig. 3A , both ubiquitin conjugates and free ubiquitin levels were markedly knocked down after the treatment of ubiquitin siRNA. We further showed that viral titers were significantly reduced in the ubiquitin siRNA-transfected cells as compared to scramble siRNA control (Fig. 3B ), suggesting that protein ubiquitination is a critical process adopted by coxsackievirus for the successful completion of its lifecycle.