Pyrrolidine dithiocarbamate inhibits enterovirus 71 replication by down-regulating ubiquitin–proteasome system

Enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD). The severe neurological complications caused by EV71 infection and the lack of effective therapeutic medicine underline the importance of searching for antiviral substances. Pyrrolidine dithiocarbamate (PDTC), an antioxidant, has been reported to inhibit the replication of coxsackievirus B (CVB) through dysregulating ubiquitin-proteasome system (UPS). In this study, we demonstrated that PDTC exerted potent antiviral effect on EV71. Viral RNA synthesis, viral protein expression, and the production of viral progeny were significantly reduced by the treatment of PDTC in Vero cells infected with EV71. Similar to the previous report about the inhibitory effect of PDTC on UPS, we found that PDTC treatment led to decreased levels of polyubiquitinated proteins in EV71-infected cells. The inhibitory effect of PDTC on UPS was further confirmed by the increased accumulation of cell cycle regulatory proteins p21 and p53, which are normally degraded through UPS, while the expression levels of both proteins remained unchanged. We also showed that PDTC had no impact on the activity of proteasome. Thus, we demonstrated that the down-regulation of PDTC on UPS was the result of its inhibition on ubiquitination. More importantly, this study provides evidence that the inhibition on UPS was required for the antiviral activity of PDTC, since MG132, a potent proteasome inhibitor, significantly inhibited the cytopathic effect and viral protein synthesis in EV71-infected cells. We also found that the antioxidant property of PDTC did not contribute to its antiviral effect, since N-acetyl-l-cysteine, a potent antioxidant, could not inhibit viral replication. In addition, CPE and viral protein synthesis were not inhibited in the cells pretreated with PDTC 2h before viral infection and then cultured in the media with no PDTC supplement, while the antioxidant effect of PDTC was retained. PDTC also showed significant inhibition on apoptosis induced by EV71 infection when it was applied at the early stage of viral infection. Our results collectively suggest that PDTC could be a potential anti-EV71 compound which possesses both antiviral and anti-apoptotic capacity.