Many viruses disrupt the host cell cycle to facilitate their own growth. We assessed the mechanism and function of enterovirus 71 (EV71), a primary causative agent for recent hand, foot, and mouth disease outbreaks, in manipulating cell cycle progression. Our results suggest that EV71 infection induces S-phase arrest in diverse cell types by preventing the cell cycle transition from the S phase into the G2/M phase. Similar results were observed for an alternate picornavirus, Coxsackievirus A16. Synchronization in S phase, but not G0/G1 phase or G2/M phase, promotes viral replication. Consistent with its ability to arrest cells in S phase, the expression of cyclin A2, CDK 2, cyclin E1, and cyclin B1 was regulated by EV71 through increasing transcription of cyclin E1, promoting proteasome-mediated degradation of cyclin A2 and regulating the phosphorylation of CDK 2. Finally, a non-structural protein of EV71, the RNA-dependent RNA polymerase 3D, was demonstrated to mediate S-phase cell cycle arrest. These findings suggest that EV71 induces S-phase cell cycle arrest in infected cells via non-structural protein 3D, which may provide favorable conditions for virus production.

Aim: To study the caspase‐3‐independent mechanisms in oridonin‐induced MCF‐7 human breast cancer cell apoptosis in vitro. Methods: The viability of oridonin‐treated MCF‐7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl‐2, caspase‐3, caspase‐9, heat shock protein (Hsp)90, p53, p‐p53, p21, Poly (ADP‐ribose) polymerase (PARP), and the inhibitor of caspase‐activated DNase (ICAD) protein expressions were detected by Western blot analysis. Results: Oridonin inhibited cell growth in a time‐ and dose‐dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z‐VAD‐fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of ΔΨmit were detected in oridonin‐induced MCF‐7 cell apoptosis, which was involved in a postmitochondrial caspase‐9‐dependent pathway. Decreased Bcl‐2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase‐3. Conclusion: DNA damage provoked alternations in the mitochondrial and caspase‐9 pathways as well as p53‐mediated cell cycle arrest, but was not related to caspase‐3 activity in oridonin‐induced MCF‐7 cells.