Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPRassociated gene, p58 IPK , which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58 IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2␣, suggesting that p58 IPK , a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58 IPK and induction/ activation of CHOP, SREBP1, and caspase-12.

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Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G 1 phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [ 3 H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G 1 progression and G 1 /S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G 1 cyclin-dependent kinase activities, accompanied by decreased levels of G 1 cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3␤ pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3␤ independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G 1 /S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.Coxsackievirus group B3 (CVB3), an enterovirus of the family Picornaviridae, is a common human pathogen associated with various diseases, such as myocarditis, meningitis, and pancreatitis (7,30,48). It has been suggested that early virus and host cell interactions can determine the degree of viral replication and the progression of target organ injury. Like most viruses, CVB3 has evolved a variety of mechanisms to optimize cellular conditions to benefit its own replication. We have previously shown that the extracellular signal-regulated kinase (ERK) is activated during CVB3 infection and that inhibition of such activity blocks CVB3 replication. It has also been reported that CVB3 modifies host gene expression to optimize its replication (34, 44, 50). As part of such manipulation of host cells, CVB3 may modify the host cell cycle regulatory machinery to facilitate its replication. Indeed, during the preparation of this study, Feuer et al. (14) showed that coxsackievirus replication and persistence were affected by cell cycle status. Although it was proposed that virus-induced shutdown of host protein synthesis led to cell growth arrest (14), the effect of CVB3 infection on cell cycle control and the precis...