Ubiquitin-Dependent Proteolysis of Cyclin D1 Is Associated with Coxsackievirus-Induced Cell Growth Arrest

Abstract

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...

Inhibition of glycogen synthase kinase 3β suppresses coxsackievirus-induced cytopathic effect and apoptosis via stabilization of β-catenin

“…We examined the b-catenin expression in the SB 415286-treated cells following 5 h virus infection. As shown in Figure 6a, CVB3-induced decreased expression of b-catenin was reversed after addition of SB 415286, which is consistent with our previous finding that LiCl induced an accumulation of b-catenin in infected HeLa cells, 22 indicating that CVB3-mediated downregulation of b-catenin is dependent on GSK3b activation. To further clarify how GSK3b inhibitors affect b-catenin expression and subsequently prevent virus-induced CPE and apoptosis, we performed immunocytochemical staining to detect the expression and localization of b-catenin.…”

Dysregulation of the Ubiquitin-Proteasome System by Curcumin Suppresses Coxsackievirus B3 Replication

“…We have previously demonstrated that proteasome activities were unchanged after CVB3 infection of HeLa cells and murine cardiomyocytes (16,17). Thus, it is unlikely that virus infection affects the role of curcumin in the regulation of UPS.…”

Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58 IPK and Activation of CHOP and SREBP1

“…1E and 3D, the cleavage product of procaspase-3 was difficult to detect by immunoblotting. It may be that the siRNA silencing of target genes contributed to CVB3-induced proteosome-mediated degradation of the procaspase form, precluding its detection (33,50). To confirm the occurrence of cleavage, we performed additional Western blot analysis on 17% acrylamide resolving gels, loading 80 g of total protein (instead of the usual 30 g), and then were better able to detect the cleaved form (17 to 19 kDa) of caspase-3 (Fig.…”