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Abstract: Proteasome inhibitor, which inhibits NF-κB activation, has been reported to activate c-Jun N-terminal kinase (JNK)-c-Jun pathway. In this study, we investigated the effects of proteasome inhibitor on the human cytomegalovirus (HCMV) major immediate early (MIE) gene expression in human central nervous system (CNS)-derived cell lines. Treatment of HCMV-infected 118MGC glioma and U373-MG astrocytoma cells with three proteasome inhibitors, MG132, clasto-lactacystin β-lactone, and epoxomicin, suppressed MIE protein expression. In contrast, in HCMV-infected IMR-32 neuroblastoma cells, the proteasome inhibitors increased MIE protein expression, even in the presence of NF-κB inhibitor SN-50. A luciferase reporter assay demonstrated that MG132 markedly elevated the MIE promoter/enhancer (MIEP) activity in IMR-32 cells, but down-regulated it in 118MGC and U373-MG cells. Mutation in five cAMP response elements (CREs) within the MIEP resulted in a loss of the ability to respond to MG132 in IMR-32 cells. Moreover, Western blotting analysis revealed that MG132 induced c-Jun phosphorylation in all three CNS-derived cell lines, whereas a high level of activating transcription factor-2 (ATF-2) phosphorylation was observed only in IMR-32 cells. Finally, MG132-induced MIE protein expression was suppressed by JNK inhibitor that reduced the phosphorylation levels of both c-Jun and ATF-2. Taken together, these results suggest that the proteasome inhibitors activate CRE binding proteins consisting of c-Jun and ATF-2 through activating the JNK-c-Jun pathway, thereby inducing MIE protein synthesis in IMR-32 cells under the condition where NF-κB activity is inhibited. [Copyright &y& Elsevier]

Abstract: The anti-human cytomegalovirus (HCMV) activity of tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative from Sasa albo-marginata, was studied in the human embryonic fibroblast cell line MRC-5. In a plaque assay, tricin and ganciclovir (GCV) showed concentration-dependent inhibitory properties from 0.05 to 3.6μM and 0.01 to 1.0μM, respectively. Tricin had no virucidal effects on cell-free HCMV. Treatment with tricin 1h before, or 1h or 3h after viral infection significantly suppressed HCMV replication. Moreover, tricin inhibited the expression of immediate early (IE) 2 mRNA and DNA polymerase (UL54) mRNA in HCMV-infected cells. Western blot analysis also demonstrated that tricin decreased the expression of IE antigen (especially IE2) and cyclooxygenase 2 (COX-2) expression in HCMV-infected cells. In the presence of tricin, prostaglandin E2 (PGE2) accumulation by HCMV infection was completely inhibited. These results suggest that tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity. [Copyright &y& Elsevier]

Abstract: Expression of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV) in the human thyroid papillary carcinoma cell line TPC-1 is repressed at the transcriptional level. However, treatment of these cells with hexamethylene bisacetamide (HMBA), a chemical inducer of differentiation, for 12 to 24 h before infection enabled the cells to support IE1 and IE2 gene expression and consequently HCMV replication. In HMBA-treated cells the transcription factor NF-κB was induced and the MIE promoter (MIEP) was activated. The presence of a NF-κB inhibitory peptide SN-50 or expression of a dominant negative IκBα protein during the HMBA pretreatment period efficiently prevented the HMBA-induced MIEP activation and MIE protein synthesis. Moreover, introduction of mutations into the NF-κB binding sites in the MIEP in a plasmid expressing the IE1 protein diminished its ability to express the protein in HMBA-treated cells. Therefore, the NF-κB activity previously induced in HMBA-treated cells and the NF-κB sites in the MIEP were shown to be essential for HCMV to respond to HMBA action and to express the MIE genes. Investigation of the mechanisms by which HMBA activates NF-κB revealed that degradation of IκBα and translocation of the phosphorylated NF-κB p65 subunit to the nucleus, both of which are known to be critical steps in NF-κB activation, are stimulated in the HMBA-treated cells. These results indicate that treatment of nonpermissive TPC-1 cells with HMBA induces MIE gene permissiveness by up-regulating NF-κB activity. [Copyright &y& Elsevier]