leupeptins and Cytomegalovirus-Infections

Cytomegalovirus(CMV) reactivation causes immunopathy, graft malfunction, and even rejection. The traditional anti-CMV drug ganciclovir is not able to prevent reactivation of endogenous virus. Recent studies have found that proteasome inhibitor (PI) is able to suppress CMV replication. In this study we investigated the influence of proteasome inhibitor MG132 and ganciclovir on the CMV-specific CD8(+) T-cell immune response. We found that interferon-γ (IFN-γ) production in response to CMV-infected fibroblasts was reduced under the influence of MG132 in a dose-dependent manner. A marked reduction was observed at 0.5 μM. Likewise, CMV-specific cytotoxicity of CD8(+) T cells was decreased in the presence of MG132. In contrast, the traditional CMV replication inhibitor ganciclovir (10 μM) had no such effect. These findings might have important implications in reducing CMV-associated immunopathy by altering epitope generation through the application of selective proteasome inhibitors.

Topics: Antiviral Agents; CD8-Positive T-Lymphocytes; Cysteine Proteinase Inhibitors; Cytomegalovirus; Cytomegalovirus Infections; Dose-Response Relationship, Drug; Fibroblasts; Ganciclovir; Humans; Immediate-Early Proteins; Immunity, Cellular; Interferon-gamma; Leukocytes, Mononuclear; Leupeptins; Virus Activation; Virus Cultivation; Virus Replication

Proteasome inhibitor, which inhibits NF-kappaB 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 beta-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-kappaB 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-kappaB activity is inhibited.

Topics: Activating Transcription Factor 2; Cell Line; Central Nervous System; Cytomegalovirus; Cytomegalovirus Infections; Gene Expression Regulation, Viral; Humans; Immediate-Early Proteins; Lactones; Leupeptins; Oligopeptides; Protease Inhibitors; Proteasome Inhibitors; Proto-Oncogene Proteins c-jun

This study provides evidence that proteasomal activity is required at multiple steps in human cytomegalovirus replication. Electron microscopy revealed that no viral particles were assembled in the presence of proteasome inhibitor MG132. Immunofluorescence and Western blot analyses using MG132 demonstrated that immediate early gene expression was suppressed at low but not high MOI. In contrast, expression of late proteins was completely blocked independent of MOI. Additionally, pulsed-field gel electrophoresis demonstrated that MG132 interferes with cleavage of HCMV DNA. Bromodeoxyuridine incorporation studies showed that de novo viral DNA synthesis is reduced in the presence of MG132. Furthermore, in contrast to previous hypotheses we demonstrated that neither the ND10 components PML and hDaxx nor NFkappaB activation represent the target for MG132.

Topics: Antiviral Agents; Cells, Cultured; Cytomegalovirus; Cytomegalovirus Infections; DNA, Concatenated; DNA, Viral; Fibroblasts; Humans; Leupeptins; NF-kappa B; Proteasome Inhibitors; Viral Proteins; Virion; Virus Assembly; Virus Replication