Proteasome inhibitors: a novel tool to suppress human cytomegalovirus replication and virus-induced immune modulation

Recently, we like others, demonstrated that systemic inflammation is the most important mechanism involved in (re)activation of human cytomegalovirus (HCMV) in both immunocompetent patients. By in vitro studies the eukaryotic transcription factor NF-kappaB could be identified as the key mediator of TNF-alpha- and IE1-dependent stimulation of the HCMV IE1/2 enhancer/promoter activity, which is crucial for initiation of viral gene expression during reactivation from latency as well as productive infection. The enzymatic proteasome complex plays a central role in regulating intracellular processes, including the activation of NF-kappaB. As present antiviral strategies target mainly late events in HCMV replication (DNA replication, virus assembly) that do not completely prevent virus mediated immunopathogenesis, we wondered whether proteasome inhibitors might be a novel tool for targeting the interaction between inflammation and HCMV (re)activation. Here, proteasome inhibitors like MG132, PSI, II and III (MG262) have been shown to block both TNF-alpha-associated up-regulation of the HCMV IE1/2 enhancer/promoter in monocytic cells in an in vitro transient transfection system and HCMV replication in permissive embryonal fibroblasts. Importantly, ganciclovir-resistant HCMV strains are sensitive to proteasome inhibitors. The effect of proteasome inhibitors on HCMV replication was found to be specific as replication of other herpes viruses, like HSV-1 and HSV-2, under identical experimental conditions was not influenced. Inhibition of HCMV replication correlated with a delayed and significantly reduced expression of IE proteins, particularly of the IE2 protein, suggesting that MG132 blocks HCMV replication at an immediate early stage of infection. Early and late protein synthesis as shown exemplary for the pp52 (DNA-binding protein) and p68 (structural protein) protein production and viral DNA synthesis were also inhibited. Suppression of HCMV replication could be correlated with an increased cytosolic accumulation of IkappaB as well as a reduced NF-kappaB binding activity in nuclear extracts of MG132-treated cells, which mainly regards NF-kappaB p50. MG132 also reduced the immune modulatory activity of the virus by abrogating virus-induced up-regulation of cellular ICAM-1. These data suggest that short-term therapy with proteasome inhibitors might be an alternative strategy to prevent (re)activation, replication and immune modulatory activity of HCMV in patients with systemic inflammation.