Proteasome inhibitor MG132 blocks viral DNA replication and assembly of human cytomegalovirus

Abstract

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 NFjB activation represent the target for MG132.

Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

“…A significant delay in early (i.e., UL57) and late (i.e., UL99) protein expression was also observed. These results are consistent with previous studies (31,46,50) that showed that the addition of protea- HCMV TRANSCRIPTION REQUIRES PROTEASOME ACTIVITY 3081…”

“…It was unclear whether the decrease in early or late gene expression could be attributed to a specific defect in viral DNA synthesis and/or gene transcription or to the decrease in IE expression, which would subsequently affect transactivation of the early genes required for viral DNA synthesis. The block in IE protein expression was later shown to be MOI dependent (31,50), while early and late protein expression remained significantly impaired at all MOIs tested (31), further indicating that the stages of viral gene expression may be differentially affected by proteasome inhibition. Another potential complication that needs to be considered when studying the effects of proteasome inhibitors on HCMV gene expression is the time at which the virus and inhibitor are added in relation to the cell cycle.…”

“…Previous studies assessing the impact of proteasome inhibitors on HCMV infection showed a decrease in viral titer and an apparent defect at all stages of the infection, with a significant reduction in viral protein expression (31,46). These studies were done with the proteasome inhibitor added after viral adsorption and maintained in culture through the time course.…”

“…HCMV has also been shown to inhibit the degradation of several cell cycle proteins by inactivating the anaphase-promoting complex, an E3 ubiquitin ligase (4,59,61,62). In general, inhibition of the proteasome appears to impact negatively on viral replication (31,46,50). Viral gene expression is temporally regulated into three different kinetic classes: immediate early (IE), early, and late (for a review, see reference 41).…”

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

“…Third, we investigated whether hnRNP K is required for IE2 protein stability by using the chemical compound MG132, a proteasome inhibitor that does not affect IE gene expression. 30 MG132 failed to restore IE2 protein levels in HCMV-infected hnRNP K-deficient HFFs (Figure 5e), indicating that hnRNP K does not protect IE2 protein from proteasomal degradation. Finally, as hnRNP K is an RNA-binding protein, we addressed the possibility that hnRNP K interacts with IE2 mRNA.…”