The 19S Proteasome Activator Promotes Human Cytomegalovirus Immediate Early Gene Expression through Proteolytic and Nonproteolytic Mechanisms

Abstract

Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. IMPORTANCEProteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection. Proteasomes mediate the majority of cellular protein turnover, promoting multiple signaling pathways, cell cycle progression, and general cellular homeostasis (1). These large, multisubunit complexes also permit immune detection of virally infected cells by generating the foreign peptides displayed by major histocompatibility complexes (MHCs) (2). In addition to this prominent antiviral role, proteasomes promote viral infection through the degradation of cellular factors that restrict viral infection. The dichotomous roles of proteasomes during viral infection and their general requirement for cellular health necessitate that viruses modulate proteasomal activities in a precise manner to promote viral infection (3-5).The barrel-like 20S catalytic core particle (CP) of the proteasome, consisting of two sets of seven distinct alpha (␣1 to ␣7) and beta (␤1 to ␤7) subunits, contains the proteolytic components responsibl...

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles

“…The model of viral protein expression predicted an increase in protein degradation at late times that followed the trend of increasing vDNA as infection progressed. This interaction could be related to an increase in proteasome activity that promotes protein degradation and is demonstrated to occur for HCMV ( 53 – 55 ), lending further experimental support to this proposed mechanism. In addition, herpesvirus capsids do undergo protease-dependent maturation ( 56 ), and numerous DNA and RNA viruses undergo late-stage maturation events involving protein cleavage; most notable is HIV ( 57 ).…”

Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein

“…ChIP assays were performed as described previously by Winkler and Kalejta ( 82 ). Precipitating DNA was analyzed by qPCR and normalized to input levels and shown relative to AD169-infected controls.…”

Non-proteolytic activity of 19S proteasome subunit RPT-6 regulates GATA transcription during response to infection

“…For example, in yeast and mammalian cells, SUG1/RPT6 and some other proteasome subunits can physically interact with transcription factors to control gene transcription in a non-proteolytically fashion [ 8 , 14 – 17 ]. Also, viral gene transcription has been associated with the non-proteolytic activity of the 19S subunits during gene expression [ 11 , 18 ], but the role of non-canonical functions of the proteasome in the control of defense against pathogens and activation of innate immunity has not been studied.…”