Human Cytomegalovirus pUL97 Regulates the Viral Major Immediate Early Promoter by Phosphorylation-Mediated Disruption of Histone Deacetylase 1 Binding

Self Cite

Gammaherpesviruses are ubiquitious pathogens that establish lifelong infection and are associated with several malignancies. All gammaherpesviruses encode a conserved protein kinase that facilitates viral replication and chronic infection and thus represents an attractive therapeutic target. In this study, we identify a novel function of gammaherpesvirus protein kinase as a regulator of class I histone deacetylases (HDAC). Mouse gammaherpesvirus 68 (MHV68)-encoded protein kinase orf36 interacted with HDAC1 and 2 and prevented association of these HDACs with the viral promoter driving expression of RTA, a critical immediate early transcriptional activator. Furthermore, the ability to interact with HDAC1 and 2 was not limited to the MHV68 orf36, as BGLF4, a related viral protein kinase encoded by Epstein-Barr virus, interacted with HDAC1 in vitro. Importantly, targeting of HDAC1 and 2 by orf36 was independent of the kinase's enzymatic activity. Additionally, orf36 expression, but not its enzymatic activity, induced changes in the global deacetylase activity observed in infected primary macrophages. Combined deficiency of HDAC1 and 2 rescued attenuated replication and viral DNA synthesis of the orf36 null MHV68 mutant, indicating that the regulation of HDAC1 and 2 by orf36 was relevant for viral replication. Understanding the mechanism by which orf36 facilitates viral replication, including through HDAC targeting, will facilitate the development of improved therapeutics against gammaherpesvirus kinases.

Section: Ementioning

confidence: 99%

A Conserved Gammaherpesvirus Protein Kinase Targets Histone Deacetylases 1 and 2 To Facilitate Viral Replication in Primary Macrophages

Mounce

Mboko

Bigley

et al. 2013

Self Cite

“…Moreover, biphasic effects on viral chromatin during lytic replication were recently reported for Kaposi's sarcomas-associated herpesvirus, a gammaherpesvirus (53). Whether or not such biphasic phenomena turn out to be a general feature of the herpesvirus lytic cycle, the influence of chromatin on the viral genome is an area of active investigation (50)(51)(52)(54)(55)(56)(57)(58)(59) and may provide new insights into how UL97 and UL133/8 impact viral gene expression.…”

Section: Discussionmentioning

confidence: 99%

An Epistatic Relationship between the Viral Protein Kinase UL97 and the UL133-UL138 Latency Locus during the Human Cytomegalovirus Lytic Cycle

Rak

Nguyen

et al. 2014

We report that UL133-UL138 (UL133/8), a transcriptional unit within the ULb= region (ULb=) of the human cytomegalovirus (HCMV) genome, and UL97, a viral protein kinase encoded by HCMV, play epistatic roles in facilitating progression of the viral lytic cycle. In studies with HCMV strain TB40/E, pharmacological blockade or genetic ablation of UL97 significantly reduced the levels of mRNA and protein for IE2 and viral early and early-late genes during a second wave of viral gene expression that commenced at between 24 and 48 h postinfection. These effects were accompanied by significant defects in viral DNA synthesis and viral replication. Interestingly, deletion of UL133/8 likewise caused significant defects in viral DNA synthesis, viral gene expression, and viral replication, which were not exacerbated upon UL97 inhibition. When UL133/8 was restored to HCMV laboratory strain AD169, which otherwise lacks the locus, the resulting recombinant virus replicated similarly to the parental virus. However, during UL97 inhibitor treatment, the virus in which UL133/8 was restored showed significantly exacerbated defects in viral DNA synthesis, viral gene expression, and production of infectious progeny virus, thus recapitulating the differences between wild-type TB40/E and its UL133/8-null derivative. Phenotypic evaluation of mutants null for specific open reading frames within UL133/8 revealed a role for UL135 in promoting viral gene expression, viral DNA synthesis, and viral replication, which depended on UL97. Taken together, our findings suggest that UL97 and UL135 play interdependent roles in promoting the progression of a second phase of the viral lytic cycle and that these roles are crucial for efficient viral replication. IMPORTANCEA unique feature of the herpesviruses, such as human cytomegalovirus (HCMV), is that they can undergo latency, a state during which the virus silences its gene expression, which allows lifelong viral persistence in immunocompetent hosts. We have uncovered an unexpected link between a cluster of HCMV genes involved in latency, UL133-UL138, and a virally encoded protein kinase, UL97, which plays crucial roles in manipulating the cell cycle during HCMV lytic replication. Although viral immediate early (IE) gene expression is essential for HCMV lytic replication, the activation of IE gene expression in latently infected cells is not sufficient to result in production of infectious virus. Our findings here and in an accompanying study (M. Umashankar, M. Rak, F. Bughio, P. Zagallo, K. Caviness, and F. D. Goodrum, J. Virol. 88:5987-6002, 2014) show that proteins expressed from the UL133-UL138 latency locus and UL97 play interdependent roles in overcoming checkpoints that restrict the viral lytic replication cycle, findings which suggest intriguing implications for establishment of and reactivation from HCMV latency.

“…In serum-starved, nondividing cells, part of the virus yield defect can be ascribed to a defect in viral DNA synthesis, stemming from UL97's role in inactivation of retinoblastoma protein (16)(17)(18). UL97 is necessary for the phosphorylation of a variety of viral and cellular proteins in infected cells (11,16,(18)(19)(20)(21)(22)(23)(24) and is sufficient for phosphorylation of some of these proteins in vitro (11,19,20,25,26). One such substrate is the nuclear lamina component lamin A/C.…”

mentioning

confidence: 99%

Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Sharma

Bender

Kamil

et al. 2015

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses. Herpesviruses replicate and package their DNA genomes into capsids in the nucleus of the host cell. The nucleocapsids are then transported out of the nucleus through an unusual process called nuclear egress. A widely accepted model for nuclear egress entails envelopment and de-envelopment of capsids as they transit the nuclear membranes (reviewed in references 1, 2, and 3). Studies of human cytomegalovirus (HCMV) nuclear egress are of particular interest, because of the medical ...