Dataset Information

The Cytomegalovirus Protein Kinase pUL97:Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting.

ABSTRACT: Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by deletion of the UL97 gene or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed in experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.

SUBMITTER: Steingruber M

PROVIDER: S-EPMC7232230 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Cytomegalovirus Protein Kinase pUL97:Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting.

Steingruber Mirjam M Marschall Manfred M

Microorganisms 20200404 4

Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pro ...[more]

PMID: 32260430

Similar Datasets

Project description:Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.

Project description:UnlabelledHuman cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1). In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21(Cip1). Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21(Cip1) in wild-type virus but not a pUL27-deficient virus. We manipulated the p21(Cip1) levels to evaluate the functional consequence to MBV. Overexpression of p21(Cip1) restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21(Cip1) in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21(Cip1) and support the idea that CDKs can complement some activities of pUL97.ImportanceHCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21(Cip1). In contrast, we provide evidence that p21(Cip1) functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21(Cip1) activators might be useful in combination with MBV to effectively inhibit HCMV infections.

Dataset Information

The Cytomegalovirus Protein Kinase pUL97:Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting.

Publications

The Cytomegalovirus Protein Kinase pUL97:Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets