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. 2023 Dec 13;24(24):17421.
doi: 10.3390/ijms242417421.

The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells

Martin Schütz  1 , Arne Cordsmeier  1 , Christina Wangen  1 , Anselm H C Horn  2 , Emanuel Wyler  3 , Armin Ensser  1 , Heinrich Sticht  2 , Manfred Marschall  1
Affiliations

The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells

Martin Schütz et al. Int J Mol Sci. .

Abstract

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.

Keywords: cyclin-dependent kinases (CDKs); first vCDK/pUL97-specific transcriptomic analysis; functional complexation with host CDK7; human cytomegalovirus; impact on RNA polymerase (RNAP II) in infected cells; vCDK/pUL97–cyclin binding; viral CDK ortholog (vCDK/pUL97).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic overview of the analyzed samples for the RNA-seq analysis. Individual steps of the procedure are indicated in the upper line: (1) HFFs were either treated under wild-type (WT) conditions or cyclin H knockout (KO) via lentiviral gene transduction. HFFs transduced with the CRISPR/Cas9 system without sgRNAs served as no KO controls. (2) Cells were infected with HCMV AD169 (MOI 0.1) or remained uninfected. (3) Inhibitors or solvent DMSO were added. (4) Cells were harvested and assayed in the RNA-seq procedure. Bold letters indicate the six analyzed treatment conditions, i.e., either uninfected control (mock-inf.), with (KO) or without cyclin H KO (no KO ctrl), or inhibitor treatment (DMSO, LDC4297, MBV).
Figure 2
Figure 2
Principal component analysis (PCA): the individual biological triplicates are closely clustered, indicating consistency in their gene expression profiles. Each point on the scatter plot represents a sample, and its coordinates are determined by gene expression levels. Samples with similar gene expression profiles are positioned close together. The PCA plot displays the most significant patterns of variance within the high-dimensional dataset. Principal component 1 accounted for 20.4% of the total variance, capturing the primary pattern in the data. Principal component 2 represents the second most significant direction of variance and accounted for 15.5% of the total variance. Black indicates uninfected/mock control samples (non-bold) or DMSO-treated samples of HCMV infection (bold); green indicates samples of inhibitor treatment (MBV, LDC4297) or cyclin H knockout (KO).
Figure 3
Figure 3
Venn-diagram-based data evaluation of differential gene expression in HCMV-infected HFFs under various conditions. Gene expression in DMSO-treated samples served as a reference and was compared with mock-infected, cyclin H KO, MBV-treated, and LDC4297-treated samples. The number of significantly differentially expressed (DE) genes (p ≤ 0.05) with at least a 1.5-fold change are listed. DE genes were categorized into cellular transcripts (A), viral transcripts (B), and a combination of both cellular and viral transcripts (C). Bold numbers indicate DE genes in MBV and cyclin H KO; underlined numbers indicate DE genes in MBV and LDC4297; asterisks indicate DE genes in MBV and cyclin H KO, but not in LDC4297.
Figure 4
Figure 4
Predicted location of pUL97 in the preinitiation complex (PIC) of RNAP II-mediated eukaryotic transcription. (A) Structure of the RNAP II-specific PIC in white-space-filled presentation with the bound template DNA in yellow. The CDK7-MAT1-cyclin H unit, located at the periphery of the PIC, in association with RNAP II (1–1487) are shown in colors. The region in the dashed box is shown as an enlargement in panel (B). (B) The putative pUL97 binding site in the PIC (ribbon presentation of the PIC proteins). Note that the main cyclin-binding interface (IF2) of pUL97, comprising amino acids 231–280, is shown in the purple-space-filled presentation and the pUL97 (329–634) kinase domain is schematically outlined as a purple oval. The C-terminal domain (CTD) of RNAP II, which is not resolved in the experimental PIC structure, is shown as an orange dashed line. Filled orange spots indicate CTD phosphorylation sites located in spatial proximity of CDK7 and pUL97.
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