Studies on the human cytomegalovirus kinase−cyclin interaction that determines host regulation and viral replication efficiency
Creators: name: Schuetz, Martin
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2024

description: The human cytomegalovirus (HCMV) is a significant opportunistic human pathogen that relies on a complex network of virus–host interaction. The viral protein kinase pUL97 represents a crucial determinant of replication efficiency since it acts as a viral cyclin-dependent kinase (vCDK) ortholog by combining structural and functional features of host CDKs. Importantly, vCDK/pUL97 has been found to interact with cyclin types T1, H, and B1. Two primary regions in pUL97 mediate the interaction with cyclins: interface 1 (IF1), a large contact region in the C-terminal kinase domain that overlaps with drug resistance mutation sites (amino acids 328-651), and interface 2 (IF2), a short linear binding motif within the unstructured N-terminus (231-280). IF2 is required for both pUL97 oligomerization and specific cyclin interactions, whereas IF1 contributes to cyclin binding in general. The aim of this thesis was to determine the functional relevance of the pUL97–cyclin interaction. Accordingly, the impact of clinically relevant UL97 drug-resistance mutations in the IF1 region was examined. Notably, cyclin interaction was found conserved in all analyzed mutants and viral replication fitness was almost indistinguishable from wildtype (WT), indicating a selection pressure preserving this regulatory interaction. Subsequently, partial and complete HCMV UL97 IF2 deletion mutants were generated. While smaller deletions maintained WT-like functional qualities, a complete IF2 deletion resulted in a loss of interaction with cyclins T1 and H (but not B1) as well as severely impaired replication efficiency. Furthermore, pUL97 kinase activity was significantly reduced, thus proving the functional importance of the pUL97–cyclin interaction. Importantly in this regard, knock-out and knock-down experiments confirmed the importance of cyclin H as a major determinant of HCMV replication efficiency, whereas cyclins B1 and T1 showed no impact on HCMV replication phenotype or pUL97 kinase activity. Moreover, a novel assay was established to quantify pUL97 kinase activity, thereby showing a significant increase of pUL97 kinase activity in the presence of cyclin H. The importance of cyclin H could be further highlighted by its strong upregulation upon HCMV infection, regardless of strain and cell type. Analysis of the total transcriptome indicated that the interaction between pUL97, cyclin H, and CDK7 regulates the transcription of numerous genes. Substantiating this regulatory interplay, bioinformatic analysis confirmed our protein-biochemical data on a potential ternary complex consisting of pUL97, cyclin H, and CDK7. As a central point, human CDK7 activity was increased by a pUL97-mediated trans-stimulation (while, vice versa, CDK7 did not trans-stimulate pUL97). A phospho-specific analysis indicated that the CDK7 T¬¬ loop may be phosphorylated by pUL97 during HCMV infection, indicating a so far undiscovered aspect of HCMV-specific transcriptional regulation. As a consequence of these investigations, a first covalently binding compound that inhibited the pUL97–cyclin H interaction was identified with potent anti-HCMV activity, pointing towards a novel antiviral targeting strategy. In conclusion, this thesis provides multilayered points of evidence for the functional importance of the pUL97–cyclin interaction and suggests future options for antiviral interference.