Interestingly, HCMV encodes the serine–threonine protein kinase pUL97 that represents a viral CDK ortholog (vCDK) combining typical structural and functional features of host CDKs. This initial Rb inactivation, followed by further viral regulatory intervention steps, ultimately results in early S-phase cell-cycle arrest accompanied by significant dysregulation of cyclin-dependent kinases (CDKs) and cyclins, termed as pseudomitosis. Notably, HCMV replication drastically interferes with cell-cycle regulation, a process in which the HCMV-encoded protein kinase pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein (Rb). High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog.
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