Highly conserved interaction profiles between clinically relevant mutants of the cytomegalovirus CDK-like kinase pUL97 and human cyclins: functional significance of cyclin H

Abstract The complex network of host interaction of the human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. 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. We further addressed the question 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. To this end, clinically relevant UL97 drug resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants still showed interaction with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231–280 and Δ236–275 lost interaction with cyclins T1 and H, showed an impaired replication efficiency and also reduced kinase activity. 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. To the contrary, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication. Thus, cyclin H proved to be a very relevant determinant of both, pUL97 kinase activity and viral replication efficiency. Combined, the results provide evidence for the functional importance of pUL97–cyclin interaction, and a high selective pressure on the formation of pUL97–cyclin complexes as identified for clinically relevant mutants.