Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97

Abstract

The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus–host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231–280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231–255, ∆256–280 and ∆231–280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231–280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231–280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231–255 and ∆256–280; and (iv) a bioinformatic assessment of the pUL97–cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97–cyclin interaction concerning kinase activity and viral replication fitness.

Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations

“…It appears possible that in the case of the HCMV-supportive function of CDK7, this kinase might switch to multiple cyclin binding properties, in particular under the situation of experimental cyclin knock-out (Marschall, Schütz et al, unpublished results). It should be mentioned in this regard that the viral CDK ortholog pUL97 is also capable of multiple cyclin binding, including the types H, T1 and B1 [ 5 , 52 , 53 ].…”

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

“…Viral pathogenesis is closely linked to the efficiency of viral replication in individual tissues. In this context, the role of host factors, especially the interaction through virus–host multiprotein complexes, needs to be deciphered in greater detail [ 7 , 8 , 9 , 10 , 11 ].…”

“…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 [ 11 , 15 ]. 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 [ 10 , 12 , 16 , 17 , 18 ]. Previously, the current authors and others identified a specific feature of pUL97, in that it associates with human cyclins of various types, most abundantly with T1, H, and B1, according to our investigations [ 10 , 11 , 19 , 20 , 21 , 22 , 23 , 24 ].…”

“…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 [ 10 , 12 , 16 , 17 , 18 ]. Previously, the current authors and others identified a specific feature of pUL97, in that it associates with human cyclins of various types, most abundantly with T1, H, and B1, according to our investigations [ 10 , 11 , 19 , 20 , 21 , 22 , 23 , 24 ]. Based on these observations, pUL97 is considered as a multiple cyclin-binding kinase, so that in addition to the types T1, B1, and H, further cyclin-binding activities, e.g., including cyclin A, might play supplementary roles [ 11 , 18 , 20 , 25 ].…”

The Complex Regulatory Role of Cytomegalovirus Nuclear Egress Protein pUL50 in the Production of Infectious Virus

“…For this reason, the central point of a future study should be the normalization of virus inocula quantities, i.e., ∆UL50N and ∆UL50C, determined by qPCR after DNase treatment for a further characterization of the functional role of pUL50. The DNase treatment appears to be a rather unusual practice, which has not been performed in our previous phenotypic characterization of mutant HCMVs so far [13,[56][57][58][59]. However, the results of the present study suggest to take into account, specifically for ∆UL50 preparations, the different options of inocula normalization, such as total virion protein, virion DNA, encapsidated virion DNA, or plaque-forming units.…”