Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

Abstract

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

“…Previous investigations led to the postulate of a substantial relevance of pUL97-cyclin interactions, as characterized by the following findings: (i) The HCMV kinase pUL97 acts as a structural CDK ortholog originally based on our bioinformatic modeling and biochemical analyses. (ii) Our initial report on pUL97-cyclin T1 interaction could be extended to additional types such as cyclins B1 and H [47,55,56,82]. (iii) The interaction pUL97-cyclins B1/T1/H was confirmed by several methods including highly sensitive mass spectrometry-based proteomics.…”

“…The three cyclins obviously possess different affinities in terms of strength of pUL97 binding detected by coimmunoprecipitation (CoIP)-and mass spectrometry (MS)-based analyses. In case of cyclin B1, a requirement of catalytic activity of pUL97 for cyclin binding was identified, whereas in case of cyclin H, pUL97 interaction was found dependent on the environment of HCMV replication [82]. Recently published data indicate a substrate-bridging function of cyclin(s) for the binding of pUL97 to its substrate pp65, as determined with a pp65 mutant lacking a putative cyclin-docking motif [83].…”

“…Inhibitors of pUL97 small molecules (<500 Da, various chemical classes) indolocarbazoles, benzimidazoles, quinazolines, others [53] (references therein) [44,64,119] [ 114,120,121] Phenotype of pUL97 inhibition or UL97 deletion strongly reduced viral replication efficiency (100-1000-fold) delayed replication kinetics; impaired genomic replication; impaired viral nuclear egress [44,51,53,94,109,122,123] [ 59,61,104,124] Microorganisms 2020, 8, 515 5 of 23 The interaction between HCMV pUL97 and human cyclins of the types B1, T1 and H has been described in our earlier reports [47,55,82]. The three cyclins obviously possess different affinities in terms of strength of pUL97 binding detected by coimmunoprecipitation (CoIP)-and mass spectrometry (MS)-based analyses.…”

“…differences in substrate binding, nuclear translocation and drug susceptibility [44,70] [ [71][72][73][74] Similarity and sequence conservation with other kinases low <35% identity with herpesviral kinases, <15% identity with cellular kinases [45,63] [ [48][49][50]75] Sequence conservation ORF-UL97 of HCMVs high no variation of translational start sites, NLS sequences or kinase domains [44] [ 76,77] Related to cell kinases cyclin-dependent kinases (CDKs), viral CDK ortholog functional overlap with CDKs, specific crosstalk with CDK9, CDK7 and CDK1, direct interaction with cyclins [47,55,56,[78][79][80][81][82][83] [57, 84,85] Coregulation of viral replication by pUL97 and cellular kinases several novel cellular kinases, including CDKs, identified to be involved in HCMV replication virus-supporting functions in signaling pathways and nuclear capsid egress [55,56,86,87] [ [88][89][90][91][92][93] Substrate proteins viral, cellular pUL44, pUL69, pp65, Rb, p32/gC1qR, nuclear lamins, EF-1δ, RNAP II, IFI16, SAMHD1 [53,79,87,[94][95][96]…”

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis

“…In total, we quantified 2,816 proteins, of which 135 were classified as candidate interaction partners to at least one kinase (Table S1). This list includes several previously found CHPK interactors and substrates, such as SAMHD1 (Zhang et al, 2019) , IRS4, CCAR2 (Steingruber et al, 2016) , RBL2 (Iwahori et al, 2017) , PPP2CA, PUM1, PRDX1 (Li et al, 2011) and NUMA1, TUBA1B, MAP7D1 (Umaña et al, 2018) .…”