Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97

Abstract

Human cytomegalovirus (HCMV) is a common ␤-herpesvirus causing lifelong latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.

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.…”

“…(iv) Specifically, the interaction pUL97-cyclin B1 was found to be phosphorylation-dependent for both proteins. In addition, cyclin B1 (but not H) was phosphorylated by pUL97 in vitro [56]. (v) Using a protein assembly-based CoIP assay, the formation of binary and ternary complexes involving pUL97, cyclin H and CDK7 was identified, thus suggesting a cyclin bridging concept [125].…”

“…Auto-phosphorylation pronounced auto-phosphorylation activity, several N-terminal Ser and Thr residues autophosphorylation most probably required for kinase activity/autoactivation [44,54,56,94,109] [65, 66,110] Nucleoside phosphorylation ganciclovir, valganciclovir, penciclovir, acyclovir, etc.…”

“…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

“…Accordingly, v-CDKs are considered immune to cellular control mechanisms (Hume et al, 2008) , despite the fact that the UL97 kinase of human cytomegalovirus (human herpesvirus 5, HHV5) can physically interact with various cyclins (Graf et al, 2013;Steingruber et al, 2019) . V-CDKs mimic CDK1 and 2 in phosphorylating Lamin A/C (Hamirally et al, 2009;Kuny et al, 2010) , retinoblastoma-associated tumor suppressor RB1 (Hume et al, 2008;Kuny et al, 2010;Prichard et al, 2008) and deoxynucleoside triphosphate (dNTP) hydrolase SAMHD1 (Businger et al, 2019;Kim et al, 2019;Zhang et al, 2019) .…”