New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97

Abstract

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9-cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69-cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.

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

“…CoIP analysis demonstrated a specific signal of interaction (Figure 2C) and no difference was noted between the two strains AD169 and TB40. This finding is consistent with our earlier observations, which also included the wild-type-like strain Merlin, stating that pUL97-cyclin interaction, similar to pUL69-cyclin interaction, appears to be independent from viral strain and host cell type [13,36,56]. …”

“…Therefore, one possible conclusion could be that these proteins collectively represent substrates of phosphorylation mediated by CDK-cyclin or pUL97-cyclin complexes, or both. It should be stressed that as an example of dual phosphorylation by CDKs and pUL97, the viral regulatory protein pUL69 has been identified by our recent studies [13,29,36]. The successful coimmunoprecipitation of almost all relevant CDKs and CDK/cyclin-specific proteins, considered as a parameter of reliability of our setting, was confirmed by the respective portions of the mass spectrometry data (see lower part in Table 3).…”

“…As a novel finding reported here by employing very sensitive mass spectrometry measurements, additional viral and cellular proteins are suggested to be associated within pUL97-cyclin complexes. Such a situation has already been discussed earlier when exploring dual phosphorylation of the viral early regulator pUL69 by both viral pUL97 and cellular CDKs, primarily CDK9 [13,14,29,36]. This finding led to the hypothesis that cyclin-bridged, higher order protein complexes might combine pUL97- and CDK-mediated phosphorylation of individual substrates.…”

“…Recently, we described the interaction between the HCMV regulatory protein pUL69 [13,14,36] as well as HCMV kinase pUL97 and human cyclins [58]. The latter phenomenon was characterized by an especially strong pUL97-cyclin B1 interaction [50].…”

“…Functional similarity to CDKs has been demonstrated by various experimental settings, such as a yeast complementation assay in which recombinantly expressed pUL97 rescued a Saccharomyces cerevisiae mutant lacking CDK activity [27]. In line with that, pUL97 and CDKs share identical substrates, such as nuclear lamins A/C, the retinoblastoma protein Rb, RNA polymerase II, translational elongation factor EF-1δ and histones, as well as the viral mRNA transporter pUL69 [13,26,27,28,29,30,31,32,33,34,35,36]. Notably, Rb is phosphorylated by CDKs and pUL97 at identical residues [15,27,34,37,38].…”