Abstract
Human cytomegalovirus (HCMV) is a common β-herpesvirus causing life-long 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 pUL97-cyclin 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.
Highlights
Human cytomegalovirus (HCMV) is a common -herpesvirus causing life-long 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 interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition
The HCMV-encoded protein kinase pUL97 represents a CDK ortholog that is essential for efficient viral replication via phosphorylation of several viral and cellular substrates
Summary
Human cytomegalovirus (HCMV) is a common -herpesvirus causing life-long latent infections. HCMV replication drastically interferes with cell cycle regulation, a process, in which the HCMV-encoded protein kinase pUL97 massively phosphorylates the checkpoint regulator retinoblastoma protein (Rb) (9 –11) This initial Rb inactivation, followed by further viral regulatory steps of intervention, results in an early S-phase cell cycle arrest [1, 12, 13]. The interaction between the HCMV-encoded protein kinase pUL97 and human cyclins of types B1, T1, and H has been described in our earlier reports (6, 14 –17) These three cyclins obviously possess different affinities in terms of strength of pUL97 binding detected by CoIP-based analyses [6], as well as a requirement of pUL97 activity (cyclin B1) [16] or dependence on HCMV replication (cyclin H) [6].
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