The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

Abstract

The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

“…In particular, Sharma and colleagues demonstrated that an inhibitormediated block of pUL97 results in a significant reduction in viral titres and, moreover, leads to a punctate distribution of both pUL50 and pUL53 (Sharma et al, 2015). Recent reports from our group suggested functionally relevant cyclin interaction of the viral kinase pUL97 (Graf et al, 2013;Steingruber et al, 2015), further substantiating the role of pUL97 as a viral CDK ortholog (Hume et al, 2008).…”

“…In this study, we looked at whether or not inhibitors of cellular kinases similarly block core NEC formation at the nuclear rim. CDK inhibitors were suspected as candidates, since CDKs and pUL97 are considered as orthologues, sharing properties of cyclin binding, substrate phosphorylation and regulatory functions in HCMV replication (Hume et al, 2008;Graf et al, 2013;Steingruber et al, 2015). For this purpose, we infected HFFs with the recombinant HCMV AD169-GFP UL50-HA expressing the green fluorescent protein (GFP) as a marker of infected cells and pUL50 fused to an HA-tag.…”

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

“…Interestingly, CDKs are also functionally integrated into the replication of herpesviruses. Notably, the HCMV-encoded protein kinase pUL97 shares important structural and functional features with CDKs, such as structural similarities in the N-and C-terminal lobe of the kinase domain, interaction with cyclins, phosphorylation of identical substrates, and functional complementation in heterologous systems (Chou et al, 2006;Romaker et al, 2006;Chou, 2008;Hume et al, 2008;Hamirally et al, 2009;Kamil et al, 2009;Thomas et al, 2009;Kuny et al, 2010;Graf et al, 2013;Iwahori et al, 2015;Oberstein et al, 2015;Steingruber et al, 2015). A combined regulatory impact of CDK and pUL97 activity on the viral mRNA export factor pUL69 was demonstrated Thomas et al, 2009;Feichtinger et al, 2011;Oberstein et al, 2015) and, very recently, additional posttranslational modification by methylation proved to be similarly important (Thomas et al, 2015).…”

“…Both CDK9 and viral kinase pUL97 proved to be involved in a phosphorylation-specific regulation of pUL69 mRNA transport activity (Thomas et al, 2009;Rechter et al, 2009;Feichtinger et al, 2011;Marschall et al, 2011). Interestingly, direct interactions between pUL69 and cyclin T1, as well as between pUL97 and several types of cyclins, have been demonstrated recently (Graf et al, 2013;Steingruber et al, 2015).…”

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

“…Early in the infection cycle it may activate the major immediate early promoter (20), phosphorylate retinoblastoma (Rb) protein (21) to influence the cell cycle as a cyclin-dependent kinase (CDK) ortholog (22)(23)(24), interact with cyclins (25), and phosphorylate the viral polymerase accessory protein pUL44 (26,27) and the nuclear mRNA export factor pUL69 (28). Later in the infection cycle, pUL97 may facilitate nuclear egress by phosphorylating the viral tegument protein pp65 (29) and nuclear lamins (30,31) and may also function through kinase-independent mechanisms (32).…”