Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1

Abstract

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269-487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69-cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication. INTRODUCTIONHuman cytomegalovirus (HCMV) is a worldwide human pathogen belonging to the beta subgroup of the family Herpesviridae. Although primary HCMV infection of the immunocompetent host typically remains asymptomatic, severe disease can occur upon infection of immunonaïve and immunocompromised individuals such as neonates, transplant recipients and cancer or AIDS patients (Mocarski et al., 2007). The efficiency of HCMV replication is dependent on the interplay between viral and hostcell functions (Marschall et al., 2011). In particular, several regulatory steps in the course of HCMV infection depend on the activity of cellular cyclin-dependent protein kinases (CDKs), mainly CDK1, -2, -7 and -9 (Bain & Sinclair, 2007;Fortunato et al., 2000;Jault et al., 1995;Kapasi & Spector, 2008;Kapasi et al., 2009;Rechter et al., 2009;Sanchez & Spector, 2006;Sanchez et al., 2004;Tamrakar et al., 2005). HCMV-induced upregulation of CDK1, -2, -7 and -9 along with the corresponding cyclin subunits is explained by the fact that CDK activity is required at various stages of virus replication, such as the regulation of gene expression and the modification as well as localization of viral proteins (Bain & Sinclair, 2007;Kapasi et al., 2009;Rechter et al., 2009). CDKs are heterodimeric serine/ threonine kinases that become activated upon bin...

CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses

“…Since CDK9/cyclin T1 complex was reported previously to be required for the replication of other viruses of Herpesviridae origin, we next verified the antiviral effect of FIT-039 on tion through phosphorylation of the CTD of RNA polymerase II and viral proteins, such as the ICP22 of HSV. The ICP22-CDK9 complex was reported previously to phosphorylate the CTD of RNA polymerase II and regulate the transcription of HSV (20,22). A previous study showed that CDK9 was localized to nuclear viral transcriptosomes at the immediate-early stage of the HCMV infection, which allowed the active transcription of HCMV IEGs (14).…”

“…CDK1 was shown to activate the expression of HSV-1 late genes (19), while CDK2 promoted the replication of adenovirus, papillomavirus, and herpes virus DNAs (7). CDK7 and CDK9 were shown to activate viral RNA transcription in HIV and HSV (7,14,15,(20)(21)(22)(23)(24). Feichtinger et al recently described the interaction between CDK9/cyclin T1 and viral mRNA export factor pUL69 in human cytomegalovirus (HCMV), which belongs to the β subgroup of Herpesviridae (20).…”

“…CDK7 and CDK9 were shown to activate viral RNA transcription in HIV and HSV (7,14,15,(20)(21)(22)(23)(24). Feichtinger et al recently described the interaction between CDK9/cyclin T1 and viral mRNA export factor pUL69 in human cytomegalovirus (HCMV), which belongs to the β subgroup of Herpesviridae (20). In mammalian cells, CDK9/ cyclin T1 (or T2) is part of the subunit of the positive transcription elongation factor b (P-TEFb) complex and reportedly initiated the transcriptional elongation of genes by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II at Ser2 (25).…”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…There is little information on the localization of viral and cellular proteins required for viral RNA transcription. In infected cells, both phosphorylated (transcriptionally active) and unphosphorylated (transcriptionally inactive) forms of RNA polymerase II concentrate within nuclear structures thought to be replication compartments (Feichtinger et al, 2011). Within these structures, unphosphorylated and phosphorylated forms of RNA polymerase II localize with the viral protein UL69 (Feichtinger et al, 2011), which is known to be required for viral RNA processing and export to the cytoplasm.…”

“…In infected cells, both phosphorylated (transcriptionally active) and unphosphorylated (transcriptionally inactive) forms of RNA polymerase II concentrate within nuclear structures thought to be replication compartments (Feichtinger et al, 2011). Within these structures, unphosphorylated and phosphorylated forms of RNA polymerase II localize with the viral protein UL69 (Feichtinger et al, 2011), which is known to be required for viral RNA processing and export to the cytoplasm. The localization of RNA polymerase II in relation to UL44 has yet to be described; however, viral proteins involved in transcription and RNA metabolism, for example IE1, IE2 and UL84 (Bender et al, 2014;Silva et al, 2010), are found throughout the nucleus and co-localize with UL44 in viral replication compartments.…”