Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strainspecific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitorinduced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69. Human cytomegalovirus (HCMV)2 is a member of the Herpesviridae family and a human pathogen with worldwide distribution. Primary HCMV infection of the immunocompetent host is usually asymptomatic, whereas severe disease can occur upon infection of the immunocompromised and immunonaive. HCMV is a leading cause of complications in transplant recipients and AIDS patients, and congenital infection may result in mental impairment and hearing loss (1).HCMV replication is differentially regulated in host cell types, and viral replication is dependent on regulation of the cell cycle (2). HCMV infection induces cell cycle arrest, while simultaneously the virus sustains an active cellular metabolic state supporting productive infection (3). Infected cells arrest in a pseudo-G 1 state with high levels of cyclin E and cyclin E-associated kinase activity (4 -6). A number of additional alterations of cyclin-dependent protein kinase (CDK) activity have also been described, such as increased synthesis and reduced degradation of cyclin B1, as well as cytoplasmic translocation of CDK1 in HCMV-infected cells (7). The up-regulation of CDK activity during HCMV replication implies that viral replication requires CDK activity to create an environment favorable for efficient viral transcription, genome replication, and assembly of viral particles. Several regulatory steps in HCMV replication are dependent on CDK activity, particularly those involving CDK1, -2, -7, and -9 (8 -12). Additionally, inhibition of CDK activity affects replication of HCMV and other herpesviruses (13). Roscovitine, a purine analog that preferentiall...

In recent studies, the nuclear domain 10 (ND10) components PML, Sp100, human Daxx (hDaxx), and ATRX were identified to be cellular restriction factors that are able to inhibit the replication of several herpesviruses. The antiviral function of ND10, however, is antagonized by viral effector proteins by a variety of strategies, including degradation of PML or relocalization of ND10 proteins. In this study, we analyzed the interplay between infection with herpesvirus saimiri (HVS), the prototypic rhadinovirus, and cellular defense by ND10. In contrast to other herpesviruses, we found that HVS specifically degraded the cellular ND10 component Sp100, whereas other factors like PML or hDaxx remained intact. We could further identify the ORF3 tegument protein of HVS, which shares homology with the cellular formylglycinamide ribotide amidotransferase (FGARAT) enzyme, to be the viral factor that induces the proteasomal degradation of Sp100. Interestingly, recent studies showed that the ORF3-homologous proteins ORF75c of murine gammaherpesvirus 68 and BNRF-1 of Epstein-Barr virus modulate the ND10 proteins PML and ATRX, respectively, suggesting that the ND10 targets of viral FGARAT-homologous proteins diversified during evolution. Furthermore, a virus with the ORF3 deletion was efficiently complemented in Sp100-depleted cells, indicating that Sp100 is able to inhibit HVS in the absence of antagonistic mechanisms. In contrast, we observed that PML, which was neither degraded nor redistributed after HVS infection, strongly restricted both wild-type HVS and virus with the ORF3 deletion. Thus, HVS may lack a factor that efficiently counteracts the repressive function of PML, which may foster latency as the outcome of infection.

Nucleocytoplasmic shuttling and interaction with the cellular mRNA export factor UAP56 are prerequisites for the mRNA export activity of human cytomegalovirus (HCMV) pUL69. Although the murine cytomegalovirus homolog pM69 shuttles, it fails to export mRNAs due to its inability to recruit UAP56. However, chimeric proteins comprising pM69 fused to N-terminal pUL69 fragments, including its UAP56 interaction motif, acquire mRNA export activity. Importantly, growth curves of recombinant HCMVs illustrate that such a chimeric protein, but not pM69, substitutes for pUL69 during HCMV infection.

Herpesviruses establish latency in suitable cells of the host organism after a primary lytic infection. Subgroup C strains of herpesvirus saimiri (HVS), a primate gamma-2 herpesvirus, are able to transform human and other primate T lymphocytes to stable growth in vitro. The viral genomes persist as nonintegrated, circular, and histone-associated episomes in the nuclei of those latently infected T cells. Epigenetic modifications of episomes are essential to restrict the transcription during latency to selected viral genes, such as the viral oncogenes stpC/tip and the orf73/LANA. In this study, we describe a genome-wide chromatin immunoprecipitation-on-chip (ChIP-on-chip) analysis to profile the occupancy of CTCF on the latent HVS genome. We then focused on two distinct, conserved CTCF binding sites (CBS) within the orf73/LANA promoter region. Analysis of recombinant viruses harboring deletions or mutations within the CBS indicated that the lytic replication of such viruses is not substantially influenced by CTCF. However, T cells latently infected with CBS mutants were impaired in their proliferation abilities and showed a significantly reduced episomal maintenance. We detected a reduced transcription of the orf73/LANA gene in the T cells, corresponding to the reduced viral genomes; this might contribute to the loss of HVS episomes, as LANA is central in the maintenance of viral episomes in the dividing T cell populations. These data demonstrate that the episomal stability of HVS genomes in latently infected human T cells is dependent on CTCF.