doi: 10

doi: 10.1128/jvi.74.17.7720-7729.2000. as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the conversation site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49. IMPORTANCE The multifunctional CBR 5884 regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that this pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular CBR 5884 mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is usually important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation. is usually pUL69 of human cytomegalovirus (HCMV). This multifunctional protein of 744 amino acids is usually expressed in several isoforms with molecular masses of 105 to 116?kDa during the early and late phases of the viral replication cycle and localizes to the nucleus of the host cell. As part of the tegument, HCMV pUL69 is usually ZPKP1 delivered to the host cell and acts as a pleiotropic transactivator of gene expression (4, 5). In addition, pUL69 acts as a posttranscriptional transactivator by facilitating the nuclear export of unspliced mRNAs (6). We reported previously that pUL69 binds to RNA and via N-terminal arginine-rich motifs comprising amino acids (aa) 17 to 50 (R1/R2 motifs) and 123 to 139 (RS motif) (7). While mRNA binding is usually dispensable for pUL69-mediated export of an artificial reporter construct, recruitment of the cellular RNA helicases UAP56 and URH49 is absolutely essential (6, 7). Previous studies suggested that this UAP56/URH49 conversation motif is located between N-terminal amino acids 18 to 30 of pUL69 and thus partially overlaps the bipartite nuclear localization signal (NLS) and the RNA-binding motif. Interestingly, UAP56/URH49 recruitment is usually well conserved within the cytomegaloviral pUL69 homologs pC69 of chimpanzee cytomegalovirus (CCMV) and pRh69 of rhesus cytomegalovirus (RhCMV) and was correlated with the mRNA export activity of the respective proteins, analogously to pUL69 (8, 9). The importance of UAP56/URH49 recruitment for efficient HCMV replication was ultimately confirmed by the observation that recombinant cytomegaloviruses that had deletions or point mutations within the UAP56 conversation motif of UL69 (UL69R1RS and UL69mutUAP) exhibited severe replication defects (8). More recently, we could demonstrate that this pUL69 N terminus is usually methylated, which involves an conversation with the cellular protein arginine methyltransferase 6 (PRMT6). Recruitment of PRMT6 critically affected pUL69-mediated mRNA export and replication of HCMV (10). Meanwhile, it is well established that this multifunctional herpesviral mRNA export factors are also regulated by phosphorylation. For instance, ICP27 phosphorylation CBR 5884 is crucial for its conversation with the cellular RNA export factors REF.