Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49

Abstract

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans 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 interaction 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 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 the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans 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 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 important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus

“…Supplementary Table S3). Shown are phosphosites found on (a) pUL32/pp150, (b) pUL83/pp65, (c) pUL82/pp71, (d) pUL25, (e) pUL97, and (f) pUL69 in virions (this study and Rieder et al [23]) and in infected cells [25,[83][84][85][86][87] (HFF, human foreskin fibroblasts).…”

“… Graphical display of phosphosites identified in this study and by previous investigations (details are given in Supplementary Table S3 ). Shown are phosphosites found on ( a ) pUL32/pp150, ( b ) pUL83/pp65, ( c ) pUL82/pp71, ( d ) pUL25, ( e ) pUL97, and ( f ) pUL69 in virions (this study and Rieder et al [ 23 ]) and in infected cells [ 25 , 83 , 84 , 85 , 86 , 87 ] (HFF, human foreskin fibroblasts). …”

“…The spectrum of currently identified pUL97 protein substrates comprises viral and host proteins. The viral targets of pUL97 comprise the DNA polymerase cofactor pUL44 [102,103], the RNA transport factor pUL69 [87,104,105], the major tegument protein pUL83/pp65 [26,100,106], and the nuclear egress core protein heterodimer pUL50-pUL53 [35,107]. The host proteins shown to be phosphorylated by pUL97 are the cellular multiligand binding protein p32/gC1qR [108], the tumor suppressor and checkpoint protein Rb [109][110][111], its associated protein E2F3 [112], the Rb-related proteins p107 and p130 [113] and their interacting protein LIN52 [112], the nuclear lamins A/C [108,114,115], the RNA polymerase II [116][117][118], the translation factor EF-1δ [119,120], the FOXM1 transcription factor [112], the interferon-inducible protein IFI16 [121], and the host restriction factor SAMHD1 [122].…”

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties

“…To illustrate this process, the strategies of pharmacological Pin1 inhibition or genetic Pin1 knockout provided conclusive evidence that Pin1 promotes the disassembly of the nuclear lamina during HCMV replication [ 10 , 12 ]. Very recently, we were able to provide additional evidence for the relevance of Pin1 in HCMV replication, in that data derived from several independent approaches demonstrated an interaction of Pin1 with the viral proteins pUL50, pUL69 and pUL44 [ 75 , 76 ]. Whether the regulatory impact of Pin1, especially its lamin-directed role in nuclear egress, is specific for HCMV, or whether Pin1 activity is likewise a regulatory cofactor for other α-, β- and γ-herpesviruses has still to be clarified.…”

Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells

“…Upon knocking-down SPT6, an approximately 90% inhibition of the wild-type replication was detected, thus highlighting the significance of SPT6-UL69 binding in HCMV replication (24,26,27). Besides, pUL69 possesses binding motifs for UAP56/URH49 known as cellular mRNA adaptor proteins (28). pUL69 binding to the cellular helicase UAP56 has a role in transcriptional elongation and also promotes the mRNA nuclear export (16,29).…”