pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

Abstract

The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same ␣-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus. IMPORTANCEThe UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy. E very mammalian or avian herpesvirus encodes a member of the ICP27 protein family which functions as a posttranscriptional activator by facilitating the nuclear export of intronless mRNAs ...

Chemical probes targeting epigenetic proteins: Applications beyond oncology

“…The regulatory protein pUL69 of HCMV acts as a viral RNA export factor. Among the 8 PRMTs (1–8), only PRMT2 and PRMT6 co-localized with pUL69 in the nucleus 164 . PRMT6 methylates pUL69 within the functionally important arginine rich R1 box.…”

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

“…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). A pronounced kinase inhibitor-induced formation of nuclear speckled aggregation of pUL69 was originally demonstrated by Sanchez & Spector (2006).…”

“…amino acid replacement mutations in putative phosphorylation sites) that were exposed to the kinases CDK9 or pUL97 under established conditions of in vitro kinase assays (IVKAs) (Marschall et al, 2002;Thomas et al, 2009;Rechter et al, 2009;Webel et al, 2014). Replacement mutants were generated on the basis of pUL69-Flag cloned in vector pcDNA3.1 by the use of a protocol for site-directed mutagenesis described previously (Lischka et al, 2006;Thomas et al, 2009Thomas et al, , 2015Schmeiser et al, 2013) (see Table S1 for PCR primers and a description of constructs; Invitrogen/Life Technologies). For IVKAs, proteins were either transiently expressed in plasmid-transfected 293T cells and immunoprecipitated by the use of mAb-Flag [pUL69-Flag and pUL97(181-707)-Flag] ( Thomas et al, 2009) or exogenously added to the reactions in the form of affinity-purified proteins (CDK9-cyclin T1, histone 2B and Rb-CTF; Proqinase) .…”

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

“…Phosphorylation at S/T residue 46, 49, 51, or 52 does not affect the secondary structure of pUL69. In previous experiments, we have used nuclear magnetic resonance (NMR) to determine the secondary structure of wild-type pUL69aa17-46 and its UAP56/PRMT6-binding-deficient derivative pUL69aa17-46-R22/23/25/26A, revealing an overall ␣-helical conformation (10) (Fig. 5A).…”

“…More recently, we could demonstrate that the pUL69 N terminus is methylated, which involves an interaction with the cellular protein arginine methyltransferase 6 (PRMT6). Recruitment of PRMT6 critically affected pUL69-mediated mRNA export and replication of HCMV (10).…”