Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

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 ...

Section: Discussionsupporting

confidence: 49%

Section: Importancementioning

confidence: 99%

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

Thomas

Sonntag

Müller

et al. 2015

“…nuclear (34). In this study, we found that hypomethylation of ICP27 did not affect import of ICP27 into the nucleus and that hypomethylation did not appear to affect the rate of import as measured by FRAP, which is in contrast to the results seen with ICP27 export.…”

contrasting

confidence: 62%

Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection

Souki

Hernandez

Sandri‐Goldin

2011

Arginine methylation can regulate protein import and export and can modulate protein interactions. Herpes simplex virus 1 (HSV-1) ICP27 is a shuttling protein involved in viral mRNA export. We previously reported that ICP27 is methylated on three arginines within its RGG box and that arginine methylation regulates ICP27 export and its interaction with SRPK1 and Aly/REF. Here, we report that ICP27 was efficiently imported into the nucleus when hypomethylated as determined by Fluorescence Recovery After Photobleaching (FRAP). Furthermore, coimmunoprecipitation of ICP27 with ␤-importin was not significantly affected by ICP27 hypomethylation. Thus, ICP27 import does not appear to be regulated by arginine methylation.

“…85,2011 ROSCOVITINE INHIBITS EBNA1 2865 late transcription (74,78). In addition, EBNA1 RG1 and RG2 recruitment of PRMT1 and -5 to oriP sites may enhance EBNA1-upregulated RNA export through enhanced RNA 3Ј-terminal cleavage, SM protein effects on splicing, RNA nuclear export, EIF4E and NXF1 binding, and ICP27-like RNA export effects (29,37,51,64,86). Compounds that inhibit or enhance PRMT1 or -5 effects would likely downregulate EBNA1-and oriP-dependent transcription but would also broadly affect protein methylation, RNA processing, and translation.…”

Section: Discussionmentioning

confidence: 99%

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Kang

Lee

Soni

et al. 2011

Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists asan episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1-and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1-and oriP-dependent transcription and do not inhibit EBNA1-and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and 486 RALL 489 and 580 KDLVM 584 as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1-and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1-and oriPCp-dependent transcription and episome persistence.Epstein-Barr virus (EBV) replicates in oropharyngeal epithelial cells (62,70,85). Subsequent latent infection of mature B lymphocytes can result in infectious mononucleosis and is essential for long-term latency and reactivation and continues to be shed in saliva (21). Latently EBV-infected B cells can emerge as Burkitt's lymphomas (BL) or Hodgkin's disease in otherwise healthy people or as lymphoproliferative disease in people who have T-lymphocyte deficiencies due to HIV infection, transplantation, advanced age, or a genetic predisposition (4,8,25,34,49,62,85). In immunocompetent people, particularly those of southern Chinese descent, latent EBV infection can also cause nasopharyngeal carcinoma, which is the most common EBV malignancy worldwide (25,34). Latent EBV infection is a significant cause of morbidity and mortality.In latent infections, including malignancies, EBV usually persists as a multicopy episome (1,47,56,60). EBV genome integration is unusual (33, 52). EBNA1 is essential for EBV episome persistence in dividing cells and enhances episome transcription (59,61,83). The EBNA1 DNA binding domain recognizes 24 cognate sites in the EBV episome OriP element, while ...