The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The adenovirus type 5 (Ad5) late region 4 (L4) 100-kDa nonstructural protein (L4-100K) mediates inhibition of cellular protein synthesis and selective translation of tripartite leader (TL)-containing viral late mRNAs via ribosome shunting. In addition, L4-100K has been implicated in the trimerization and nuclear localization of hexon protein. We previously proved that L4-100K is a substrate of the protein arginine methylation machinery, an emergent posttranslational modification system involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing, and DNA repair. As understood at present, L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. To identify the methylated arginine residues and assess the role of L4-100K arginine methylation, we generated amino acid substitution mutations in the RGG and GAR motifs to examine their effects in Adinfected and plasmid-transfected cells. Arginine-to-glycine exchanges in the RGG boxes significantly diminished L4-100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation in RGG motifs is an important host cell function required for efficient Ad replication. Our data further indicate that PRMT1-catalyzed arginine methylation in the RGG boxes regulates the binding of L4-100K to hexon and promotes the capsid assembly of the structural protein as well as modulating TL-mRNA interaction. Furthermore, substitutions in GAR, but not RGG, regions affected L4-100K nuclear import, implying that the nuclear localization signal of L4-100K is located within the GAR sequence.With the onset of the late phase, one of the first adenovirus type 5 (Ad5) late proteins translated, the late region 4 (L4) 100-kDa protein (L4-100K), starts to perform a number of functions that are essential for efficient completion of lytic virus infection. This Ad nonstructural late protein alters the cellular machinery in favor of translating large amounts of virus products, leading to their subsequent nuclear accumulation for capsid assembly. L4-100K achieves this by contributing to the transport and selective translation of late viral mRNAs (12, 13, 21), acting as a chaperone for hexon trimerization and being involved in its transport (8,9,24) and also playing a role in preventing apoptosis of the infected cell by interacting with granzyme B and inhibiting its activity (1). However, most of the mechanisms underlying these processes, and how L4-100K is regulated to accomplish these, remain unclear.One of the most striking features of L4-100K is promoting viral mRNA translation through ribosome shunting and preventing cellular mRNA translation by eliminating the capdependent translation pathway (12,13,15,21,(49)(50)(51). Underlying these processes is the interaction of L4-100K with both the tripartite leader (TL) sequence possessed by all...

Section: Construction Of L4-100k Mutant Virus H5pm4151supporting

confidence: 74%

Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production

Koyuncu

Dobner

2009

“…Yu and collaborators demonstrated that a total loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to DNA damage, cell cycle progression delay, checkpoint defects, as well as cell division aberration (59). PRMT1 controls the DNA damage response pathway in part through the methylation of MRE11 and 53BP1 (60,61). Interestingly, HBx contributes to HBV-induced hepatocarcinogenesis, and it is now well established that HBx expression correlates with mitotic aberrations such as chromosome segregation defects and polyploidy and impairs DNA damage checkpoint control (2,(62)(63)(64).…”

Section: Discussionmentioning

confidence: 99%

Methyltransferase PRMT1 Is a Binding Partner of HBx and a Negative Regulator of Hepatitis B Virus Transcription

Benhenda

Ducroux

Rivière

et al. 2013

104

The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.

“…Studies are also ongoing to determine if methylation of the RGG box affects RNA binding by ICP27 during viral infection. For example, arginine methylation has been shown to alter the DNA binding specificity of some cellular transcription factors (5,43).…”

Section: Vol 83 2009 Arginine Methylation Regulates Icp27 Export 5317mentioning

confidence: 99%

Arginine Methylation of the ICP27 RGG Box Regulates ICP27 Export and Is Required for Efficient Herpes Simplex Virus 1 Replication

Souki

Gershon

Sandri‐Goldin

2009

The herpes simplex virus 1 (HSV-1) multifunctional regulatory protein ICP27 shuttles between the nucleus and cytoplasm in its role as a viral mRNA export factor. Arginine methylation on glycine-and arginine-rich motifs has been shown to regulate protein export. ICP27 contains an RGG box and has been shown to be methylated during viral infection. We found by mass spectrometric analysis that three arginine residues within the RGG box were methylated. Viral mutants with substitutions of lysine for arginine residues were created as single, double, and triple mutants. Growth of these mutants was impaired and the viral replication cycle was delayed compared to wild-type HSV-1. Most striking was the finding that under conditions of hypomethylation resulting from infection with arginine substitution mutants or treatment of wild-type HSV-1-infected cells with the methylation inhibitor adenosine dialdehyde, ICP27 export to the cytoplasm occurred earlier and was more rapid than wild-type ICP27 export. We conclude that arginine methylation of the ICP27 RGG box regulates its export activity and that early export of ICP27 interferes with the performance of its nuclear functions.