Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function

Invernizzi, Cédric; Xie, Baode; Frankel, Fernando; Feldhammer, Matthew; Roy, Bibhuti Bhusan; Richard, Stéphane; Wainberg, Mark A.

doi:10.1097/qad.0b013e32803277ae

Cited by 44 publications

(37 citation statements)

References 48 publications

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“…ity. PRMT6 has been shown to inhibit HIV-1 transcription through the methylation of Tat, Rev, and the nucleocapsid proteins (69,70,71). PRMT1 has been shown to methylate and inhibit hepatitis C virus (HCV) NS3 protein, but in turn, HCV counteracts PRMT1 repressive activity by activating protein phosphatase 2, which inhibits PRMT1 (72,73).…”

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

J Virol

101

110

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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.

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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

J Virol

101

110

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“…To establish a linear range of enzyme activity, 50 M R1 or R1(MMA) substrates were incubated with 200 M AdoMet at 37°C for 0 -300 min. (10,20,30, and 50 M) AdoMet. Complementary assays with the product inhibitor R1(aDMA) at concentrations of 50, 100, and 300 M were also performed with the same constant or variable concentrations of R1 and AdoMet as above.…”

Section: Methodsmentioning

confidence: 99%

“…For the specific case of PRMTs, radioactively methylated protein products have been hydrolyzed, and the component-methylated arginine amino acids are separated by ion exchange chromatography (42), TLC (8), or reversed-phase high-performance liquid chromatography (5). Radioactively methylated proteins are also frequently separated by SDS-PAGE, and the resultant dried gels are exposed to film or storage phosphor screen for detection and quantitation (27,30,49,61). The present study contains a method that utilizes the separation of 14 Cmethylated amino acids from hydrolyzed peptides by TLC.…”

Section: Productmentioning

confidence: 99%

“…Most recently several groups have demonstrated that PRMT6 methylation of Arg-2 on histone H3 in vivo exists in an exclusive relationship with Lys-4 methylation, thus adding to the mounting evidence that PRMT6 is a negative regulator of cellular as well as viral transcriptional activation (31)(32)(33). PRMT6 and other PRMTs are now a focus of attention for the development of inhibitors targeting methyl transfer activity (20,29,30,(35)(36)(37).…”

mentioning

confidence: 99%

“…PRMT6 localizes exclusively to the cell nucleus, exhibits automethylation, and methylates in vitro glycine-and arginine-rich (GAR) sequences in proteins (6). Despite its overlapping substrate specificity with PRMT1, some PRMT6-specific cellular targets do not contain the GAR consensus sequence (1,24), including high mobility group proteins HMGA1a and HMGA1b (25,26), DNA polymerase ␤ (27), HIV-1 trans-activator of transcription (Tat) protein (28), HIV-1 regulator of virion (Rev) protein (29), HIV-1 nucleocapsid protein (30), and histone H3 (31)(32)(33). Methylation of polymerase ␤ by PRMT6 was shown to increase its repair activity of damaged DNA, implicating PRMT6 as a regulator of base excision repair (27).…”

mentioning

confidence: 99%

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A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Lakowski

Frankel

2008

Journal of Biological Chemistry

128

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Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-Lmethionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as -N G -monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K m and higher V max values than what are observed for the unmethylated peptide. This difference in substrate K m and V max , as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K m values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.Human protein arginine N-methyltransferases (PRMTs) 2 are a family of enzymes that transfer methyl groups from the co-substrate S-adenosyl-L-methionine (AdoMet) to the terminal nitrogen atoms on the guanidino groups of arginine residues within proteins, forming S-adenosyl-L-homocysteine

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Assays for S‐Adenosylmethionine (AdoMet/SAM)‐Dependent Methyltransferases

2008

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Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and adenine by recombinant AdoHcy nucleosidase. Subsequently, the adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant adenine deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput.

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Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function

Abstract: NC is an in-vivo target of PRMT6, and arginine methylation of NC reduces RNA annealing and the initiation of reverse transcription. These findings may lead to ways of driving HIV-infected cells out of latency with drugs that inhibit PRMT6.

Cited by 44 publications

References 48 publications

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

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

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Assays for S‐Adenosylmethionine (AdoMet/SAM)‐Dependent Methyltransferases

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