Abstract
BackgroundProtein arginine methyltransferase 6 (PRMT6) can methylate the HIV-1 Tat, Rev and nucleocapsid proteins in a manner that diminishes each of their functions in in vitro assays, and increases the stability of Tat in human cells. In this study, we explored the relationship between PRMT6 and HIV-1 Tat by determining the domains in each protein required for interaction.MethodsThrough domain mapping and immunoprecipitation experiments, we determined that both the amino and carboxyl termini of PRMT6, and the activation domain within Tat are essential for interaction. Mutation of the basic domain of Tat did not affect the ability of PRMT6 to interact with Tat.ResultsWe next used the A549 human alveolar adenocarcinoma cell line, which naturally expresses undetectable levels of PRMT6, as a model for testing the effects of PRMT6 on Tat stability, transactivation, and HIV-1 replication. As previously observed, steady state levels and the protein half-life of Tat were increased by the ectopic expression of PRMT6. However, no down regulation of Tat transactivation function was observed, even with over 300-fold molar excess of PRMT6 plasmid. We also observed no negative effect on HIV-1 infectivity when A549 producer cells overexpressed PRMT6.ConclusionsWe show that PRMT6 requires the activation domain, but surprisingly not the basic domain, of Tat for protein interaction. This interaction between Tat and PRMT6 may impact upon pathogenic effects attributed to Tat during HIV-1 infection other than its function during transactivation.
Highlights
The HIV-1 Tat protein is a transcriptional activator that plays an essential role during the late phase of viral replication
The termini of Protein arginine methyltransferase 6 (PRMT6) interact with Tat Little is known about the functional domains of PRMT6 apart from a catalytic methyltransferase domain that is conserved among all members of the protein arginine methyltransferase family [14,21]
The mechanism of action they proposed involves PRMT6catalyzed methylation of Tat within its basic domain, which subsequently disrupts formation of the Tat-transactivation response element (TAR)-cyclin T1 ternary structure critically required for activating transcription at the HIV-1 proviral promoter [7,12]
Summary
The HIV-1 Tat protein is a transcriptional activator that plays an essential role during the late phase of viral replication. PRMT6 has been shown to down regulate transactivation by a proposed mechanism in which methylation of Tat arginine interferes with the interaction with TAR [7,12], even though overexpression of PRMT6 results in the increased stability of Tat through inhibition of proteasome-dependent degradation [13]. PRMT6 is a type I enzyme of the protein arginine methyltransferase family [14] which, among other functions, regulates cellular gene expression by methylating arginine-2 of histone H3. Deposition of a methyl group at arginine-2 interferes with the trimethylation of lysine-4 in histone H3, a post-translational modification associated with chromatin remodeling amiable to gene expression [15,16,17,18]. Protein arginine methyltransferase 6 (PRMT6) can methylate the HIV-1 Tat, Rev and nucleocapsid proteins in a manner that diminishes each of their functions in in vitro assays, and increases the stability of Tat in human cells. We explored the relationship between PRMT6 and HIV-1 Tat by determining the domains in each protein required for interaction
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