Nuclear Targeting of Protein Phosphatase-1 by HIV-1 Tat Protein

Abstract

Transcription of human immunodeficiency virus (HIV)-1 genesis activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val 36 and Phe 38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/ F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat. Human immunodeficiency virus type 1 (HIV-1)3 is a retrovirus that encodes transcriptional activator (Tat) protein. The activation domain of Tat (amino acids 1-48) interacts with host cell factors, whereas the positively charged RNA-binding domain (amino acids 49 -57) interacts with HIV-1 transactivation response (TAR) RNA (1). In cell-free transcription assays Tat induces elongation of transcription (2, 3). In vivo, Tat also induces initiation of transcription from the integrated HIV-1 promoter (4 -6). In a recent study Tat was shown to stimulate formation of a transcription complex containing TATA box-binding protein but not TATA box-binding protein-associated factors, thus indicating that Tat may enhance initiation of transcription (4). This finding apparently agrees with the early observation that Tat binds directly to the TATA box-binding protein-containing basal transcription factor TFIID (7). Tat activates HIV-1 transcription by recruiting transcriptional coactivators that include positive transcription elongation factor b, containing CDK9/cyclin T1, an RNA polymerase II CTD kinase (3,8,9) and histone acetyltransferases (10 -12). Whereas positive transcription elongation factor b induces HIV-1 transcription from non-integrated HIV-1 template (3,8,9), histone acetyltransferases allow induction of integrated HIV-1 provirus (10 -12). In contrast to the well defined role of protein kinases, the role of protein phosphatases in Tat-mediated HIV-1 transcription is less well understood. FCP1, a CTD phosphatase that dephosphorylates Ser-2 during elongation of transcription (13), is inhibited by Tat and this inhibition may alleviate FCP1-mediated pausing of transcription (14,15). In addition to FCP1, PP2A and PP1 were also shown to be involved in the regulation of HIV-1 transcription. Disregulation of cellular enzymatic activity of PP2A inhibited Tat-induced HIV-1 transcription (16). Expression of the catalytic subunit of PP2A enhanced activa...

Expression of a Protein Phosphatase 1 Inhibitor, cdNIPP1, Increases CDK9 Threonine 186 Phosphorylation and Inhibits HIV-1 Transcription

“…We previously hypothesized that during HIV-1 infection, the viral Tat protein serves as a PP1-targeting subunit. Here, we found that the expression of cdNIPP1 disrupts the association of Tat with PP1; this disruption could be a mechanism for inhibiting HIV-1 transcription, similar to the previously reported HIV-1 inhibitory effect of Tat mutations that prevented binding of Tat to PP1 (24). Accordingly, we observed the inhibition of HIV-1 transcription by a short peptide containing the RVTF sequence, although the effect was relatively small likely due to the problem with cell permeability and inefficient delivery.…”

“…One of the major nuclear regulators of PP1 is NIPP1 (nuclear inhibitor of PP1), which inhibits the dephosphorylation of a wide range of PP1 substrates (22,23). Tat-mediated HIV-1 transcription is blocked by the expression of NIPP1, and the inhibition is reversed by the co-expression of PP1␥ (21 motif and potentially functions as a PP1 regulatory subunit (24). Mutation of residues in the PP1-binding motif (V36A and F38A) prevents Tat from inducing HIV-1 transcription and the translocation of PP1 to the nucleus (24).…”

“…Tat-mediated HIV-1 transcription is blocked by the expression of NIPP1, and the inhibition is reversed by the co-expression of PP1␥ (21 motif and potentially functions as a PP1 regulatory subunit (24). Mutation of residues in the PP1-binding motif (V36A and F38A) prevents Tat from inducing HIV-1 transcription and the translocation of PP1 to the nucleus (24). We showed that in cultured cells, PP1, but not PP2A, likely dephosphorylates CDK9 and contributes to the regulation of activated HIV-1 transcription (25).…”

Enforcing the pause: Transcription factor Sp3 limits productive elongation by RNA polymerase II

“…Protein phosphatases are recruited to genes as part of complexes containing histone-modifying or chromatin-remodeling activities, 70 for example HDAC complexes, 71,72 and by transcription factors such as REST/ NRSF 73 or the HIV-1 Tat protein. 74 We found that both protein phosphatases PP1 and PP2A interact with the transcription factor Sp3. We have demonstrated that PP1 is recruited to the p21 CIP1 promoter and other Sp3-repressed genes in a Sp3-dependent manner.…”

A novel anticancer agent ARC antagonizes HIV-1 and HCV

“…We also compared ARC with poly(I-C), that inhibits protein translation by activating PKR and inducing the phosphorylation of eukaryotic initiation factor 2 a (Nekhai et al, 2000). We observed similar inhibition of protein translation with 100 mM ARC and with (Ammosova et al, 2005) were expressed in Escherichia coli and purified on an Aquapore RP-300 column (Applied Biosystems, Foster City, CA, USA) by reversed-phase chromatography as we described previously (Deng et al, 2002). CEM-GFP cells were treated in a 96-well plate with purified recombinant Tat added at 0.8 mg per 300 000 cells per well along with 100 mM chloroquine (Ammosova et al, 2003), and with the indicated concentrations of ARC.…”