Human immunodeficiency virus type 1 Tat prevents dephosphorylation of Sp1 by TCF-4 in astrocytes

Abstract

Previous examination of the effect of TCF-4 on transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in human astrocytic cells found that TCF-4 affects the HIV-1 promoter through the GC-rich domain (nt "80 to nt "68). Here, the physical interaction and a functional consequence of TCF4-Sp1 contact were characterized. It was shown that expression of TCF-4 in U-87 MG (human astrocytic) cells decreased basal and Sp1-mediated transcription of the HIV-1 promoter. Results from a GST pull-down assay, as well as combined immunoprecipitation and Western blot analysis of protein extracts from U-87 MG cells, revealed an interaction of Sp1 with TCF-4. Using in vitro protein chromatography, the region of Sp1 that contacts TCF-4 was mapped to aa 266-350. It was also found that, in cell-free extracts, TCF-4 prevented dsDNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation. Surprisingly, TCF-4 failed to decrease Sp1-mediated transcription of the HIV-1 long terminal repeat (LTR) and Sp1 phosphorylation in cells expressing HIV-1 Tat. Results from immunoprecipitation/Western blotting demonstrated that TCF-4 lost its ability to interact with Sp1, but not with Tat, in Tat-transfected cells. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1, which is affected by Tat and DNA-PK. Interactions among TCF-4, Sp1 and/or Tat may determine the level of viral gene transcription in human astrocytic cells.

Human Immunodeficiency Virus-Restricted Replication in Astrocytes and the Ability of Gamma Interferon To Modulate This Restriction Are Regulated by a Downstream Effector of the Wnt Signaling Pathway

“…It is also still likely that TCF-4 may bind Tat and that this complex binds to or near TAR, altering the stereochemistry of Tat-TAR interaction, leading to HIV inhibition or the formation of a multicomplex among TCF-4/␤-catenin, Tat, and TAR that leads to HIV inhibition. Recently it was also shown that TCF-4 interacts with the Sp1 transcription factor, which is one of the transcription factors that activate the HIV LTR, interfering with Sp1 LTR transactivation ability, in the absence of Tat (40). This interference could therefore result in lowered HIV gene transcription.…”

“…TCF-4 was shown to form a complex with Tat, presumably inhibiting Tat binding to transactivation response (TAR). More recently, TCF-4 was shown to interfere with Sp1 transcription factor, leading to modulation of its capability to activate the HIV LTR by binding to its cognate sites (40).…”

Functions of Tat: the versatile protein of human immunodeficiency virus type 1

“…Sp1 plays a critical role in many cellular events by regulating the expression of cellular genes. Tat activates Sp1 by phosphorylation of this protein, and thereby Sp1 proteins bind to LTR to upregulate the HIV-1 promoter (Chang et al, 1994;Rossi et al, 2006). The interaction of Tat with the HIV-1 accessory protein Vpr has also been shown to enhance the transcriptional activity of the HIV-1 LTR.…”

“…Besides the RNA polymerase II complex, the phosphorylation of additional transcription factors, including Sp1, CREB, the alpha subunit of eukaryotic initiation factor 2 (eIF2a) and NF-kB have also been reported to be triggered by Tat (Demarchi et al, 1999;Li et al, 2005;Rossi et al, 2006;Zauli et al, 2001). Tat has been indicated to influence the HIV-1 promoter by phosphorylation of Sp1.…”

tatExon 1 Exhibits Functional Diversity during HIV-1 Subtype C Primary Infection

“…Corresponding acetylation of key Tat residues by the CBP/p300 complex enhances the subsequent engagement of Tat with the positive transcription elongation factor P-TEFb complex (12,18,19), composed of cyclin T1 and CDK9 subunits, permitting crucial phosphorylation of serine residues in the C-terminal domain (CTD) of RNAPII, resulting in complete viral gene expression (20). Additional phosphorylation events occur between Tat and cellular transcription factors, such as NF-B and Sp-1 (21,22), which play critical roles in the regulation of viral gene transcription. Subtype differences exist in the configuration of these and other transcription factor binding sites within the LTR, which have been reported to alter the responsiveness of HIV transcription to external stimuli: a single NF-B binding site is present in the promoter of HIV-1 subtype E (HIV-1E), yet subtype B contains two with subtype C containing at least three, but as many as four, NF-B consensus sequences (23).…”