Interplay between cdk9 and NF-κB factors determines the level of HIV-1 gene transcription in astrocytic cells

Abstract

Basal transcription of the HIV-1 genome is controlled by a variety of ubiquitous and inducible regulatory factors, some with the ability to associate with the viral DNA sequences within the promoter spanning the long terminal repeat (LTR). In this report we demonstrate that activation of the HIV-1 promoter through the inducible DNA binding NF-kB transcription factors can be affected by cdk9 in human astrocytic cells. Our results show that ectopic expression of cdk9, but not its mutant variant which lacks the domain responsible for its kinase activity, augments transcription of the LTR. Moreover, we demonstrate that induction of the NF-kB pathway by PMA, or overexpression of its subunits including p50/p65 have a negative effect on the ability of cdk9 to stimulate viral gene transcription in these cells. Results from bandshift experiments demonstrated significant suppression of p50/p65 association to its DNA target motif by cdk9. Further, data from GST pull-down and combined immunoprecipitation/Western blot analysis of the protein extracts from cells expressing cdk9, p50 and p65 have revealed the interaction of cdk9 with both p50 and p65 in the absence of DNA containing the kB motif. All of these observations led us to conclude that the interaction of cdk9 with the NF-kB factors can determine the ability of NF-kB to modulate HIV-1 gene transcription.

p73 Interacts with Human Immunodeficiency Virus Type 1 Tat in Astrocytic Cells and Prevents Its Acetylation on Lysine 28

“…GST, GST-Tat, and GST-p73, and the deletion mutant fusion proteins were prokaryotically expressed and purified as described previously (2). Radiolabeled Tat, p73␣, and p73␤ proteins were synthesized with a TNT-coupled wheat germ extract system according to the manufacturer's recommendations (Promega, Madison, WI).…”

“…The arrows depict the positions of the 73-and 66-kDa p73␣ and p73␤ (B) and the 13-kDa (C) Tat. The Western or IP/Western assays were carried out according to the procedure described previously (2)…”

Specification of DNA Binding Activity of NF- B Proteins

“…Inhibition of NF-kB DNA binding activity by b-catenin could be important in oncogenesis, as hinted at by an inverse correlation between the b-catenin expression levels and levels of the NF-kB target gene Fas in colon and breast tumor tissues . Furthermore, the inhibitory effects on NF-kB DNA binding by Cyclin-dependent kinase 9 (Cdk9), the PIAS1 ( protein inhibitor of activated STAT [signal transducers and activator of transcription] 1) protein, and myocardin were also shown using EMSA, in which these proteins significantly reduced the DNA binding activity of NF-kB (Amini et al 2002;Liu et al 2005;Tang et al 2008). Cdk9 markedly suppressed the association of the p50 -p50, p65 -p65, and p50 -p65 complexes to kB DNA, and the ability of NF-kB to modulate HIV-1 gene transcription was controlled by this inhibitory function (Amini et al 2002).…”

Interplay between HIV-1 Vpr and Sp1 Modulates p21WAF1 Gene Expression in Human Astrocytes

“…Cells were transfected with 0.5 g of reporter plasmid (p21-LUC or p21-CAT) or co-transfected with 2.5 g of various expression cDNAs as described in Amini et al (43). The amount of DNA used for transfection was normalized with pCDNA3 or with pCEP-4 plasmids.…”

“…Gel Electrophoretic Mobility Shift Assay-Gel electrophoretic mobility shift assay was performed as described previously (43). Oligonucleotides corresponding to the p21 promoter region were synthesized, annealed, labeled with [␥-32 P]ATP, and incubated at 4°C for 30 min with 10 g of nuclear extracts or with 100 ng of translated Vpr protein.…”