Acetylation of HIV-1 Tat by CBP/P300 Increases Transcription of Integrated HIV-1 Genome and Enhances Binding to Core Histones

Abstract

The HIV-1 Tat protein is required for viral replication and is a potent stimulator of viral transcription. Although Tat has been extensively studied in various reductive paradigms, to date there is little information as to how this activator mediates transcription from natural nucleosomally packaged long terminal repeats. Here we show that CREB-binding protein (CBP)/p300 interacts with the HIV-1 Tat protein and serves as a coactivator of Tat-dependent HIV-1 gene expression on an integrated HIV-1 provirus. The site of acetylation of Tat was mapped to the double-lysine motif in a highly conserved region, (49)RKKRRQ(54), of the basic RNA-binding motif of Tat. Using HLM1 cells (HIV-1(+)/Tat(-)), which contain a single copy of full-length HIV-1 provirus with a triple termination codon at the first AUG of the Tat gene, we find that only wild type, and not K50A, K51A, or K50A/K51A alone or in combination of ectopic CBP/p300, is able to produce full-length infectious virions, as measured by p24 gag ELISAs. Tat binds CBP/p300 in the minimal histone acetyltransferase domain (1253-1710) and the binding is stable up to 0.85 M salt wash conditions. Interestingly, wild-type peptide 41-54, and not other Tat peptides, changes the conformation of the CBP/p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB, indicating that Tat may influence the transcription machinery by helping CBP/p300 to recruit new partners into the transcription machinery. Finally, using biotinylated wild-type or acetylated peptides, we find that acetylation decreases Tat's ability to bind the TAR RNA element, as well as to bind basal factors such as TBP, CBP, Core-Pol II, or cyclin T. However, the acetylated Tat peptide is able to bind to core histones on a nucleosome assembled HIV-1 proviral DNA.

Enhancement of Nuclear Factor-κB Acetylation by Coactivator p300 and HIV-1 Tat Proteins

“…HIV-LTR-CAT reporter and eukaryotic Tat expression vectors (pcTat) have been described previously (27)(28)(29). GSTp300 (HAT) construction from amino acids 1,197-1,735 has been described previously (25). A 500-ml LB ϩ ampicillin flask was inoculated with the overnight culture and was grown for 4 h at 37°C.…”

“…After binding, the column was washed with buffer D (20 mM Hepes, pH 7.9, 0.5 mM EDTA, 1 mM dithiothreitol, 0.2% (v/v) Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 15% (v/v) glycerol) containing 150 mM NaCl, and bound protein eluted with buffer D containing 400 mM NaCl, and dialyzed against buffer D (30). Tat peptides used in this study, Tat 36 -72 and Tat 41-54, have been described previously (25).…”

“…This superinduction in activated transcription on chromatin DNA has been attributed to the HAT activity of CBP/p300 on the integrated HIV-1 promoter. It is known that CBP/p300 acetylates Tat at a double lysine motif in a highly conserved region (24,25,50). In the absence of CBP/p300 and Tat crystal structures, it was shown previously that Tat can change the conformation of the CBP/ p300 in vitro such that basal transcription factors such as TATA-binding protein (TBP) and TFIIB could bind with higher affinity to CBP/p300 and influence the activation process by aiding CBP/p300 to recruit new partners into the transcription machinery (25).…”

The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax

“…His-taggedTax proteins were detected with a mouse secondary antibody using SuperSignal West Dura (Pierce, Brebie`res, France). The various GST-CBP and GST-p300 constructs have previously been described (Kashanchi et al, 1998;Deng et al, 2000). Briefly, GST-CBP 1-770 encompasses the KIX and the CH/1 domains, GST-CBP 451-682 corresponds to the KIX domain.…”

Acetyltransferase machinery conserved in p300/CBP-family proteins

“…They not only use nucleosomal histones as substrates but also many other transcription factors (Boyes et al, 1998;Deng et al, 2000;Gu and Roeder, 1997;Imhof et al, 1997;Naar et al, 1998;Zhang and Bieker, 1998;Cress and Seto, 2000). p300/CBP can also acetylate itself (Kraus and Kadonaga, 1998).…”