Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription

Abstract

Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction.

Figure 1

Efficient phosphorylation of RNA pol II CTD by stable human-rodent hybrid P-TEFb complexes. Cell lysates were prepared from two human 293-based cell lines and two CHO-derived cell lines expressing hCycT1-HA or hCdk9-HA. Four different P-TEFb complexes (hCdk9-HA/hCycT1, hCdk9-HA/rCycT1, hCdk9/hCycT1-HA, and rCdk9/hCycT1-HA) were affinity-purified from these lysates by anti-HA immunoprecipitation followed by HA peptide elution from the antibody column. (A) Western blotting with antibodies specific for hCdk9 and hCycT1 was carried out to examine the levels of Cdk9 and CycT1 in these complexes. (B) After normalization by Western blotting, equal amounts of the four P-TEFb complexes were analyzed in in vitro kinase reactions containing purified calf thymus RNA pol II as a substrate. The abilities of increasing amounts (two-fold increase at each step) of the four complexes to phosphorylate pol II as well as Cdk9 and CycT1 endogenous to the complexes were examined. In control reactions (lanes 1 and 2), anti-HA immunoprecipitates prepared from the parental 293 and CHO cell lysates were analyzed.

Figure 2

Human but not rodent CycT1 is required for Tat activation of HIV-1 transcription in vitro. Transcription reactions containing P-TEFb-depleted HeLa nuclear extract as well as DNA templates pHIV+TAR-G400 and pHIVΔTAR-G100 (23) were performed in the absence (−) or presence (+) of Tat. Equal amounts of the four P-TEFb complexes were added to transcription reactions as indicated. +TAR-G400 and ΔTAR-G100 represent RNA fragments transcribed from the two G-less DNA cassettes (400 bp and 100 bp) inserted, respectively, into the two DNA templates at a position ≈1 kb downstream of the HIV-1 promoter region.

Figure 3

Human but not rodent CycT1 mediates a specific and high-affinity interaction of P-TEFb with Tat. Four different P-TEFb complexes as indicated were incubated with glutathione-Sepharose beads coupled with equal amounts of wild-type GST-Tat(1–48) or a mutant GST-Tat(C22G, 1–48). After washes, the amount of P-TEFb bound to Tat was examined by Western blotting with anti-CycT1 (A) or anti-Cdk9 (B) antibodies. For each complex, 20% of the sample used in the binding reaction was shown as input.

Figure 4

Requirement of hCycT1 and to a lesser extent hCdk9 for formation of a TAR loop-dependent P-TEFb–Tat–TAR ribonucleoprotein complex. Equal amounts of the indicated four P-TEFb complexes were bound to ³²P-labeled wild-type HIV-1 TAR (labeled w) or loop mutant +31/+34 (labeled m) in the presence (+) or absence (−) of Tat. In lane 22, α-Cdk9 antibodies, Tat, TAR, and hCdk9-HA/hCycT1 were present in the binding reaction. The exposure time for lanes 1′–6′ was ≈20% of that for lanes 5–22. Compared with the previous condition (13, 14), the presence of Zn²⁺ in the binding reaction increased the efficiency of the formation of P-TEFb–Tat–TAR complexes. Formation of these complexes, however, was still less efficient than that of the Tat–TAR complex and the hCycT1–Tat–TAR complex containing recombinant hCycT1 (13). The reason is unclear.

Figure 5

Cdk9 and Tat interact with mainly the cyclin-box region of cyclin T1. (A) A diagram showing the domain structures of the full-length hCycT1 and it C-terminal truncation mutants Δ1-Δ5. (B) Different P-TEFb complexes consisting of Cdk9/CycT1Δ1-Δ5 were affinity-purified from human 293T cells transfected with the HA-tagged CycT1Δ1-Δ5 constructs (lanes 2–6). After normalization of their Cdk9 levels, equal amounts of Cdk9/CycT1Δ1-Δ5 were tested for their abilities to phosphorylate GST-CTD (lanes 2–6). Lane 1 contains anti-HA immunoprecipitates derived from 293T cells transfected with an empty vector. P-TEFb complexes containing the HA-tagged wild-type Cdk9 (K⁺, lane 8) or a kinase-defective Cdk9 (K⁻, lane 7) were also obtained from transfected 293T cells (13) and tested for GST-CTD phosphorylation. These control experiments demonstrated that GST-CTD was indeed phosphorylated by Cdk9 rather than an unknown factor that coimmunoprecipitated with the HA tag. (C) Equal amounts of Cdk9/CycT1Δ1-Δ5 were incubated with wild-type GST-Tat(1–48) and mutant GST-Tat(1–48, C22G) bound to glutathione-Sepharose beads. After extensive washes, the bound proteins were analyzed by Western blotting for the levels of Cdk9. For each complex, 20% of the material used in the binding reaction was also shown (input).