Analysis of the effect of natural sequence variation in Tat and in cyclin T on the formation and RNA binding properties of Tat-cyclin T complexes

Abstract

The biological activity of the human immunodeficiency virus type 1 (HIV-1) Tat (Tat1) transcriptional activator requires the recruitment of a Tat1-CyclinT1 (CycT1) complex to the TAR RNA target encoded within the viral long terminal repeat (LTR). While other primate immunodeficiency viruses, such as HIV-2 and mandrill simian immunodeficiency virus (SIVmnd), also encode Tat proteins that activate transcription via RNA targets, these proteins differ significantly, both from each other and from Tat1, in terms of their ability to activate transcription directed by LTR promoter elements found in different HIV and SIV isolates. Here, we show that CycT1 also serves as an essential cofactor for HIV-2 Tat (Tat2) and SIVmnd Tat (Tat-M) function. Moreover, the CycT1 complex formed by each Tat protein displays a distinct RNA target specificity that accurately predicts the level of activation observed with a particular LTR. While Tat2 and Tat-M share the ability of Tat1 to bind to CycT1, they differ from Tat1 in that they are also able to bind to the related but distinct CycT2. However, the resultant Tat-CycT2 complexes fail to bind TAR and are therefore abortive. Surprisingly, mutation of a single residue in CycT2 (asparagine 260 to cysteine) rescues the ability of CycT2 to bind Tat1 and also activates not only TAR binding by all three Tat-CycT2 complexes but also Tat function. Therefore, the RNA target specificity of different Tat-CycT1 complexes is modulated by natural sequence variation in both the viral Tat transcriptional activator and in the host cell CycT molecule recruited by Tat. Further, the RNA target specificity of the resultant Tat-CycT1 complex accurately predicts the ability of that complex to activate transcription from a given LTR promoter element.

FIG. 1

Comparison of primate immunodeficiency virus Tat proteins and TAR elements. A sequence alignment of the HIV-1 (strain IIIB), HIV-2 (strain ROD), and SIV_mnd Tat proteins is shown (8, 26). While very different in terms of length, and in the more amino- and carboxyl-terminal sequences, all three proteins contain recognizable cysteine-rich, core, and basic domains, as indicated. A critical cysteine residue (C22 in Tat1) that is required for biological activity is indicated by an asterisk. The predicted secondary structures of the viral TAR elements suggest the presence of one Tat target site in HIV-1 TAR and duplicated target sites in both HIV-2 and SIV_mnd TAR. The critical functional elements in TAR are the terminal hexanucleotide loops and the 5′ proximal bulges.

FIG. 2

Interactions between Tat proteins and cyclins in vivo. The ability of the indicated lentiviral Tat proteins and human and murine cyclins to interact was assayed by yeast two-hybrid analysis, as previously described (4). Tat proteins were expressed as GAL4 fusions, whereas CycT variants were expressed fused to the VP16 transcription activation domain.

FIG. 3

RNA binding activities of different cyclin T-Tat complexes. The ability of different human and murine cyclins to bind to the indicated primate immunodeficiency virus TAR elements, in the presence or absence of the cognate Tat protein, was assayed by the yeast three-hybrid assay as previously described (4). Cyclins were expressed as VP16 activation domain fusions, as shown in Fig. 2, while Tat proteins were expressed in their wild-type, nonfused form. Induced β-galactosidase activity was measured by light absorption at 595 nm and is expressed in milli-optical density (mOD) units.

FIG. 4

Rescue of Tat function in murine cells. Murine LmTK⁻ cells were transfected with indicator constructs consisting of the HIV-2 or SIV_mnd LTR promoter element linked to the CAT indicator gene together with expression plasmids encoding HIV-2 or SIV_mnd Tat and one of the indicated cyclins. At ∼48 h after transfection, CAT expression levels were determined as described previously (4).

FIG. 5

A single-amino-acid substitution in hCycT2B induces Tat1 binding and permits TAR recruitment by divergent Tat proteins. (A) Sequence comparison between the hCycT1 TRM (residues 250 to 262) and the corresponding sequences in mCycT1 and hCycT2. (B) Interactions between the indicated GAL4-Tat fusion proteins and either VP16-hCycT1 or wild-type or mutant (N260) forms of the VP16-hCycT2B fusion protein measured by the yeast two-hybrid assay. (C) Recruitment of hCycT2B(N260) but not wild-type hCycT2B to TAR. The yeast three-hybrid assay was performed with plasmids expressing Tat proteins and MS2-TAR hybrid RNAs derived from the indicated primate immunodeficiency virus along with the indicated VP16-CycT proteins. OD595, optical density at 595 nm, given in milli-optical density (mOD) units.

FIG. 6

The hCycT2B(N260C) mutant can support the function of divergent lentiviral Tat proteins. Murine LmTK− cells were transfected with the indicated viral LTR-based reporter and Tat expression plasmid along with plasmids expressing the indicated hCycT proteins, as described for Fig. 4.

FIG. 7

The LTR promoter specificity of diverse Tat proteins is predicted by the TAR binding affinity of the relevant hCycT1-Tat complex. (A) The ability of hCycT1 to bind to the indicated viral TAR elements in the presence of each viral Tat protein was determined by the yeast three-hybrid assay, as shown in Fig. 3. The negative control (Neg) reflects the level of β-galactosidase activity obtained in the absence of any TAR element. (B) Dose response analysis in transfected human 293T cells of the response of CAT-based indicator constructs containing the HIV-1, HIV-2, or SIV_mnd LTR promoter to increasing levels of the indicated Tat proteins.

FIG. 8

Mutational definition of an hCycT1 sequence motif required to mediate Tat function. The protein sequence of hCycT1 between residues 235 and 280 is shown, with the critical cysteine 261 indicated. Alanine scanning mutants X1 to X8 were derived by substitution of alanine for the indicated contiguous stretches of two to three residues in the context of the full-length hCycT1 sequence. The bar graph shows the ability of each of these hCycT1 mutants to rescue the function of the indicated primate immunodeficiency virus LTR promoter-Tat combination in mouse cells.