Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat

Background The positive transcription elongation factor b (P-TEFb) is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1). The cyclin T1 (CycT1) subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies. Results To create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins co-expressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, the expression levels of CycT1-U7 and Tat were restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors. Conclusion These results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could potentially be used in the design of anti-viral therapy.


Background
The transcription of human immunodeficiency virus type 1 (HIV-1) is a highly regulated process in which several host cellular co-factors and the viral transactivator protein Tat are involved [1,2]. Tat stimulates the elongation of transcription with the aid of the positive transcription elongation factor b (P-TEFb), a heterodimer comprised of cyclin T1 (CycT1) and cyclin dependent kinase 9 (Cdk9). Tat and CycT1 bind to the transactivation response element (TAR), an RNA stem loop structure located at the 5'-end (+1 to +59) of all viral transcripts [3][4][5]. This interaction results in the recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat [6]. Among three distinct P-TEFb complexes (CycT1/Cdk9, CycT2/ Cdk9, and CycK/Cdk9), only the CycT1/Cdk9 complex can support Tat transactivation [7][8][9].
Since P-TEFb is the essential cellular host co-factor of the viral Tat protein, this interaction serves as a potential target for anti-HIV therapeutics. Several approaches have been taken to block HIV transcription by targeting P-TEFb. First, mutant Cdk9 proteins defective in kinase activity have been shown to inhibit HIV transcription in cell culture systems [20]. A number of small compounds that inhibit Cdk9 activities or disrupt the Tat/TAR/P-TEFb interaction have also been tested [20][21][22][23][24][25][26][27][28]. Another approach by Napolitano et al. aimed to inactivate Cdk9 by an oligomerization chain reaction [29]. Additionally, our group has constructed chimeric proteins containing wild type (wt) CycT1 and mutant Cdk9 which inhibited HIV replication up to 90% [30]. Moreover, several CycT1binding proteins and their truncation mutants have been used as inhibitors of Tat transactivation [31][32][33]. Finally, Bai et al. demonstrated that intrabodies against CycT1 inhibited Tat stimulated transactivation [34]. It is important to note, however, that because P-TEFb is involved in the transcription of many cellular genes [35], it is critical to exclusively block HIV-specific pathways in order to develop safe and effective anti-HIV therapies.
In this study, we sought to construct dominant negative CycT1 mutant proteins capable of blocking HIV transcription. A sequence alignment between the cyclin proteins CycT1, T2 and K revealed ten very well-conserved regions that are essential for the formation of the alpha-helical cyclin box repeat domain. We introduced random mutations in the nine most conserved amino acid clusters in these regions and tested the resulting mutant CycT1 proteins for their ability to block HIV transcription. One of the mutant proteins, called CycT1-U7, showed a potent, yet specific, dominant negative effect on HIV transcription, although the steady-state expression level of CycT1-U7 was remarkably low. Western blot analysis indicated that the expression level of the Tat proteins co-expressed with CycT1-U7 was also significantly lower than those coexpressed with wt CycT1. Proteasome inhibitors restored the expression of CycT1-U7 and Tat proteins. As a consequence, these inhibitors diminished the dominant negative effect elicited by over-expression of CycT1-U7. Our results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat proteins. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription, which might be further utilized in development of safe and effective anti-HIV therapies.

Construction and screening of CycT1 mutants
CycT1 is a member of the C-type cyclin family [36]. Its Nterminal 250 amino acids form two cyclin repeat boxes that are essential for the interaction with, and the activation of, Cdk9. Recently, we have determined the three dimensional crystal structure of CycT1 [18]. The cyclin boxes consist of two repeats, each containing five α-helices ( Figure. 1A and 1B). Sequence alignment of three P-TEFb-forming cyclins T1, T2, and K from different species revealed that the secondary structure elements are well conserved among these cyclins, indicating that they play important roles in P-TEFb functions ( Figure. 1B). Based on this secondary structure alignment, we selected the nine most conserved amino acid clusters in the cyclin box domain of CycT1 and introduced random mutations into a C-terminal truncation mutant of CycT1 (CycT1 ). This truncation is sufficient to support Tat transactivation as described before [4,5,9] Table  1).
Mutations were introduced by oligonucleotides containing degenerate nucleotides corresponding to each conserved region. In total, 115 CycT1 mutants were constructed and tested for their activities on Tat transactivation by co-transfecting murine NIH 3T3 cells with an HIV LTR-Luciferase (Luc) reporter gene and Tat (Table 1). Since murine endogenous CycT1 (mCycT1) cannot support Tat transactivation, Tat activated the LTR-driven Luc expression only by approximately 10-fold ( Figure. 2A, lane 2). Over-expression of the wt human CycT1 further activated the gene expression up to 70-fold ( Figure. 2A, lanes 3 and 4). The luciferase activities obtained by overexpressing any of the pool of mutant CycT1 proteins ranged from five to 70-fold. Fifteen mutants showed an equal or a higher activity than the wt, 45 mutants showed modest (50-100% of wt) activity and 55 had less than 50% of the activity of wt CycT1 in these cells (summarized in Table 1). These 55 mutants were further sequenced and tested for their dominant negative effect on HIV transcription by co-transfecting HeLa cells stably expressing the HIV-Luc reporter gene (HeLa/HR-Luc cells) with Tat (Figure. 2B and data not shown).

An N-Terminal CycT1 mutant exhibited the strongest dominant negative effect on Tat transactivation by promoting the degradation of Tat proteins
Amongst the 55 clones tested for their ability to block Tat transactivation in HeLa cells, one mutant containing four amino acid substitutions and one deletion in the second helix H2 of the N-terminal cyclin box repeat (residues HRFYM at a.a. position 67-71 to IIWE; Figure.  These results suggested that Tat transactivation in CycT1-U7 expressing cells is kept at a low level because the steady state Tat expression is diminished in these cells. Since CycT1-U7 retains the wild type sequence of Tat-TAR recognition motif ( Figure. 1C), we hypothesized that CycT1-U7 forms a complex with Tat, and this complex is rapidly degraded in cells.

Expression of CycT1-U7 and Tat can be rescued by proteasome inhibitors
To further prove our hypothesis that CycT1-U7, together with Tat, is rapidly transferred to proteasomal degradation pathways, cells expressing Tat and either wt CycT1 (1-280) or mutant CycT1-U7 were incubated with the proteasome inhibitors, MG-132 (50 μM) or Epoxomicin (50 μM) for 1, 3, and 5 hours prior to cell lysis. MG-132 showed a strong cytopathic effect when incubated for 5 hours (data not shown). The expression of both CycT1-U7 and Tat was partially restored in the presence of MG-132   Since it has been demonstrated that CycT1 is ubiquitinated in cells [37], we sought to examine whether CycT1-U7 is ubiquitinated by co-immunoprecipitation analysis ( Figure. 5A). Ubiquitinated CycT1-U7 proteins were detected in HeLa/ CycT1-U7 cells treated with 50 μM Epoxomicin for 60 min (Figure. 5A, lane 2). Also, in this condition, the interaction between CycT1-U7 and Tat was detected by coimmunoprecipitation ( Figure. 5B, lane 4). These results suggest that CycT1-U7 inhibits Tat-transactivation by rapidly recruiting Tat proteins into an ubiquitin-dependent proteasomal degradation pathway.

Discussion
Although P-TEFb is a potential target for the development of novel anti-HIV therapies, it had been extremely difficult to construct dominant negative CycT1 mutants that block HIV transcription [30]. This is presumably due to the high stability and the complex regulatory mechanism of the endogenous P-TEFb complex. In the present study, we the cyclin box repeat fold ( Figure. 1B). We have previously demonstrated that a CycT1 variant lacking this region (CycT1 (119-280)) is also unstable in cells [38]. This particular mutant exhibited a potent dominant negative effect on HIV transcription, potentially by a similar mechanism (data not shown). Therefore, H2 of CycT1 appears very important for maintaining the structural stability of CycT1 and the interface in between the two repeats. In addition, a residue directly preceding the first helix of the cyclin box repeat that varies between human and equine CycT1 has been previously identified as responsible for differences in the recognition of Tat/TAR complexes from HIV and EIAV [39]. Together, these data point towards the importance of the integrity of the first cyclin box repeat for the interaction with Tat. This region also appears to be essential for the interaction with Cdk9 [18,30]. Interestingly, CycT1-U7 does not promote degradation of endogenous Cdk9. On the other hand, this mutant does bear the wild type sequence of Tat/TAR recognition motif (a.a. 251-272). Indeed, the complex between CycT1-U7 and CycT1-U7 promotes the degradation of Tat Tat was detected when the cells were treated with proteasome inhibitors (Figure. 5). The mutant CycT1-U7 proteins can form a complex with Tat and this complex would be immediately degraded because of the instability of CycT1-U7. Therefore, we conclude that CycT1-U7 exhibits a strong dominant negative effect on Tat transactivation by specifically degrading the co-expressed Tat protein, without disturbing the endogenous P-TEFb complex ( Figure.  6).
It has been demonstrated that CycT1 interacts with other cellular transcription factors through its N-terminal cyclin box regions [40,41]. It is of importance to examine whether CycT-U7 can also inhibit cellular transcription mediated by these factors via a similar pathway. Additionally, the TRM region of CycT1 also interacts with HEXIM1, the endogenous inhibitory protein of P-TEFb which interacts with this region [11], and it is possible that CycT1-U7 affects P-TEFb activity by reducing HEXIM1 levels. More detailed studies are required to assess the effect of CycT1-U7 on cellular transcription.
Our results indicate that CycT1-U7/Tat is recruited to the ubiquitin-dependent degradation pathway. CycT1 seems to be ubiquitinated not only on its C-terminal PEST region (a.a. 706-726) but also at other regions [37]. It is to be noted that wt CycT1 (1-280) is resistant to degradation ( Figure. 3). Although we have not identified the CycT1-U7 is ubiquitinated potential ubiquitination site(s) of CycT1-U7 in this study, it is possible that the cyclin box structure stabilizes the protein by preventing ubiquitination. Conformational changes induced by post-translational modifications such as phosphorylation may expose any additional ubiquitination sites in this region, which would represent a novel pathway to regulate P-TEFb function.
Since the mechanism by which CycT1-U7 inhibits HIV transcription seems not to be through blocking the normal function of P-TEFb, but rather through a "gain-offunction" pathway, it represents a novel class of inhibitory molecules. Moreover, since the steady-state expression of CycT-U7 is very low, it may be an excellent candidate for gene therapy because the mutant proteins would not persist for a prolonged period of time, thereby avoiding induction of unwanted immune responses. Additionally, these proteins would work only when Tat is actively expressed in cells.
HIV utilizes the cellular transcriptional machinery for its own replication. Therefore, it is important to inhibit this Proposed model for the mechanism of dominant negative effect elicited by CycT1-U7 Figure 6 Proposed model for the mechanism of dominant negative effect elicited by CycT1-U7. Wild type CycT1 forms a complex with Cdk9 as an active P-TEFb, and interacts with Tat and TAR RNA. Alternatively, CycT1-U7 associates with Tat but the CycT-U7/Tat complex is immediately degraded via an ubiquitin-dependent proteasomal pathway. This degradation can be prevented using proteasome inhibitors.
step without disturbing cellular functions. Since CycT1 interacts with Tat and TAR, it can be an excellent target to develop safe and effective anti-HIV therapies. Here we present an example of a dominant negative CycT1 molecule which specifically blocks HIV transcription. Studying the precise mechanism by which this mutant CycT1 protein inhibits HIV transcription could unveil novel regulatory pathways of the HIV life cycle and therefore provide reliable clues for designing anti-HIV agents.

Conclusion
In this study, we constructed and evaluated dominant negative CycT1 mutant proteins that specifically block HIV transcription by promoting a rapid degradation of Tat proteins. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription, which can be further utilized to develop a safe and effective anti-HIV therapy.

Materials
HeLa, 293T or NIH 3T3 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal bovine serum at 37°C with 5% CO 2 . HeLa cells stably carrying an HIV-LTR-driven luciferase reporter gene (HeLa-HR-Luc cells) were established using pHR lentiviral vector expressing the luciferase gene under the control of the HIV-LTR, as described previously [42,43]. Anti-myc, anti-HA, anti-CycT1, anti-Cdk9, and anti-Ub antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-Tubulin was purchased from Sigma Aldrich (St. Louis, MO). Proteasome inhibitors, MG-132 and Epoxomicin were purchased from EMD Bioscience (San Diego, CA) and Alexis (San Diego, CA), respectively.

Construction of CycT1 mutants
A structure-based sequence alignment resulting from the crystal structure of the cyclin box repeat of human CycT1 [18] revealed highly conserved α-helical structures in the P-TEFb-forming cyclins T1, T2 and K ( Figure. 1B). Based on this alignment, we selected the nine most conserved regions in the cyclin box repeat domain of CycT1 and introduced random mutations into a C-terminal truncation mutant of CycT1 (1-280) by using oligonucleotides that contain degenerated nucleotides at positions corresponding to each conserved helix and the Transformer Site Directed Mutagenesis Kit (Clontech) ( Figure. 1 and Table 1). The resulting 115 CycT1 mutants were tested for their ability to support Tat transactivation in murine cells as described previously [4]. The CycT1 mutants that failed to activate HIV-transcription in murine cells were sequenced and further tested for their ability to block Tat transactivation in HeLa cells as described previously [30].
The mutant CycT1 (termed CycT1-U7) that exhibited the strongest inhibitory effect on Tat-dependent HIV transcription was used in this study. Sequences of the mutagenic oligonucleotides are shown in Additional file 1.

Generation of stable cell lines
CycT-U7 was subcloned downstream of a CMV promoter in a modified pHR'-SIN lentiviral vector [44,45]. The VSV-G pseudotyped lentiviruses were produced by co-transfection with packaging plasmids (pMDG and p8.9I, [46]), and used to infect Hela cells and HeLa-HR Luc cells.

Transfection and reporter assays
HeLa or NIH 3T3 cells were transfected with 0.5 μg of pEF-CycT1 (wt or mutant constructs) and an HIV-Luciferase reporter construct, in the presence or absence of pTat (0.01 μg) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Twenty-four hours after transfection, cells were harvested and lysed. The protein concentrations of the cell lysates were determined by Protein Assay kit (BioRad). Luciferase activities in the cell lysates were measured as described previously [43].

Ubiquitination assays
HeLa cells stably expressing myc-epitope tagged mutant CycT1 proteins were expressed and, when indicated, treated with 50 μM Epoxomicin for 1 hour. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% Sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM DTT [pH 7.4]) in the presence of protease inhibitors. After preclearing with protein-G sepharose coupled with normal mouse IgG, cell lysates were incubated with 0.5 μg of monoclonal antibody against c-Myc (F-7; Santa Cruz Biotechnology) overnight at 4°C. After the cell lysates were allowed to bind to the antibody, reaction mixtures were incubated with protein-G sepharose beads (Roche) for 1 hour at 4°C. The beads were washed extensively with RIPA buffer and the proteins remaining on the beads were eluted by incubation with SDS loading buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 2 mM EDTA, 0.1 M DTT and 0.01% bromophenol blue, pH 6.8) and subjected to SDS-PAGE, followed by Western blotting with anti-Ub antibody (Santa Cruz Biotechnology).