- Open Access
Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat
© Jadlowsky et al; licensee BioMed Central Ltd. 2008
- Received: 09 April 2008
- Accepted: 11 July 2008
- Published: 11 July 2008
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.
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.
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.
- Proteasome Inhibitor
- Dominant Negative Effect
- Ubiquitination Site
- Strong Dominant Negative Effect
- Potent Dominant Negative Effect
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 [3–5]. This interaction results in the recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat . Among three distinct P-TEFb complexes (CycT1/Cdk9, CycT2/Cdk9, and CycK/Cdk9), only the CycT1/Cdk9 complex can support Tat transactivation [7–9].
The interaction between Tat, TAR, and CycT1 has been extensively studied [2–5, 8, 10]. Tat binds to the bulge region (+23 to +25) of TAR and the CycT1 subunit of P-TEFb through its central arginine-rich motif (ARM; a.a. 49–60) and its N-terminal activation domain (a.a. 1–48), respectively. CycT1, in turn, is thought to bind to the central loop (+30 to +35) of TAR through its Tat-TAR recognition motif (TRM; a.a. 251–271) in the presence of Tat [1, 2]. Human CycT1 is comprised of 726 amino acids and contains a cyclin box repeat domain (from positions 31 to 250), a coiled-coil sequence (from positions 379 to 530), and a PEST sequence (from positions 709 to 726). The N-terminal cyclin boxes are important for binding and activation of Cdk9. Residues from positions 251 to 272 are essential for the zinc ion-mediated binding between Tat and TAR . This region also interacts with the HEXIM1 protein and 7SK small nuclear RNA, which negatively regulate the kinase activity of P-TEFb [11–15]. The C-terminal region (a.a. 273–726) of CycT1 is dispensable for Tat transactivation since the N-terminal cyclin repeats (a.a. 1–250) and TRM (a.a. 251–272) of CycT1 interact with Cdk9, Tat and TAR [3–5, 9, 16, 17]. Recently, we have determined the crystal structure of the N-terminal region (a.a. 1–280) of human CycT1  and its interacting dimeric Cyclin T-binding domain in HEXIM1 .
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 . A number of small compounds that inhibit Cdk9 activities or disrupt the Tat/TAR/P-TEFb interaction have also been tested [20–28]. Another approach by Napolitano et al. aimed to inactivate Cdk9 by an oligomerization chain reaction . Additionally, our group has constructed chimeric proteins containing wild type (wt) CycT1 and mutant Cdk9 which inhibited HIV replication up to 90% . Moreover, several CycT1-binding proteins and their truncation mutants have been used as inhibitors of Tat transactivation [31–33]. Finally, Bai et al. demonstrated that intrabodies against CycT1 inhibited Tat stimulated transactivation . It is important to note, however, that because P-TEFb is involved in the transcription of many cellular genes , 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 co-expressed 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
Overview of CycT1 mutants used in this study.
Amino acid positions
# of clones
Activity in murine cells (*1)
An N-Terminal CycT1 mutant exhibited the strongest dominant negative effect on Tat transactivation by promoting the degradation of Tat proteins
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 (Figure. 3B, lanes 2 and 3 compared with lane 1), and much more efficiently restored in the presence of Epoxomicin (Figure. 3B, lanes 5 to 7 compared with lane 4). In contrast, the expression of wt CycT1 (1–280) and Tat remained virtually unchanged in the presence of these inhibitors (lanes 9 and 10 compared with lane 8).
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 , 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 . It is to be noted that wt CycT1 (1–280) is resistant to degradation (Figure. 3). Although we have not identified the 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.
Constructing CycT1 mutants based on C-terminal truncated forms of wt CycT1 (CycT1(1–280)) is particularly beneficial in terms of HIV transcription. CycT1 (1–280) has been demonstrated to be sufficient for supporting Tat-transactivation [4, 5, 9]. In addition, Tat competes with HEXIM1 to increase active P-TEFb complexes [14, 15, 19]. CycT1 (1–280) can therefore bypass the 7SK/HEXIM-mediated complex regulatory pathway and be exclusively directed towards Tat-dependent transactivation (Jadlowsky et al., unpublished data), making CycT1 (1–280) proteins highly specific for Tat.
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-of-function" 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 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.
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.
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% CO2. 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  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 . 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 . 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, ), 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 .
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).
Proteasome inhibitor treatment
293T cells (2 × 105) were transfected with 1 μg of plasmids encoding HA-tagged wt CycT1 (1–280) or CycT1-U7 in the presence or absence of the plasmid encoding HA-tagged HIV-1 Tat (0.2 μg) using calcium phosphate. Twenty-four hours post-transfection, cells were treated with MG-132 (50 μM), Epoxomicin (50 μM) or DMSO (solvent control) at 37°C for 1, 3 and 5 hours. Cells were then harvested and lysed in RIPA buffer. The protein concentrations in the cell lysates were determined by Protein Assay Kits (BioRad, Palo Alto, CA). The same amounts of cellular proteins (20 μg) were separated by SDS-PAGE followed by Western blot analysis to detect the HA-epitope tagged CycT1 proteins and myc-epitope tagged Tat proteins.
Immunofluorescence (IF) assay
HA-tagged wt and mutant CycT1 and myc-tagged Tat proteins were co-expressed in HeLa/HR-Luc cells using Lipofectin (Invitrogen). Twenty-four hours after transfection, cells were untreated or treated with 25 μM Epoxomicin for 3 hours. Cells were fixed with 4% formaldehyde and blocked with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1% sodium azide and 10% normal donkey serum. After washing with PBS, HA-CycT1 and were probed with mouse anti-HA monoclonal antibody (Santa Cruz) and Cy2-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). Myc-Tat proteins were probed with anti-Myc monoclonal antibody (Santa Cruz) pre-labeled with Zennon ® Texas Red ® anti-Mouse IgG1 (Invitrogen). Nuclei were stained with Hoechst (Sigma). Fixed cell images were captured on a Deltavision DV-RT (Applied Precision, Inc. Issaquah, WA.) microscopy system using the Deltavision Softworx program.
We thank Keith Olszens, Lindsey McGowen, Adam Heath, Satsumi Roos, Laura Paszkowsky, Michael Zane, Renee Devor, and Yehong Huang for technical assistance, Drs. Mudit Tyagi and Monica Montano for reagents, Antonia Fraser Fujinaga for proofreading the manuscripts, and Drs. Jonathan Karn, Matija Peterlin, Eric Arts, Catherine Patterson, Erik Andrulis, David McDonald, Ran Taube and members of Fujinaga lab for helpful discussions.
This work was supported by NIH R21 AI62516 (KF) and Cell and Molecular Biology training grant (5TG32 GM-08056-24) (JKJ) from NIH, American Foundation of AIDS Research (AmFAR) 106386-33-RGGN (KF), the Deutsche Forschungsgemeinschaft (GE 976/5, MG), Japan Human Science Program (TO), and the Center for AIDS Research (CFAR) at Case Western Reserve University.
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