Tat Cofactor Research Articles

The lysine methyltransferase SMYD5 amplifies HIV-1 transcription and is post-transcriptionally upregulated by Tat and USP11

A successful HIV-1 cure strategy may require enhancing HIV-1 latency to silence HIV-1 transcription. Modulators of gene expression show promise as latency-promoting agents invitro and invivo. Here, we identify Su(var)3-9, enhancer-of-zeste, and trithorax (SET) and myeloid, Nervy, and DEAF-1 (MYND) domain-containing protein 5 (SMYD5) as a host factor required for HIV-1 transcription. SMYD5 is expressed in CD4+ Tcells and activates the HIV-1 promoter with or without the viral Tat protein, while knockdown of SMYD5 decreases HIV-1 transcription in cell lines and primary Tcells. SMYD5 associates invivo with the HIV-1 promoter and binds the HIV trans-activation response (TAR) element RNA and Tat. Tat is methylated by SMYD5 invitro, and in cells expressing Tat, SMYD5 protein levels are increased. The latter requires expression of the Tat cofactor and ubiquitin-specific peptidase 11 (USP11). We propose that SMYD5 is a host activator of HIV-1 transcription stabilized by Tat and USP11 and, together with USP11, a possible target for latency-promoting therapy.

PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription

Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire.

Control of HIV latency by epigenetic and non-epigenetic mechanisms.

Intensive antiretroviral therapy successfully suppresses viral replication but is unable to eradicate the virus. HIV persists in a small number of resting memory T cells where HIV has been transcriptionally silenced. This review will focus on recent insights into the HIV transcriptional control mechanisms that provide the biochemical basis for understanding latency. There are no specific repressors of HIV transcription encoded by the virus, instead latency arises when the regulatory feedback mechanism driven by HIV Tat expression is disrupted. Small changes in transcriptional initiation, induced by epigenetic silencing, lead to profound restrictions in Tat levels and force the entry of proviruses into latency. In resting memory T cells, which carry the bulk of the latent viral pool, additional restrictions, especially the limiting cellular levels of the essential Tat cofactor P-TEFb and the transcription initiation factors NF-κB and NFAT ensure that the provirus remains silenced unless the host cell is activated. The detailed understanding of HIV transcription is providing a framework for devising new therapeutic strategies designed to purge the latent viral pool. Importantly, the recognition that there are multiple restrictions imposed on latent proviruses suggest that proviral reactivation will not be achieved when only a single reactivation step is targeted and that any optimal activation strategy will require both removal of epigenetic blocks and the activation of P-TEFb.

The molecular biology of HIV latency: breaking and restoring the Tat-dependent transcriptional circuit

Despite the remarkable success of intensive antiretroviral drug therapy in blocking the HIV replication, the virus persists in a small number of cells in which HIV has been transcriptionally silenced. This review will focus on recent insights into the HIV transcriptional control mechanisms that provide the biochemical basis for understanding latency. Latency arises when the regulatory feedback mechanism driven by HIV Tat expression is disrupted. Small changes in transcriptional initiation, induced by epigenetic silencing, can lead to restrictions in Tat levels and entry of proviruses into latency. In resting memory T-cells, which carry the bulk of the latent viral pool, additional restrictions limiting cellular levels of the essential Tat cofactor P-TEFb and the transcription initiation factors nuclear factor kappa B and nuclear factor of activated T cells ensure that the provirus remains silenced unless the host cell is activated. Strategies to purge the latent proviral pool require nontoxic activator molecules. The multiple restrictions imposed on latent proviruses that need to be overcome suggest that proviral reactivation will not be achieved when only a single reactivation step is targeted but will require both removal of epigenetic blocks and the activation of P-TEFb. Alternatively, new inhibitors that block proviral reactivation could be developed.

Progranulin (granulin/epithelin precursor) and its constituent granulin repeats repress transcription from cellular promoters

Progranulin (also known as granulin/epithelin precursor, GEP) is composed of seven granulin/epithelin repeats (granulins) and functions both as a full-length protein and as individual granulins. It is a secretory protein but a substantial amount of GEP is found inside cells, some in complexes with positive transcription elongation factor b (P-TEFb). GEP and certain granulins interact with the cyclin T1 subunit of P-TEFb, and with its HIV-1 Tat co-factor, leading to repression of transcription from the HIV promoter. We show that GEP lacking the signal peptide (GEPspm) remains inside cells and, like wild-type GEP, interacts with cyclin T1 and Tat. GEPspm represses transcription from the HIV-1 promoter at the RNA level. Granulins that bind cyclin T1 are phosphorylated by P-TEFb in vivo and in vitro on serine residues. GEPspm and those granulins that interact with cyclin T1 also inhibit transcription from cellular cad and c-myc promoters, which are highly dependent on P-TEFb, but not from the PCNA promoter. In addition, GEPspm and granulins repress transcriptional activation by VP16 or c-Myc, proteins that bind and recruit P-TEFb to responsive promoters. These data suggest that intracellular GEP is a promoter-specific transcriptional repressor that modulates the function of cellular and viral transcription factors.

Involvement of the p53 and p73 transcription factors in neuroAIDS

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.

Potent and Selective Inhibition of Tat-Dependent HIV-1 Replication in Chronically Infected Cells by a Novel Naphthalene Derivative JTK-101

In search for effective human immunodeficiency virus type 1 (HIV-1) transcription inhibitors, we have evaluated more than 100,000 compounds for their inhibitory effects on HIV-1 long terminal repeat (LTR)-driven reporter gene expression, and identified a novel naphthalene derivative, JTK-101. This compound could suppress tumour necrosis factor (TNF)-alpha-induced HIV-1 production in latently infected OM-10.1 cells at nanomolar concentrations. JTK-101 could also potently inhibit constitutive HIV-1 production in MOTL-4/IIIB. However, the antiviral activity of JTK-101 was found to be much weaker in acutely infected cells and the chronically infected cells U937/IIIB cells than in OM-10.1 and MOLT-4/IIIB cells. JTK-101 selectively suppressed TNF-alpha-induced HIV-1 mRNA synthesis in OM-10.1 cells in a dose-dependent fashion. JTK-101 modestly inhibited TNF-alpha-induced HIV-1 LTR-driven reporter gene expression, but potently inhibited Tat-induced gene expression. Immunoblot analysis revealed that low-level expression of the Tat cofactors CDK9 and cyclin T1 might contribute to the diminished antiviral activity in U937/IIIB cells. Furthermore, JTK-101 could not inhibit HIV-1 replication in chronically infected monocytes/macrophages, in which CDK9 and cyclin T1 were undetectable. These results suggest that JTK-101 exerts its anti-HIV-1 activity through the inhibition of known or unknown Tat cofactors, presumably CDK9/cyclin T1.

Suppression of MicroRNA-Silencing Pathway by HIV-1 During Virus Replication

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.

Induction of the HIV-1 Tat co-factor cyclin T1 during monocyte differentiation is required for the regulated expression of a large portion of cellular mRNAs

BackgroundP-TEFb, a general RNA polymerase II elongation factor, is composed of CDK9 (cyclin-dependent kinase 9) as a catalytic unit and either cyclin T1, T2 or K as a regulatory subunit. The cyclin T1/P-TEFb complex is targeted by HIV to mediate Tat transactivation. Cyclin T1 protein expression is induced during early macrophage differentiation, suggesting a role in regulation of mRNA expression during the differentiation process. To study the functional significance of cyclin T1 induction during differentiation, we utilized the human Mono Mac 6 (MM6) monocytic cell line.ResultsWe found that cyclin T1 protein expression is induced by a post-transcriptional mechanism following PMA treatment of MM6 cells, similar to its induction in primary monocytes and macrophages. Also in agreement with findings in primary cells, cyclin T2a is present at relatively high levels in MM6 cells and is not induced by PMA. Although the knock-down of cyclin T1 in MM6 cells by shRNA inhibited HIV-1 Tat transactivation, MM6 cell growth was not affected by the depletion of cyclin T1. Using DNA microarray technology, we found that more than 20% of genes induced by PMA require cyclin T1 for their normal level of induction, and approximately 15% of genes repressed by PMA require cyclin T1 for their normal level of repression. Gene ontology analysis indicates that many of these cyclin T1-dependent genes are related to immune response and signal transduction.ConclusionThese results suggest that cyclin T1 serves a critical role in the program of macrophage differentiation, and this raises questions about the feasibility of cyclin T1 serving as an antiviral therapeutic target.

Recent status of HIV-1 gene expression inhibitors

Human immunodeficiency virus type 1 (HIV-1) gene expression and transcription is a crucial step in the viral replication cycle, which is considered to be a potential target for inhibition of HIV-1. Among the factors involved in this step, the cellular protein nuclear factor (NF)-κB is the most powerful inducer of HIV-1 gene expression. On the other hand, the viral protein Tat plays a central role in sustaining a high level of HIV-1 replication. Several compounds have been reported to selectively inhibit the functions of Tat and NF-κB. Tat inhibitors target either the Tat/TAR RNA interaction or the Tat cofactor cyclin-dependent kinase 9/cyclin T1. Antioxidants, protein kinase C inhibitors, and IκB kinase inhibitors are known to suppress the activation of NF-κB. Although some of the compounds inhibit HIV-1 replication in cell cultures at low concentrations, they also have considerable toxicity to the host cells. Considering the increase of treatment failure cases in highly active antiretroviral therapy due to the emergence of multidrug resistance, HIV-1 gene expression inhibitors should be extensively studied as alternative approach to effective anti-HIV-1 chemotherapy.

Soluble HIV-1 gp120 enhances HIV-1 replication in non-dividing CD4+ T cells, mediated via cell signaling and Tat cofactor overexpression

The soluble HIV-1 gp120 envelope glycoprotein, after being shed from infected cells, can cross-link its receptors on both HIV-1 infected and non-infected target cells, leading to their activation. We have assessed the impact of soluble gp120 on viral replication in CD4+/CXCR4+ T cells, via its effects on Tat-mediated transactivation of the HIV-1/LTR. Primary cord blood-derived CD4+/CXCR4+ T cells were stimulated with soluble recombinant gp120 (rgp120) from the HIV-1/HXB2 clone. The level of gene or protein expression was assessed by serial analysis gene expression (SAGE), reverse transcriptase-polymerase chain reaction, western blotting or flow-cytometry analysis. Cellular division of rgp120-stimulated T cells was assessed by CFDA-SE labeling. Long terminal repeat (LTR) activity and HIV infection level were respectively measured by a chemiluminescent beta-gal Reporter Gene Assay and by p24 determination. We have demonstrated that rgp120 activates both PKCepsilon and its upstream effector PI3K/Akt, involved in the HIV-1 replication process. Moreover, rgp120 enhances the gene, as well as protein expression of the cellular Tat cofactors Tat-Sf1 and SPT5 in primary CD4+/CXCR4+ T cells. Finally, stimulation of HIV-1 infected T cells with rgp120 was found to result in both a higher LTR-activity and an increased production of viral particles. Taken together, these results show that soluble gp120 contributes to HIV-1 replication and dissemination, via the activation of multiple cell signaling pathways and the induction of Tat-cofactor expression, underscoring its potential as a therapeutic target in HIV-1-mediated pathogenesis.

Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication.

Sequence analysis of bacterial redox enzyme maturation proteins (REMPs).

The twin-arginine protein transport (Tat) system is a remarkable molecular machine dedicated to the translocation of fully folded proteins across energy-transducing membranes. Complex cofactor-containing Tat substrates acquire their cofactors prior to export, and substrate proteins actually require to be folded before transport can proceed. Thus, it is very likely that mechanisms exist to prevent wasteful export of immature Tat substrates or to curb competition between immature and mature substrates for the transporter. Here we assess the primary sequence relationships between the accessory proteins implicated in this process during assembly of key respiratory enzymes in the model prokaryote Escherichia coli. For each respiratory enzyme studied, a redox enzyme maturation protein (REMP) was assigned. The main finding from this review was the hitherto unexpected link between the Tat-linked REMP DmsD and the nitrate reductase biosynthetic protein NarJ. The evolutionary link between Tat transport and cofactor insertion processes is discussed.

Increased association of 7SK snRNA with Tat cofactor P-TEFb following activation of peripheral blood lymphocytes.

This study was undertaken to determine whether 7SK small nuclear RNA (snRNA), which has been proposed to function as an inhibitor of Tat cofactor P-TEFb, plays a role in transcriptional latency in T cells. The association of 7SK snRNA with P-TEFb was investigated in resting and activated peripheral blood lymphocytes (PBLs). Primary PBLs were isolated by standard methods and activated with phytohemagglutinin (PHA). Levels of 7SK snRNA were determined by Northern blotting and levels of the P-TEFb subunits cyclin-dependent kinase 9 and cyclin T1 were analyzed by immunoblotting. The association of 7SK snRNA with P-TEFb complexes was specific. Following activation of PBLs, the levels of 7SK snRNA increased in a manner similar to U1 and U6 snRNA, sn RNAs involved in positive aspects of cellular gene expression. Unexpectedly, the association of 7SK snRNA with P-TEFb increased dramatically following lymphocyte activation. Increased association of 7SK snRNA with P-TEFb in activated lymphocytes correlates with increased global transcription. This suggests that 7SK snRNA is unlikely to promote transcriptional latency in lymphocytes through an association with P-TEFb; it also suggests that the proposal that the association of 7SK snRNA with P-TEFb acts to inhibit transcriptional elongation needs to be re-evaluated.

Acetylation of Tat Defines a CyclinT1-Independent Step in HIV Transactivation

The HIV transcriptional activator Tat is acetylated by p300 at a single lysine residue in the TAR RNA binding domain. We have generated monoclonal and polyclonal antibodies specific for the acetylated form of Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the p300 inhibitor Lys-CoA and siRNA specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA with the same affinity as unacetylated Tat, but formation of a Tat-TAR-CyclinT1 ternary complex was completely inhibited in the presence of AcTat. We propose that Tat acetylation may help in dissociating the Tat cofactor CyclinT1 from TAR RNA and serve to transfer Tat onto the elongating RNA polymerase II.

A Human Immunodeficiency Virus Type 1 (HIV-1) Tat Cofactor Absent in Rodent Cells is a TAR-associated Factor

Background: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. Methods: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. Results: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. Conclusion: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors. (Immune Network 2002;2(3):150-157)

The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription.

The human positive transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T1 heterodimer, functions as both a general and an HIV-1 Tat-specific transcription factor. P-TEFb activates transcription by phosphorylating RNA polymerase (Pol) II, leading to the formation of processive elongation complexes. As a Tat cofactor, P-TEFb stimulates HIV-1 transcription by interacting with Tat and the transactivating responsive (TAR) RNA structure located at the 5' end of the nascent viral transcript. Here we identified 7SK, an abundant and evolutionarily conserved small nuclear RNA (snRNA) of unknown function, as a specific P-TEFb-associated factor. 7SK inhibits general and HIV-1 Tat-specific transcriptional activities of P-TEFb in vivo and in vitro by inhibiting the kinase activity of CDK9 and preventing recruitment of P-TEFb to the HIV-1 promoter. 7SK is efficiently dissociated from P-TEFb by treatment of cells with ultraviolet irradiation and actinomycin D. As these two agents have been shown to significantly enhance HIV-1 transcription and phosphorylation of Pol II (refs 6,7,8), our data provide a mechanistic explanation for their stimulatory effects. The 7SK/P-TEFb interaction may serve as a principal control point for the induction of cellular and HIV-1 viral gene expression during stress-related responses. Our studies demonstrate the involvement of an snRNA in controlling the activity of a Cdk-cyclin kinase.

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

Human GLI-2 is a tat activation response element-independent Tat cofactor.

Zinc finger-containing GLI proteins are involved in the development of Caenorhabditis elegans, Xenopus, Drosophila, zebrafish, mice, and humans. In this study, we show that an isoform of human GLI-2 strongly synergizes with the Tat transactivating proteins of human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and markedly stimulates viral replication. GLI-2 also synergizes with the previously described Tat cofactor cyclin T1 to stimulate Tat function. Surprisingly, GLI-2/Tat synergy is not dependent on either a typical GLI DNA binding site or an intact Tat activation response element but does require an intact TATA box. Thus, GLI-2/Tat synergy results from a mechanism of action which is novel both for a GLI protein and for a Tat cofactor. These findings link the GLI family of transcriptional and developmental regulatory proteins to Tat function and HIV replication.

Cell Cycle-Dependent Stimulation of the HIV-1 Promoter by Tat-Associated CAK Activator

Activation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat–TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436–7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 → G1 progression with the expression of dominant negative mutant RasAsn17 or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor.