Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by interactions between both viral and host factors. These interactions are also responsible for changes in the expression of many host cell genes, including cytokines and other immune regulators, which may account for the state of immunological dysregulation that characterizes HIV-1 infection. We have investigated the role of a host cell protein, the transcription factor NFAT1, in HIV-1 pathogenesis. We show that NFAT1 interacts with Tat and that this interaction, which involves the major transactivation domain of NFAT1 and the amino-terminal region of Tat, results in a reciprocal modulatory interplay between the proteins: whereas Tat enhances NFAT1-driven transcription in Jurkat T cells, NFAT1 represses Tat-mediated transactivation of the HIV-1 long terminal repeat (LTR). Moreover, NFAT1 binds to the κB sites on the viral LTR and negatively regulates NF-κB-mediated activation of HIV-1 transcription, by competing with NF-κB1 for its binding sites on the HIV-1 LTR. Tat-mediated enhancement of NFAT1 transactivation may explain the upregulation of interleukin 2 and other cytokines that occurs during HIV-1 infection. We discuss the potentially opposing roles of NFAT1 and another family member, NFAT2, in regulating gene transcription of HIV-1 and endogenous cytokine genes.
Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory protein that is critical for viral gene expression and replication, transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation response element (TAR) and, along with other cellular factors, increases viral transcription initiation and elongation. Tat also superactivates the HIV-1 promoter through a TAR-independent mechanism, including tumor necrosis factor alpha-induced and protein kinase C (PKC)-dependent activation of NF-kappa B, and inhibitors of Tat and NF-kappa B cooperatively down-regulate this Tat-mediated LTR superactivation. In this study, a combined pharmacologic and genetic strategy using two PKC (NF-kappa B) inhibitors, pentoxifylline (PTX) and Gö-6976, and a stably expressed anti-Tat single-chain intracellular antibody (sFv intrabody) was employed to obtain cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment of cells with PTX and Gö-6976 resulted in cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. In addition, the combined use of anti-Tat sFv intrabodies and the two NF-kappa B inhibitors retained the virus in the latent state for as long as 45 days. The combined treatment resulted in more durable inhibition of HIV-1 replication than was seen with the NF-kappa B inhibitors alone or the anti-Tat sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival of transduced cells and prolong clinical benefit.
The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1α. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1α associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1α complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis.
Human immunodeficiency virus type 1 (HIV-1) encodes the transactivator protein Tat, which is essential for viral replication and progression to disease. Here we demonstrate that transcriptional activation by HIV-1 Tat involves p300 or the related cellular transcriptional coactivator CREB binding protein (CBP). Tat transactivation was inhibited by the 12S form of the adenovirus E1A gene product, which inhibits p300 function, and this inhibition was independent of its effect on NF-κB transcription. A biochemical interaction of p300 with Tat was demonstrated in vitro and in vivo by coimmunoprecipitation. The carboxy-terminal region of p300, which binds to E1A, was shown to bind specifically to the highly conserved basic domain of Tat, which also mediates binding to the Tat-responsive region RNA stem-loop structure. The ability of Tat to interact physically and functionally with this coactivator provides a mechanism to assemble a basal transcription complex which may subsequently respond to the effect of Tat on transcriptional elongation and represents a novel interaction between an RNA binding protein and a transcriptional coactivator.
Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.
Arginine methylation has been shown to regulate signal transduction, protein subcellular localization, gene transcription, and protein-protein interactions that ultimately alter gene expression. Although the role of cellular protein arginine methyltransferases (PRMT) in viral gene expression is largely unknown, we recently showed that the Tat protein of human immunodeficiency virus type 1 (HIV-1) is a substrate for one such enzyme, termed PRMT6. However, the mechanism by which arginine methylation impairs the transactivation potential of Tat and the sites of arginine methylation within Tat remain obscure. We now show that Tat is a specific in vitro and in vivo substrate of PRMT6 which targets the Tat R52 and R53 residues for arginine methylation. Such Tat methylation led to decreased interaction with the Tat transactivation region (TAR) of viral RNA. Furthermore, arginine methylation of Tat negatively affected Tat-TAR-cyclin T1 ternary complex formation and diminished cyclin T1-dependent Tat transcriptional activation. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, reduced levels of Tat transactivation of HIV-1 long terminal repeat chloramphenicol acetyltransferase and luciferase reporter plasmids in a dose-dependent manner. In cell-based assays, knockdown of PRMT6 resulted in increased HIV-1 production and faster viral replication. Thus, PRMT6 can compromise Tat transcriptional activation and may represent a form of innate cellular immunity in regard to HIV-1 replication. Finding a way of inhibiting or stimulating PRMT6 activity might help to drive quiescently infected cells out of latency or combat HIV-1 replication, respectively.
Jembrana disease virus (JDV) encodes a potent regulatory protein Tat that strongly stimulates viral expression by transactivating the long terminal repeat (LTR) promoter. JDV Tat (jTat) promotes the transcription from its own LTR as well as non-cognate LTRs, by recruiting host transcription factors and facilitating transcriptional elongation. Here, we compared the sequence requirements of jTat for transactivation of JDV, bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV) LTRs.
In this study, we identified the minimal protein sequence for LTR activation using jTat truncation mutants. We found that jTat N-terminal residues were indispensable for transactivating the HIV LTR. In contrast, transactivation of BIV and JDV LTRs depended largely on an arginine-rich motif and some flanking residues. Competitive inhibition assay and knockdown analysis showed that P-TEFb was required for jTat-mediated LTR transactivation, and a mammalian two-hybrid assay revealed the robust interaction of jTat with cyclin T1. In addition, HIV LTR transactivation was largely affected by fusion protein at the jTat N-terminus despite the fact that the cyclin T1-binding affinity was not altered. Furthermore, the jTat N-terminal sequence enabled HIV Tat to transactivate BIV and JDV LTRs, suggesting the flexibility at the jTat N-terminus.
This study showed the distinct sequence requirements of jTat for HIV, BIV and JDV LTR activation. Residues responsible for interaction with cyclin T1 and transactivation response element are the key determinants for transactivation of its cognate LTR. N-terminal residues in jTat may compensate for transactivation of the HIV LTR, based on the flexibility.
The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA.
This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion.
These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication.
The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5′ end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatin-remodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef. In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.
The human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription protein Tat is an important factor in viral pathogenesis. In addition to its function as the key trans-activator of viral transcription, Tat is also secreted by the infected cell and taken up by neighboring cells where it has an effect both on infected and uninfected cells. In this review we will focus on the relationship between the structure of the Tat protein and its function as a secreted factor. To this end we will summarize some of the exogenous functions of Tat that have been implicated in HIV-1 pathogenesis and the impact of structural variations and viral subtype variants of Tat on those functions. Finally, since in some patients the presence of Tat-specific antibodies or CTL frequencies are associated with slow or non-progression to AIDS, we will also discuss the role of Tat as a potential vaccine candidate, the advances made in this field, and the importance of using a Tat protein capable of eliciting a protective or therapeutic immune response to viral challenge.
The major group of human immunodeficiency virus type 1 (HIV-1) strains that comprise the current global pandemic have diversified during their worldwide spread into at least 10 distinct subtypes, or clades. Subtype C predominates in sub-Saharan Africa and is responsible for the majority of worldwide HIV-1 infections, subtype B predominates in North America and Europe, and subtype E is prevalent in Southeast Asia. Significant amino acid variations have been observed among the clade-specific Tat proteins. For the present study, we examined clade-specific interactions between Tat, transactivation-responsive (TAR) element, and P-TEFb proteins and how these interactions may modulate the efficiency of HIV-1 transcription. Clade-specific Tat proteins significantly modified viral gene expression. Tat proteins derived from HIV-1 clades C and E were strong transactivators of long terminal repeat (LTR) activity; Tat E also had a longer half-life than the other Tat proteins and interacted more efficiently with the stem-loop TAR element. Chimeric Tat proteins harboring the Tat E activation domain were strong transactivators of LTR expression. While Tat B, C, and E were able to rescue a Tat-defective HIV-1 proviral clone, Tat E was significantly more efficient at rescue than Tat C, possibly due to the relative stability of the Tat protein. Swapping the activation domains of Tat B, C, and E identified the cyclin T1 association domain as a critical determinant of the transactivation efficiency and of Tat-defective HIV-1 provirus rescue.
The human immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV.
Human immunodeficiency viruses (HIVs) and the related bovine lentiviruses bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) utilize the viral Tat protein to activate viral transcription. The arginine-rich RNA-binding domains of the Tat proteins bind to their cognate transactivation response element (TAR) RNA hairpins located at the 5′ ends of the viral mRNAs, resulting in enhanced processivity of RNA polymerase II. It has previously been shown that HIV type 1 (HIV-1) Tat requires the cellular cyclin T1 protein for high-affinity RNA binding whereas BIV Tat and JDV Tat bind with high affinity on their own and adopt distinct β-hairpin conformations when complexed to RNA. Here we have engineered the BIV and JDV Tat-TAR interactions into HIV-1 and show that the heterologous interactions support viral replication, correlating well with their RNA-binding affinities. Viruses engineered with a variant TAR able to bind all three Tat proteins replicate efficiently with any of the proteins. In one virus containing a noncognate Tat-TAR pair that neither interacts nor efficiently replicates (HIV-1 TAR and BIV Tat), viral revertants were isolated in which TAR had become mutated to generate a functional BIV Tat binding site. Our results support the view that incremental changes to TAR structure can provide routes for evolving new Tat-TAR complexes while maintaining active viral replication.
Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).
The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.
HIV-1 gene expression requires both viral and cellular factors to control and coordinate transcription. While the viral factor Tat is known for its transcriptional transactivator properties, we present evidence for an unexpected function of Tat in viral splicing regulation. We used a series of HIV-1 reporter minigenes to demonstrate that Tat’s role in splicing is dependent on the cellular co-transcriptional splicing activators Tat-SF1 and CA150. Surprisingly, we show that this Tat-mediated splicing function is independent from transcriptional activation. In the context of the full-length viral genome, this mechanism promotes an autoregulatory feedback that decreases expression of tat and favors expression of the env-specific mRNA. Our data demonstrate that Tat-mediated regulation of transcription and splicing can be uncoupled and suggest a mechanism for the involvement of specific transcriptional activators in splicing.
Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.
Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.
Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.
The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.
Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.
Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.
The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites for the NF-kappa B/Rel family of transcription factors which are required for the transcriptional activation of viral genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1. In the present study, we examined the effect of transdominant mutants of I kappa B alpha on the synergistic activation of the HIV-1 LTR by TNF-alpha and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I kappa B alpha inhibited Tat-TNF-alpha activation of HIV LTR in a dose-dependent manner. Transdominant forms of I kappa B alpha, mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or constitutive (S283A, T291A, T299A) phosphorylation of I kappa B alpha were tested for their capacity to block HIV-1 LTR transactivation. I kappa B alpha molecules mutated in the N-terminal sites were not degraded following inducer-mediated stimulation (t1/2, > 4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I kappa B alpha (S32A, S36A) transdominant mutant was at least five times as effective as wild-type I kappa B alpha in inhibiting synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1 gene expression by interfering with the NF-kappa B/Rel signaling pathway.
For productive replication of human immunodeficiency virus type 1 (HIV-1) in host cells, the viral genome-encoded transactivator Tat and several cellular transcription factors are required for efficient viral gene transcription. However, it remains unclear how the viral genome initiates transcription before Tat is transcribed or when Tat is at suboptimal levels. Here, we utilized the human T-cell leukemia type 1 Tax protein as a molecular tool to investigate the mechanism of viral gene transcription that initiates the early phase of infection of HIV-1. Tax alone does not significantly increase the activity of HIV-1 long terminal repeat (LTR) in T lymphocytes, but it markedly enhanced the replication of an infectious HIV-1 provirus with a truncated nef gene. This enhancement is preferentially mediated by the cooperation of Tax and Tat which is dependent on TAR and duplicated κB cis elements of the HIV-1 LTR as well as the NF-κB activation domain of Tax. Furthermore, phorbol myristate acetate and membrane-targeted HIV-1 Nef also enhanced the LTR activity in the presence of Tat in the TAR- and κB cis element-dependent manner. These data suggest that activated NF-κB can functionally interact with a suboptimal amount of Tat and the HIV-1 LTR for efficient initiation of viral gene transcription.
Arginine methylation of human immunodeficiency virus type 1 (HIV-1) Tat protein downregulates its key function in viral-gene transactivation. The fate of methylated Tat is unknown, so it is unclear whether methylated Tat is degraded or persists in the cell for additional functions. Here we show that the arginine methyltransferase PRMT6 increases Tat protein half-life by 4.7-fold. Tat stabilization depends on the catalytic activity of PRMT6 and requires arginine methylation within the Tat basic domain. In contrast, HIV-1 Rev, which is also methylated by PRMT6, is completely refractory to the stabilizing effect. Proteasome inhibition and silencing experiments demonstrated that Tat can be degraded by a REGγ-independent proteasome, against which PRMT6 appears to act to increase Tat half-life. Our data reveal a proteasome-dependent Tat degradation pathway that is inhibited by arginine methylation. The stabilizing action of PRMT6 could allow Tat to persist within the cell and the extracellular environment and thereby enable functions implicated in AIDS-related cancer, neurodegeneration, and T-cell death.
The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3′-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.
The transcriptional activity of the integrated HIV provirus is dependent on the chromatin organization of the viral promoter and the transactivator Tat. Tat recruits the cellular pTEFb complex and interacts with several chromatin-modifying enzymes, including the histone acetyltransferases p300 and PCAF. Here, we examined the interaction of Tat with activation-dependent histone kinases, including the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 and treatment with a small-molecule inhibitor of RSK2 kinase activity inhibited the transcriptional activity of Tat, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from subjects with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. Tat interacted with RSK2 and activated RSK2 kinase activity in cells. Both properties were lost in a mutant Tat protein (F38A) that is deficient in HIV transactivation. Our data identify a novel reciprocal regulation of Tat and RSK2 function, which might serve to induce early changes in the chromatin organization of the HIV LTR.