Latent HIV-1 TAR Regulates 7SK-responsive P-TEFb Target Genes and Targets Cellular Immune Responses in the Absence of Tat.
ABSTRACT: The transactivating response element (TAR) structure of the nascent HIV-1 transcript is critically involved in the recruitment of inactive positive transcription elongation factor b (P-TEFb) to the promoter proximal paused RNA polymerase II. The viral transactivator Tat is responsible for subsequent P-TEFb activation in order to start efficient viral transcription elongation. In the absence of the viral transactivator of transcription (Tat), e.g., during latency or in early stages of HIV transcription, TAR mediates an interaction of P-TEFb with its inhibitor hexamethylene bis-acetamide-inducible protein 1 (HEXIM1), keeping P-TEFb in its inactive form. In this study, we address the function of HIV-1 TAR in the absence of Tat by analyzing consequences of HIV-1 TAR overexpression on host cellular gene expression. An RNA chimera consisting of Epstein-Barr virus-expressed RNA 2 (EBER2) and HIV-1 TAR was developed to assure robust overexpression of TAR in HEK293 cells. The overexpression results in differential expression of more than 800 human genes. A significant proportion of these genes is involved in the suppression of cellular immune responses, including a significant set of 7SK-responsive P-TEFb target genes. Our findings identify a novel role for HIV-1 TAR in the absence of Tat, involving the interference with host cellular immune responses by targeting 7SK RNA-mediated gene expression and P-TEFb inactivation.
Project description:The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.
Project description:Transcription of HIV provirus is a key step of the viral cycle, and depends on the recruitment of the cellular positive transcription elongation factor b (P-TEFb) to the HIV promoter. The viral transactivator Tat can displace P-TEFb from the 7SK small nuclear ribonucleoprotein, where it is bound and inactivated by HEXIM1, and bring it to TAR, which allows the stalled RNA polymerase II to transition to successful transcription elongation. In this study, we designed a chimeric inhibitor of HIV transcription by combining functional domains from HEXIM1 and Tat. The chimera (HT1) potently inhibited gene expression from the HIV promoter, by competing with Tat for TAR and P-TEFb binding, while keeping the latter inactive. HT1 inhibited spreading infection as well as viral reactivation in lymphocyte T cell line models of HIV latency, with little effect on cellular transcription and metabolism. This proof-of-concept study validates an innovative approach to interfering with HIV transcription via peptide mimicry and competition for RNA-protein interactions. HT1 represents a new candidate for HIV therapy, or HIV cure via the proposed block and lock strategy.
Project description:Efficient transcription of the HIV-1 genome is regulated by Tat, which recruits P-TEFb from the 7SK small nuclear ribonucleoprotein (snRNP) and other nucleoplasmic complexes to phosphorylate RNA polymerase II and other factors associated with the transcription complex. Although Tat activity is dependent on its binding to the viral TAR sequence, little is known about the cellular factors that might also assemble onto this region of the viral transcript. Here, we report that the splicing factor SRSF1 (SF2/ASF) and Tat recognize overlapping sequences within TAR and the 7SK RNA. SRSF1 expression can inhibit Tat transactivation by directly competing for its binding to TAR. Additionally, we provide evidence that SRSF1 can increase the basal level of viral transcription in the absence of Tat. We propose that SRSF1 activates transcription in the early stages of viral infection by recruiting P-TEFb to TAR from the 7SK snRNP. Whereas in the later stages, Tat substitutes for SRSF1 by promoting release of the stalled polymerase and more efficient transcriptional elongation.
Project description:Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.
Project description:The transition from transcription initiation to elongation at the HIV-1 promoter is controlled by Tat, which recruits P-TEFb to TAR RNA to phosphorylate RNA polymerase II. It has long been unclear why the HIV-1 promoter is incompetent for elongation. We report that P-TEFb is recruited to the promoter in a catalytically inactive state bound to the inhibitory 7SK small nuclear ribonucleoprotein (snRNP), thereby preventing elongation. It also has long been believed that TAR functions to recruit Tat to the promoter, but we find that Tat is recruited to the DNA template before TAR is synthesized. We propose that TAR binds Tat and P-TEFb as it emerges on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase. Recruitment of an inhibitory snRNP complex at an early stage in the transcription cycle provides a new paradigm for controlling gene expression with a noncoding RNA.
Project description:The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5'-terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.
Project description:The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.
Project description:Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.
Project description:HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Project description:BACKGROUND:HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein-protein interactions and RNA and ATP binding. RESULTS:We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. CONCLUSION:DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.