Arginine rich short linear motif of HIV-1 regulatory proteins inhibits dicer dependent RNA interference.
ABSTRACT: BACKGROUND: Arginine Rich Motif (ARM) of HIV-1 Tat and Rev are extensively studied linear motifs (LMs). They are already established as an inefficient bipartite nuclear localisation signal (NLS). The unusual passive diffusion of HIV-1 NLS tagged reporter proteins across the nucleus is due to an unknown competing functionality of ARM. Recent findings about the role of retroviral proteins as a suppressor of RNA interference (RNAi) involving their basic residues hint an interesting answer to this alternate functionality. The present work explores the role of HIV-1 ARM as a uniquely evolved viral motif to combat Dicer dependent RNAi. RESULTS: We show that RNA binding ARM of both HIV-1 Tat and Rev is a LM with a pattern RXXRRXRRR unique to viruses. Extending the in silico results to wet lab, we proved both HIV-1 Tat and Rev can suppress Dicer dependent RNA silencing process involving ARM. We show, HIV-1 Tat and Rev and their corresponding ARM can bind the RISC loading complex (RLC) components TRBP and PACT confirming ARM as an independent RNAi suppression motif. Enhancement of RNAi in infection scenario through enoxacin increases HIV-1 replication as indicated by p24 levels. Except Dicer, all other cytoplasmic RNAi components enhance HIV-1 replication, indicating crucial role of Dicer independent (Ago2 dependent) RNAi pathway in HIV-1 infection. Sequence and structural analysis of endo/exo-microRNA precursors known to be regulated in HIV-1 infection highlights differential features of microRNA biogenesis. One such set of miRNA is viral TAR encoded HIV-1-miR-TAR-5p (Tar1) and HIV-1-miR-TAR-3p (Tar2) that are known to be present throughout the HIV-1 life cycle. Our qPCR results showed that enoxacin increases Tar2 miRNA level which is interesting as Tar2 precursor shows Ago2 dependent processing features. CONCLUSIONS: We establish HIV-1 ARM as a novel viral motif evolved to target the Dicer dependent RNAi pathway. The conservation of such motif in other viral proteins possibly explains the potent suppression of Dicer dependent RNAi. Our model argues that HIV-1 suppress the processing of siRNAs through inhibition of Dicer while at the same time manipulates the RNAi machinery to process miRNA involved in HIV-1 replication from Dicer independent pathways.
Project description:The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.
Project description:HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.
Project description:Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.
Project description:We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.
Project description:The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.
Project description:Superelongation complexes (SECs) are essential for transcription elongation of many human genes, including the integrated HIV-1 genome. At the HIV-1 promoter, the viral Tat protein binds simultaneously to the nascent TAR RNA and the CycT1 subunit of the P-TEFb kinase in a SEC. To understand the preferential recruitment of SECs by Tat and TAR, we determined the crystal structure of a quaternary complex containing Tat, P-TEFb, and the SEC scaffold, AFF4. Tat and AFF4 fold on the surface of CycT1 and interact directly. Interface mutations in the AFF4 homolog AFF1 reduced Tat-AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 binding in the presence of Tat partially orders the CycT1 Tat-TAR recognition motif and increases the affinity of Tat-P-TEFb for TAR 30-fold. These studies indicate that AFF4 acts as a two-step filter to increase the selectivity of Tat and TAR for SECs over P-TEFb alone.DOI: http://dx.doi.org/10.7554/eLife.02375.001.
Project description:HIV-1 Tat transactivates viral genes through strong interaction with TAR RNA. The stem-loop bulged region of TAR consisting of three nucleotides at the position 23-25 and the loop region consisting of six nucleotides at the position 30-35 are essential for viral transactivation. The arginine motif of Tat (five arginine residues on subtype TatC) is critically important for TAR interaction. Any mutations in this motif could lead to reduce transactivation ability and pathogenesis. Here, we identified structurally important residues (arginine and lysine residues) of Tat in this motif could bind to TAR via hydrogen bond interactions which is critical for transactivation. Natural mutant Ser46Phe in the core motif could likely led to conformational change resulting in more hydrogen bond interactions than the wild type Tat making it highly potent transactivator. Importantly, we report the possible probabilities of number of hydrogen bond interactions in the wild type Tat and the mutants with TAR complexes. This study revealed the differential transactivation of subtype B and C Tat could likely be due to the varying number of hydrogen bonds with TAR. Our data support that the N-terminal and the C-terminal domains of Tat is involved in the TAR interactions through hydrogen bonds which is important for transactivation. This study highlights the evolving pattern of structurally important determinants of Tat in the arginine motif for viral transactivation.
Project description:Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.
Project description:HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat-CycT1-TAR complexes by enhancing an interaction with the CycT1 Tat-TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA-protein complex, thereby tuning the levels of transcription elongation.
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.