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Ping, Yueh Hsin; Chu, Chia Ying; Cao, Hong; Jacqué, Jean Marc; Stevenson, Mario; Rana, Tariq M.

doi:10.1186/1742-4690-1-46

Cited by 43 publications

(12 citation statements)

References 64 publications

(81 reference statements)

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“…sient basal transcription from the HIV LTR (20). Both DSIF and NELF contribute to Tat-dependent activation of HIV transcription and are phosphorylated by P-TEFb (20,21). Phosphorylation alters DSIF so that it stimulates elongation.…”

Section: Human Immunodeficiency Virus (Hiv)mentioning

confidence: 99%

Negative Elongation Factor NELF Represses Human Immunodeficiency Virus Transcription by Pausing the RNA Polymerase II Complex

Zhang¹,

Klatt

Gilmour³

et al. 2007

Journal of Biological Chemistry

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Human immunodeficiency virus (HIV) transcription requires virally encoded Tat and the P-TEFb protein complex, which together associate with the Tat-activating region, a structured region in the nascent transcript. P-TEFb phosphorylates proteins in the transcription elongation complex, including RNA polymerase II (pol II), to stimulate elongation and to overcome premature termination. However, the status of the elongation complex on the HIV long terminal repeat (LTR) in a repressed state is not known. Chromatin immunoprecipitation demonstrated that NELF, a negative transcription elongation factor, was associated with the LTR. Depleting NELF increased processive HIV transcription and replication. Mapping pol II on the LTR showed that pol II was paused and that NELF depletion released pol II. Decreasing NELF also correlated with displacement of a positioned nucleosome and increased acetylation of histone H4, suggesting coupling of transcription elongation and chromatin remodeling. Previous work has indicated that the Tat-activating region plays a critical role in regulating transcription from the LTR. Our results reveal an earlier stage, mediated by NELF, when repression occurs at the HIV LTR. Human immunodeficiency virus (HIV)3 proviral expression is regulated at the transcriptional level. Virus transcription is controlled by the upstream long terminal repeat (LTR), which includes cis-elements that recruit both cellular and viral factors. The HIV-1 LTR is often divided into functional elements: the Tat-activating region (TAR), the promoter, the enhancer, and the negative/modulatory regulatory element. The promoter, enhancer, and modulatory elements recruit host transcription factors, such as Sp-1, NF-B and CCAAT/enhancer-binding protein, necessary to initiate transcription (1, 2). These factors also recruit coactivators, including histone acetyltransferases and SWI/SNF complexes, that influence the chromatin structure of integrated provirus (3-7). For example, the 5Ј-untranslated leader region located downstream of the transcriptional start site is associated with a nucleosome in latent HIV proviruses, which is displaced upon induction of virus transcription. Furthermore, agents that inhibit histone deacetylases, such as trapoxin and trichostatin A, activate HIV provirus transcription, suggesting a critical role for chromatin remodeling in the repression of HIV transcription (7,8).Transcription elongation has also been demonstrated to be a limiting step for HIV expression. HIV encodes a transcriptional activator (Tat) that binds the RNA stem-loop structure formed by TAR, and by recruiting P-TEFb to the LTR, HIV enhances processive transcription. P-TEFb, which is composed of cyclin T1 and CDK9, modifies RNA polymerase II (pol II) activity by hyperphosphorylating the C-terminal domain of pol II. In the absence of Tat, transcription elongation by RNA pol II from the HIV promoter is very inefficient (9). In vitro transcription analyses in the absence of chromatin revealed that the majority of elongation complexes init...

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Section: Human Immunodeficiency Virus (Hiv)mentioning

confidence: 99%

Negative Elongation Factor NELF Represses Human Immunodeficiency Virus Transcription by Pausing the RNA Polymerase II Complex

Zhang¹,

Klatt

Gilmour³

et al. 2007

Journal of Biological Chemistry

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“…Tat binds the RNA stem loop structure formed by TAR (transactivation-responsive region) 9; 10 and by recruiting P-TEFb to the LTR, enhances processive transcription 11 . P-TEFb, which is composed of cyclin T1 and cyclin-dependent kinase 9 (Cdk9), modifies RNA polymerase II (RNAPII) activity by hyperphosphorylating the carboxy-terminal domain (CTD) of RNAPII as well as negative elongation factors, NELF and DSIF 12; 13; 14; 15 . In the absence of Tat, transcription elongation by RNAPII from the HIV promoter is very inefficient leading to the accumulation of short transcripts 16; 17 .…”

Section: Introductionmentioning

confidence: 99%

Celastrol Inhibits Tat-Mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

Narayan

Ravindra

Chiaro

et al. 2011

Journal of Molecular Biology

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Current drugs used for anti-retroviral therapy against HIV have a narrow spectrum of activity, and more often have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, Tat, an early virus encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription, by targeting its cysteine (Cys) rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from T. wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel by covalently modifying the cysteine thiols inhibits Tat transactivation function. Using circular dichroism (CD) spectroscopy, we show that alklylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat-TAR interaction. Our results demonstrate the underlying mechanism of anti-retroviral activity of the plant-derived MAEs, and suggest that Cel could serve as a lead compound to develop novel anti-viral therapeutics.

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“…Cap synthesis entails three enzymatic reactions: (i) the 5′ triphosphate end of the pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; (ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase; and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (1). RNA guanylyltransferase is essential for cell growth in all organisms where its function has been tested, including: the fungi Saccharomyces cerevisiae (2–4), Schizosaccharomyces pombe (5) and Candida albicans (6); the nematode Caenorhabditis elegans (7,8); and cultured human cells (9). Cap guanine-N7 methyltransferase is essential for the viability of S. cerevisiae (10–13), but is reported to be nonessential in C. albicans (6).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe

Hausmann

Ramı́rez

Schneider

et al. 2007

Nucleic Acids Research

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RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential. Alanines at Pro142, Tyr148 and Pro185 reduced activity to 7–12% of wild-type. Structure–activity relationships at Phe18, Thr40, Asp76, Asn103, Asp140 and Tyr148, and at three other essential residues defined previously (Asp68, Glu91 and Trp143) were gleaned by testing the effects of 18 conservative substitutions. Our results engender a provisional map of the Tgs2 active site, which we discuss in light of crystal structures of related methyltransferases. A genetic analysis of S. pombe Tgs1 showed that it is nonessential. An S. pombe tgs1Δ strain grows normally, notwithstanding the absence of 2,2,7-trimethylguanosine caps on its U1, U2, U4 and U5 snRNAs. However, we find that S. pombe requires cap guanine-N7 methylation catalyzed by the enzyme Pcm1. Deletion of the pcm1+ gene was lethal, as were missense mutations in the Pcm1 active site. Thus, whereas m7G caps are essential in both S. pombe and S. cerevisiae, m2,2,7G caps are not.

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Untitled

Cited by 43 publications

References 64 publications

Negative Elongation Factor NELF Represses Human Immunodeficiency Virus Transcription by Pausing the RNA Polymerase II Complex

Negative Elongation Factor NELF Represses Human Immunodeficiency Virus Transcription by Pausing the RNA Polymerase II Complex

Celastrol Inhibits Tat-Mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe

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