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

2007 ◽  
Vol 282 (23) ◽  
pp. 16981-16988 ◽  
Author(s):  
Zhiqiang Zhang ◽  
Alicia Klatt ◽  
David S. Gilmour ◽  
Andrew J. Henderson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Virus Transcription ◽  
Immunodeficiency Virus ◽  
Negative Elongation Factor

scholarly journals The Ability of Positive Transcription Elongation Factor b To Transactivate Human Immunodeficiency Virus Transcription Depends on a Functional Kinase Domain, Cyclin T1, and Tat

1998 ◽  
Vol 72 (9) ◽  
pp. 7154-7159 ◽  
Author(s):  
Koh Fujinaga ◽  
Thomas P. Cujec ◽  
Junmin Peng ◽  
Judit Garriga ◽  
David H. Price ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Response Element ◽  
Factor B ◽  
Cyclin T1 ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Immunodeficiency Virus

ABSTRACT By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat–P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome.

scholarly journals Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

2002 ◽  
Vol 22 (4) ◽  
pp. 1079-1093 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Young Kyeung Kim ◽  
Mark J. Churcher ◽  
Michelle J. West ◽  
Jonathan Karn
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dna Sequences ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.

scholarly journals Dynamics of Human Immunodeficiency Virus Transcription: P-TEFb Phosphorylates RD and Dissociates Negative Effectors from the Transactivation Response Element

2004 ◽  
Vol 24 (2) ◽  
pp. 787-795 ◽  
Author(s):  
Koh Fujinaga ◽  
Dan Irwin ◽  
Yehong Huang ◽  
Ran Taube ◽  
Takeshi Kurosu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Dominant Negative ◽  
Rna Recognition Motif ◽  
Transcription Elongation Factor ◽  
Virus Transcription ◽  
Immunodeficiency Virus ◽  
Cellular Cofactor ◽  
Negative Effect

ABSTRACT The elongation of transcription is a highly regulated process that requires negative and positive effectors. By binding the double-stranded stem in the transactivation response (TAR) element, RD protein from the negative transcription elongation factor (NELF) inhibits basal transcription from the long terminal repeat of the human immunodeficiency virus type 1 (HIVLTR). Tat and its cellular cofactor, the positive transcription elongation factor b (P-TEFb), overcome this negative effect. Cdk9 in P-TEFb also phosphorylates RD at sites next to its RNA recognition motif. A mutant RD protein that mimics its phosphorylated form no longer binds TAR nor represses HIV transcription. In sharp contrast, a mutant RD protein that cannot be phosphorylated by P-TEFb functions as a dominant-negative effector and inhibits Tat transactivation. These results better define the transition from abortive to productive transcription and thus replication of HIV.

scholarly journals DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression

2011 ◽  
Vol 92 (7) ◽  
pp. 1710-1720 ◽  
Author(s):  
Shilpi Tyagi ◽  
Alex Ochem ◽  
Mudit Tyagi
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dependent Protein Kinase ◽  
Immunodeficiency Virus ◽  
Target Sites ◽  
Dependent Protein ◽  
Rnap Ii

DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat–DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription.

scholarly journals Positive Transcription Elongation Factor b Phosphorylates hSPT5 and RNA Polymerase II Carboxyl-terminal Domain Independently of Cyclin-dependent Kinase-activating Kinase

2001 ◽  
Vol 276 (15) ◽  
pp. 12317-12323 ◽  
Author(s):  
Jae B. Kim ◽  
Phillip A. Sharp
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Elongation Factor ◽  
Rna Pol Ii ◽  
Factor B ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Immunodeficiency Virus ◽  
Carboxyl Terminal

The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol IIin vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.

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