scholarly journals CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

1997 ◽  
Vol 17 (10) ◽  
pp. 6029-6039 ◽  
Author(s):  
C Suñé ◽  
T Hayashi ◽  
Y Liu ◽  
W S Lane ◽  
R A Young ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Nuclear Protein ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Rna Polymerase Ii Holoenzyme ◽  
Hiv 1

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

scholarly journals CD4-Specific Transgenic Expression of Human Cyclin T1 Markedly Increases Human Immunodeficiency Virus Type 1 (HIV-1) Production by CD4+ T Lymphocytes and Myeloid Cells in Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV-1 Isolate

2006 ◽  
Vol 80 (4) ◽  
pp. 1850-1862 ◽  
Author(s):  
Jinglin Sun ◽  
Timothy Soos ◽  
Vineet N. KewalRamani ◽  
Kristin Osiecki ◽  
Jian Hua Zheng ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Transgenic Mice ◽  
Transcriptional Activation ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Mouse Cells ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4+ T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1JR-CSF provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4+ T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.

scholarly journals Unexpected Mutations in HIV-1 That Confer Resistance to the Tat Inhibitor Didehydro-Cortistatin A

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Andrew P. Rice
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Latent Infection ◽  
Pol Ii ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Didehydro-cortistatin A (dCA) is a human immunodeficiency virus type 1 (HIV-1) Tat inhibitor that functions by selectively binding to the RNA binding domain of Tat. In addition to inhibiting viral replication, dCA can drive HIV-1 into a state of “deep latency” in which latent viruses are refractory to reactivation. Mousseau et al. (G. Mousseau, R. Aneja, M. A. Clementz, S. Mediouni, et al., mBio 10:e01750-18, 2019, https://doi.org/10.1128/mBio.01750-18) have now selected dCA-resistant (dCAr) viruses in vitro. Remarkably, dCAr viruses do not contain mutations in Tat or the viral transactivation-responsive element (TAR) RNA element that is targeted by Tat. Rather, the viruses contain a combination of mutations in the viral long terminal repeat (LTR) and Nef and Vpr proteins that result in an increase in basal RNA polymerase II (Pol II) transcription of integrated HIV-1. Interestingly, dCAr viruses may be deficient in the establishment of latent infection because of their elevated basal Pol II transcription. dCA holds promise for strategies to achieve a functional cure of HIV-1 infection and justifies efforts to develop additional Tat inhibitors.

scholarly journals The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme.

1997 ◽  
Vol 17 (4) ◽  
pp. 1817-1823 ◽  
Author(s):  
T P Cujec ◽  
H Cho ◽  
E Maldonado ◽  
J Meyer ◽  
D Reinberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Step ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus ◽  
Rna Hairpin ◽  
Rna Polymerase Ii Holoenzyme

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.

scholarly journals NF-κB-Repressing Factor Inhibits Elongation of Human Immunodeficiency Virus Type 1 Transcription by DRB Sensitivity-Inducing Factor

2005 ◽  
Vol 25 (17) ◽  
pp. 7473-7483 ◽  
Author(s):  
Ursula Dreikhausen ◽  
Kirsten Hiebenthal-Millow ◽  
Myriam Bartels ◽  
Klaus Resch ◽  
Mahtab Nourbakhsh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase Ii ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Transcription Elongation ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is able to establish a latent infection during which the integrated provirus remains transcriptionally silent. In response to specific stimuli, the HIV-1 long terminal repeat (LTR) is highly activated, enhancing both transcriptional initiation and elongation. Here, we have identified a specific binding sequence of the nuclear NF-κB-repressing factor (NRF) within the HIV-1 LTR. The aim of this work was to define the role of NRF in regulating the LTR. Our data show that the endogenous NRF is required for transcriptional activation of the HIV-1 LTR in stimulated cells. In unstimulated cells, however, NRF inhibits HIV-1 LTR activity at the level of transcription elongation. Binding of NRF to the LTR in unstimulated cells prevents recruitment of elongation factor DRB sensitivity-inducing factor and formation of processive elongation complexes by hyperphosphorylated RNA polymerase II. Our data suggest that NRF interrupts the regulatory coupling of LTR binding factors and transcription elongation events. This inhibitory mechanism might contribute to transcriptional quiescence of integrated HIV-1 provirus.

scholarly journals A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb

2004 ◽  
Vol 24 (12) ◽  
pp. 5094-5105 ◽  
Author(s):  
Jasper H. N. Yik ◽  
Ruichuan Chen ◽  
Andrea C. Pezda ◽  
Craig S. Samford ◽  
Qiang Zhou
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Binding Motif ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT 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.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes

2003 ◽  
Vol 77 (1) ◽  
pp. 291-300 ◽  
Author(s):  
L. Musey ◽  
Y. Ding ◽  
J. Cao ◽  
J. Lee ◽  
C. Galloway ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytotoxic T Lymphocyte ◽  
Infected Individual ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8+ cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8+ CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRβ VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.

scholarly journals Gag sequence variation in a human immunodeficiency virus type 1 transmission cluster influences viral replication fitness

2013 ◽  
Vol 94 (2) ◽  
pp. 354-359 ◽  
Author(s):  
Esther F. Gijsbers ◽  
Ad C. van Nuenen ◽  
Hanneke Schuitemaker ◽  
Neeltje A. Kootstra
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Sequence Variation ◽  
Clinical Course ◽  
Immunodeficiency Virus ◽  
Compensatory Mutations ◽  
Replication Fitness ◽  
Hiv 1

Three men from a proven homosexual human immunodeficiency virus type 1 (HIV-1) transmission cluster showed large variation in their clinical course of infection. To evaluate the effect of evolution of the same viral variant in these three patients, we analysed sequence variation in the capsid protein and determined the impact of the observed variation on viral replication fitness in vitro. Viral gag sequences from all three patients contained a mutation at position 242, T242N or T242S, which have been associated with lower virus replication in vitro. Interestingly, HIV-1 variants from patients with a progressive clinical course of infection developed compensatory mutations within the capsid that restored viral fitness, instead of reversion of the T242S mutation. In HIV-1 variants from patient 1, an HLA-B57+ elite controller, no compensatory mutations emerged during follow-up.

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