scholarly journals Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF-κB through the Cellular Interferon-Inducible, Double-Stranded RNA-Dependent Protein Kinase, PKR

1999 ◽  
Vol 73 (8) ◽  
pp. 7080-7086 ◽  
Author(s):  
Francesca Demarchi ◽  
Maria Ines Gutierrez ◽  
Mauro Giacca
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protein Kinase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-κB (NF-κB), acting through degradation of the inhibitor IκB-α (F. Demarchi, F. d’Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427–4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-κB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-κB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction.

scholarly journals Human Ubc9 Contributes to Production of Fully Infectious Human Immunodeficiency Virus Type 1 Virions

2009 ◽  
Vol 83 (20) ◽  
pp. 10448-10459 ◽  
Author(s):  
Tareq Jaber ◽  
Christopher R. Bohl ◽  
Gentry L. Lewis ◽  
Charles Wood ◽  
John T. West ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Normal Numbers ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, trafficking, and Env incorporation. The Gag-mediated assembly of virus particles and protease-mediated processing of Gag and Gag-Pol were unchanged in the absence of Ubc9. However, the stability of the cell-associated Env glycoprotein was decreased and Env incorporation into released virions was altered. Interestingly, overexpression of the Ubc9 trans-dominant-negative mutant C93A, which is a defective E2-SUMO-1 conjugase, suggests that this activity may not be required for interaction with Gag, virion assembly, or infectivity. This finding demonstrates that Ubc9 plays an important role in the production of infectious HIV-1 virions.

scholarly journals Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

2010 ◽  
Vol 84 (13) ◽  
pp. 6590-6597 ◽  
Author(s):  
Elena Popova ◽  
Sergei Popov ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Leucine Zipper ◽  
Particle Production ◽  
Dominant Negative ◽  
Dimerization Domain ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed ZWT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, ZWT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.

scholarly journals Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in Macrophages through a C/EBP-Dependent Mechanism

2001 ◽  
Vol 75 (20) ◽  
pp. 9703-9712 ◽  
Author(s):  
Eileen S. Lee ◽  
Huiyu Zhou ◽  
Andrew J. Henderson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dominant Negative ◽  
Cell Contact ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Macrophages are early targets of human immunodeficiency virus type 1 (HIV-1) infection and serve as potential reservoirs for long-term infection. Through inflammatory mediators and direct cell contact, infected macrophages interact with neighboring cell populations, such as the endothelium, which create a microenvironment favorable for HIV-1 replication. We hypothesize that the transcriptional activator C/EBPβ is critical for macrophages to respond to endothelial cell-derived signals. We show that endothelial cells significantly enhance C/EBPβ binding activity and HIV-1 replication in macrophages. This increase in HIV-1 transcription is due to cell-cell contact as well as the production of soluble factors, mediated in part by ICAM-1 and interleukin 6, respectively. Furthermore, C/EBP factors are necessary for endothelial cell-dependent activation of HIV-1 transcription in macrophages, and HIV-1 induction can be inhibited by a C/EBP dominant-negative protein. In addition, C/EBP binding sites are necessary for efficient LTR activity and HIV-1 replication in the presence of endothelial cells. Taken together, these results indicate that endothelial cells, through the activation of C/EBPβ, provide a microenvironment that supports HIV-1 replication in monocytes/macrophages.

scholarly journals Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs

1999 ◽  
Vol 80 (8) ◽  
pp. 1965-1974 ◽  
Author(s):  
Anne Marie Szilvay ◽  
Stig-Ove Bøe ◽  
Karl-Henning Kalland
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Dominant Negative ◽  
Structural Proteins ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Negative Mutant

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the trans-dominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

scholarly journals Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

2002 ◽  
Vol 22 (9) ◽  
pp. 2965-2973 ◽  
Author(s):  
Guocheng He ◽  
David M. Margolis
Keyword(s):  
Human Immunodeficiency Virus ◽  
Histone Deacetylase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Trichostatin A ◽  
Dominant Negative Mutant ◽  
Histone Deacetylase 1 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Repression of human immunodeficiency virus type 1 (HIV-1) transcription may contribute to the establishment or maintenance of proviral quiescence in infected CD4+ cells. The host factors YY1 and LSF cooperatively recruit histone deacetylase 1 (HDAC1) to the HIV-1 long terminal repeat (LTR) and inhibit transcription. We demonstrate here regulation of occupancy of HDAC1 at a positioned nucleosome (nuc 1) near the transcription start site of integrated LTR. We find that expression of YY1 increases occupancy by HDAC1, decreases acetylation at nuc 1, and downregulates LTR expression. HDAC1 recruitment and histone hypoacetylation were also seen when Tat activation was inhibited by the overexpression of YY1. A YY1 mutant without an HDAC1 interaction domain and incompetent to inhibit LTR activation fails to recruit HDAC1 to LTR or decrease nuc 1 acetylation. Further, expression of a dominant-negative mutant of LSF (dnLSF), which inhibits LSF occupancy and LTR repression, results in acetylation and decreased HDAC1 occupancy at nuc 1. Conversely, exposure of cells to the histone deacetylase inhibitor trichostatin A or activation of LTR expression by HIV-1 Tat results in the displacement of HDAC1 from nuc 1, in association with increased acetylation of histone H4. Recruitment of HDAC1 to the LTR nuc 1 can counteract Tat activation and repress LTR expression. Significantly, when repression is overcome, LTR activation is associated with decreased HDAC1 occupancy. Since the persistence of integrated HIV-1 genomes despite potent suppression of viral replication is a major obstacle for current antiretroviral therapy, strategies to selectively disrupt the quiescence of chromosomal provirus may play a role in the future treatment of AIDS.

scholarly journals Caffeine Inhibits Human Immunodeficiency Virus Type 1 Transduction of Nondividing Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2058-2065 ◽  
Author(s):  
René Daniel ◽  
Elena Marusich ◽  
Elias Argyris ◽  
Richard Y. Zhao ◽  
Anna Marie Skalka ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dominant Negative ◽  
Cell Cycle Checkpoints ◽  
Retroviral Transduction ◽  
Human Neurons ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Caffeine is an efficient inhibitor of DNA repair and DNA damage-activated checkpoints. We have shown recently that caffeine inhibits retroviral transduction of dividing cells, most likely by blocking postintegration repair. This effect may be mediated at least in part by a cellular target of caffeine, the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase. In this study, we present evidence that caffeine also inhibits efficient transduction of nondividing cells. We observed reduced transduction in caffeine-treated growth-arrested cells as well as caffeine-treated terminally differentiated human neurons and macrophages. Furthermore, this deficiency was observed with a human immunodeficiency virus type 1 (HIV-1) vector lacking Vpr, indicating that the effect is independent of the presence of this viral protein in the infecting virion. Finally, we show that HIV-1 transduction of nocodazole-arrested cells is reduced in cells that express an ATR dominant-negative protein (kinase-dead ATR [ATRkd]) and that the residual transduction of ATRkd-expressing cells is relatively resistant to caffeine. Taken together, these data suggest that the effect(s) of caffeine on HIV-1 transduction is mediated at least partly by the inhibition of the ATR pathway but is not dependent on the caffeine-mediated inhibition of cell cycle checkpoints.

scholarly journals Characterization of a family of related cellular transcription factors which can modulate human immunodeficiency virus type 1 transcription in vitro.

1994 ◽  
Vol 14 (3) ◽  
pp. 1776-1785 ◽  
Author(s):  
J B Yoon ◽  
G Li ◽  
R G Roeder
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Tata Box ◽  
Immunodeficiency Virus ◽  
Hiv 1

LBP-1 is a cellular protein which binds strongly to sequences around the human immunodeficiency virus type 1 (HIV-1) initiation site and weakly over the TATA box. We have previously shown that LBP-1 represses HIV-1 transcription by inhibiting the binding of TFIID to the TATA box. Four similar but distinct cDNAs encoding LBP-1 (LBP-1a, -b, -c, and -d) have been isolated. These are products of two related genes, and each gene encodes two alternatively spliced products. Comparison of the amino acid sequence of LBP-1 with entries in the available protein data bases revealed the identity of LBP-1c to alpha-CP2, an alpha-globin transcription factor. These proteins are also homologous to Drosophila melanogaster Elf-1/NTF-1, an essential transcriptional activator that functions during Drosophila embryogenesis. Three of the recombinant LBP-1 isoforms show DNA binding specificity identical to that of native LBP-1 and bind DNA as a multimer. In addition, antisera raised against recombinant LBP-1 recognize native LBP-1 from HeLa nuclear extract. Functional analyses in a cell-free transcription system demonstrate that recombinant LBP-1 specifically represses transcription from a wild-type HIV-1 template but not from an LBP-1 mutant template. Moreover, LBP-1 can function as an activator both in vivo and in vitro, depending on the promoter context. Interestingly, one isoform of LBP-1 which is missing the region of the Elf-1/NTF-1 homology is unable to bind DNA itself and, presumably through heteromer formation, inhibits binding of the other forms of LBP-1, suggesting that it may function as a dominant negative regulator.

scholarly journals The TAR Hairpin of Human Immunodeficiency Virus Type 1 Can Be Deleted When Not Required for Tat-Mediated Activation of Transcription

2007 ◽  
Vol 81 (14) ◽  
pp. 7742-7748 ◽  
Author(s):  
Atze T. Das ◽  
Alex Harwig ◽  
Martine M. Vrolijk ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Stem Loop ◽  
Activation Of Transcription ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome contains a terminal repeat (R) region that encodes the transacting responsive (TAR) hairpin, which is essential for Tat-mediated activation of gene expression. TAR has also been implicated in several other processes during viral replication, including translation, dimerization, packaging, and reverse transcription. However, most studies in which replication of TAR-mutated viruses was analyzed were complicated by the dominant negative effect of the mutations on transcription. We therefore used an HIV-1 variant that does not require TAR for transcription to reinvestigate the role of TAR in HIV-1 replication. We demonstrate that this virus can replicate efficiently upon complete deletion of TAR. Furthermore, evolution of a TAR-deleted variant in long-term cultures indicates that HIV-1 requires a stable stem-loop structure at the start of the viral transcripts in which the 5′-terminal nucleotides are base paired. This prerequisite for efficient replication can be fulfilled by the TAR hairpin but also by unrelated stem-loop structures. We therefore conclude that TAR has no essential function in HIV-1 replication other than to accommodate Tat-mediated activation of transcription.

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