scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Rev Function by a Dominant-Negative Mutant of Sam68 through Sequestration of Unspliced RNA at Perinuclear Bundles

2001 ◽  
Vol 75 (17) ◽  
pp. 8203-8215 ◽  
Author(s):  
Vanessa B. Soros ◽  
Héctor Valderrama Carvajal ◽  
Stéphane Richard ◽  
Alan W. Cochrane
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Localization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hiv Rna ◽  
Immunodeficiency Virus ◽  
Translation Apparatus ◽  
Localization Signal ◽  
Stimulation Of

ABSTRACT Human immunodeficiency virus (HIV) type 1 encodes an essential protein, Rev, which functions to transport unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm to allow expression of the viral structural proteins. It has previously been reported that Sam68 synergistically stimulates Rev activity (T. Reddy et al., Nat. Med. 5:635–642, 1999). Here we report that the Sam68-like mammalian proteins SLM1 and SLM2 also stimulate Rev activity. Their stimulation ability cannot be attributed to a shuttling property, since Sam68, SLM1, and SLM2 do not display significant shuttling activity alone or in the presence of Rev. In addition, Sam68, SLM1, and SLM2 do not affect the equilibrium between unspliced and completely spliced HIV RNA. The C-terminally truncated Sam68 mutant (Sam68ΔC) previously observed to inhibit the Sam68-mediated stimulation of Rev activity (Reddy et al., 1999) also inhibits SLM1- and SLM2-mediated stimulation of Rev activity. This suggests that the mechanism by which Sam68, SLM1, and SLM2 stimulate Rev activity may be common. Sam68ΔC does not inhibit Rev activity by inhibiting Rev from shuttling between the nucleus and cytoplasm. Inhibition by Sam68ΔC is a consequence of its mislocalization to the cytoplasm, as evidenced by the fact that addition of an exogenous nuclear localization signal to Sam68ΔC restores nuclear localization and stimulation of Rev activity. We demonstrate that Sam68ΔC causes perinuclear accumulation of unspliced HIV env RNA and propose that Sam68ΔC inhibits Rev activity by sequestering Rev-responsive RNA away from the translation apparatus.

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 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 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 Genotypic Changes in Human Immunodeficiency Virus Type 1 Associated with Loss of Suppression of Plasma Viral RNA Levels in Subjects Treated with Ritonavir (Norvir) Monotherapy

1998 ◽  
Vol 72 (6) ◽  
pp. 5154-5164 ◽  
Author(s):  
P. Scott Eastman ◽  
John Mittler ◽  
Reed Kelso ◽  
Chris Gee ◽  
Eric Boyer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Resistance Mutation ◽  
Virologic Response ◽  
Hiv Rna ◽  
Immunodeficiency Virus ◽  
Plasma Rna ◽  
Doubling Times ◽  
Rna Levels

ABSTRACT Ten subjects received 600 to 1,200 mg of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor ritonavir per day. Following 2 weeks of therapy, plasma HIV RNA levels decreased by a mean of 1.57 (range, 0.89 to 1.96) log units. With continued therapy, HIV RNA levels began to rise in eight subjects. The initial rise in plasma RNA levels was temporally associated with the development and quantitative increase in the V82 resistance mutation. Doubling times of the V82A mutant virus were estimated to be 2.4 to 4.8 days. An L63P/A mutation was commonly present at baseline even in subjects with a durable virologic response. The concomitant acquisition of an L63P/A mutation with the V82A/F mutation at the time when plasma RNA levels rebounded suggests a role for the L63P/A mutation in improving the fitness of the V82A/F mutation. Subsequent additional genotypic changes at codons 54 and 84 were often associated with further increases in plasma RNA levels. Ongoing viral replication in the presence of drugs resulted in the appearance of additional genotypic changes, including the L90M saquinavir resistance mutation, and decreased phenotypic susceptibility. The relative fitness of the protease V82A ritonavir resistance mutation and reverse transcriptase T215Y/F zidovudine resistance mutation following drug withdrawal were estimated to be 96 to 98% that of the wild type. Durability of the virologic response was associated with plasma RNA levels at the nadir. A virologic response beyond 60 days was not observed unless plasma HIV RNA levels were suppressed below 2,000 copies/ml, consistent with estimates from V82A doubling times for selection of a single resistance mutation to dominate the replicating population.

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