Site-Directed Mutagenesis of Two Trans-Regulatory Genes (
            <i>tat</i>
            -III,
            <i>trs</i>
            ) of HIV-1

Sadaie, M. Reza; Benter, T.; Wong‐Staal, Flossie

doi:10.1126/science.3277284

Cited by 178 publications

(117 citation statements)

References 28 publications

(25 reference statements)

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“…É ric A. Cohen (IRCM, Canada) provided the HxBRUDp6 in which a stop codon was introduced following the p6 ORF (Gottlinger et al 1991). David Ott (NIH/NCI, Frederick, MD) provided the pNL4-XX and pNL4-X proviral constructs as described (Poon et al 2002); and the NIH AIDS Reference and Reagent Program provided the pMRev(À) contruct (Sadaie et al 1988). Anne Gatignol (McGill University) provided the pMAL and pADA Mtropic proviral constructs.…”

Section: Cell Lines and Transfectionsmentioning

confidence: 99%

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

Ajamian

Abrahamyan²,

Milev³

et al. 2008

RNA

152

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The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55Gag , viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55Gag . Pr55 Gag synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.

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Section: Cell Lines and Transfectionsmentioning

confidence: 99%

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

Ajamian

Abrahamyan²,

Milev³

et al. 2008

RNA

152

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“…Several mechanisms have been proposed to explain its action, including enhancement of HIV-1 LTR-directed transcription rate (Jakobovits et al 1988;Jeang et al 1988; Rice and Mathews 1988;Laspia et al 1989), increase in mRNA stability (Muesing et al 1987), anti-termination of transcription within the R region of the LTR (Kao et al 1987;Selby et al 1989;Toohey and Jones 1989), and enhancement of translation of HIV-1 mRNAs (Rosen et al 1986a;Feinberg et al 1986;Braddock et al 1989). Bimodal mechanisms involving transcriptional and post-transcriptional events have also been suggested (Cullen 1986;Wright et al 1986;Sadaie et al 1988).…”

mentioning

confidence: 99%

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.

Roy¹,

Delling²,

Chen³

et al. 1990

Genes Dev.

416

388

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The Tat protein of human immunodeficiency virus type 1 (HIV-I) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions + 23 to + 25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position + 23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions + 31 to + 34) and the stem (positions + 9 to + 12 and + 49 to + 52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.

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“…Tat produces a large stimulation of HIV-1-promoted RNA levels (CuUen 1986;Peterlin et al 1986;Wright et al 1986;Hauber et al 1987;Muesing et al 1987;Rice and Mathews 1988a) so the predominant mode of regulation appears to be transcriptional (Hauber et al 1987;Kao et al 1987;Jakobovits et al 1988;Jeang et al 1988;Rice and Mathews 1988a;Sadaie et al 1988;Laspia et al 1989), but evidence for posttranscriptional regulation also exists (Cullen 1986;Fein-Cold Spring Harbor Laboratory Press on April 4, 2019 -Published by genesdev.cshlp.org Downloaded from berg et al 1986;Rosen et al 1986;Wright et al 1986;Braddock et al 1989Braddock et al , 1990). In a model system consisting of replicating plasmids in COS cells, Kao et al (1987) found that Tat does not increase the initiation of transcription but act to stimulate transcriptional elongation.…”

mentioning

confidence: 99%

Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation.

Laspia¹,

Rice²,

Mathews³

1990

Genes & Development

107

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We studied the combined effects of Tat and general trans-activators, such as ElA and phorbol esters, on human immunodeficiency virus-1 (HIV-1) gene expression. Interaction between these two types of trans-activators may be involved in the transition from transcriptional quiesence during viral latency to active gene expression during productive infection. ElA cooperated with Tat to produce a fourfold greater increase in accumulation of full-length, cytoplasmic HIV-1-directed RNA than is expected if they were acting additively to increase RNA accumulation. Similarily, phorbol 12-myristate 13-acetate (PMA) also cooperated with Tat to elevate HIV RNA levels synergistically. Analysis of transcription rates across the HIV-1-directed transcription unit indicated, unexpectedly, that synergy between Tat and ElA could not be accounted for by increased promoter proximal transcription rates that were merely additive. However, Tat and ElA produced a greater than additive increase in transcription rates in the 3' end of the gene. These findings imply that synergy between Tat and ElA (or other general transcriptional activators) is due principally to stabilization of transcriptional elongation. Furthermore, the observation that Tat elicits only a small increase in promoter proximal transcription in the presence of ElA suggests that the magnitude of the effect of Tat on initiation is decreased when the basal level of transcription is increased. These findings underscore the importance of the ability of Tat to stabilize elongation, as well as to stimulate initiation, in an HIV-1-directed transcription unit.

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Site-Directed Mutagenesis of Two Trans-Regulatory Genes ( tat -III, trs ) of HIV-1

Cited by 178 publications

References 28 publications

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.

Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation.

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