Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants

Despite extensive in vitro studies identifying a myriad of cellular transcription factors that bind the human immunodeficiency virus type 1 5 long terminal repeat (LTR), the relative contribution of these factors to human immunodeficiency virus type 1 replication in infected individuals remains obscure. To address this question, we investigated 478 proviral quasispecies derived from uncultured peripheral blood mononuclear cells of 42 patients representing all stages of infection. In addition to highly conserved TATA box, SP-1, and NF-B sites, the Ets core and an adjacent 5-ACYGCTGA-3 motif were extremely conserved. Importantly, the most frequent naturally occurring length polymorphism (MFNLP) duplicated 5-ACYGCTGA-3 motifs in LTRs in which this same motif was disrupted or in LTRs in which a single point mutation to the Ets core ablated binding of c-Ets 1 and another factor distinct from both c-Ets 1 and Elf 1. The MFNLP's location was precise (position ؊121) and surprisingly frequent (38% of patients) and demarcated LTR Nef-coding sequences from LTR noncoding sequences that appear to be evolving independently. Aside from these features, we found no definitive clinical or transcription phenotype common to all MFNLP LTRs. We also found previously described and novel point polymorphisms, including some conferring TAR-dependent and TARindependent Tat unresponsiveness, and showed that differential binding of nuclear factor(s) to a TCTAA TATA box variant may be the mechanism for the latter.

Section: Discussionsupporting

confidence: 87%

mentioning

confidence: 99%

Human immunodeficiency virus type 1 long terminal repeat variants from 42 patients representing all stages of infection display a wide range of sequence polymorphism and transcription activity

Estable

Bell

Merzouki

et al. 1996

“…(i) Transient-transfection experiments may not reflect the regulation found in vivo with the intact provirus since transiently transfected DNA is not assembled into physiological chromatin. Similar discrepancies between transient transfection studies and in vivo functional studies have been reported previously for HIV (25,42,82). (ii) The Sp1 mutations might disrupt RNA packaging and cause a replication defect independent of transcription.…”

Section: Role Of Transcription Factor Binding Sites Downstream Of Thesupporting

confidence: 75%

“…These differences may be due to different levels of specific transcription factors in different cell types examined. Such cell-type-specific differences in the replication properties of HIV-1 have been reported by others studying Tat activation response element (TAR) and LTR mutant viruses (25,42).…”

Section: Ii) Infections Of Human Pbmcs and T-cell Lines With Wt Or Musupporting

confidence: 53%

Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity

Lint¹,

Amella²,

Emiliani³

et al. 1997

138

When transcriptionally active, the human immunodeficiency virus (HIV) promoter contains a nucleosomefree region encompassing both the promoter/enhancer region and a large region (255 nucleotides [nt]) downstream of the transcription start site. We have previously identified new binding sites for transcription factors downstream of the transcription start site (nt 465 to 720): three AP-1 sites (I, II, and III), an AP3-like motif (AP3-L), a downstream binding factor (DBF) site, and juxtaposed Sp1 sites. Here, we show that the DBF site is an interferon-responsive factor (IRF) binding site and that the AP3-L motif binds the T-cell-specific factor NF-AT. Mutations that abolish the binding of each factor to its cognate site are introduced in an infectious HIV-1 molecular clone to study their effect on HIV-1 transcription and replication. Individual mutation of the DBF or AP3-L site as well as the double mutation AP-1(III)/AP3-L did not affect HIV-1 replication compared to that of the wild-type virus. In contrast, proviruses carrying mutations in the Sp1 sites were totally defective in terms of replication. Virus production occurred with slightly delayed kinetics for viruses containing combined mutations in the AP-1(III), AP3-L, and DBF sites and in the AP3-L and DBF-sites, whereas viruses mutated in the AP-1(I,II,III) and AP3-L sites and in the AP-1(I,II,III), AP3-L, and DBF sites exhibited a severely defective replicative phenotype. No RNA-packaging defect could be measured for any of the mutant viruses as determined by quantification of their HIV genomic RNA. Measurement of the transcriptional activity of the HIV-1 promoter after transient transfection of the HIV-1 provirus DNA or of long terminal repeatluciferase constructs showed a positive correlation between the transcriptional and the replication defects for most mutants.

“…Tat activation requires the preservation of the TAR RNA secondary structure in addition to the maintenance of a 3-nucleotide bulge and a 6-nucleotide loop element (reviewed in references 20 and 33). Viruses containing mutations that either disrupt the TAR RNA stem-loop structure, delete the bulge element, or alter the primary sequences of the loop exhibit marked decreases in gene expression and replication (24). Using a genetic selection with these TAR viral mutants, we recently isolated revertants that defined minimal TAR RNA sequences required for efficient HIV-1 gene expression (25).…”

mentioning

confidence: 99%

A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription

Harrich

Ulich

Gaynor

1996

Self Cite

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on the transactivator protein Tat and an RNA element extending from the transcription initiation site to ؉57 known as TAR. TAR forms a stable RNA secondary structure which is critical for high levels of HIV-1 gene expression and efficient viral replication. Using a genetic approach, we isolated HIV-1 mutants in TAR that were competent for high levels of gene expression but yet were markedly defective for viral replication. Single-cycle infections with these viruses demonstrated that they were defective in the initiation of reverse transcription. Additional mutational analysis revealed a variety of other HIV-1 TAR mutants with the same defective phenotype. Thus, in addition to the well-characterized role of the primer binding site, other RNA elements within the HIV-1 genome are also critical in the regulation of reverse transcription. These studies demonstrate that HIV-1 TAR RNA is a key regulator of the reverse transcription and illustrate how a unique RNA structure can modulate diverse regulatory processes in the HIV-1 life cycle crucial for efficient viral replication.