An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators

Li, Y. C., Ross, J., Scheppler, J. A., Franza, B. R. (April 1991) An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators. Mol Cell Biol, 11 (4). pp. 1883-93. ISSN 0270-7306 (Print)0270-7306 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2005886
DOI: 10.1128/MCB.11.4.1883

Abstract

In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.

Item Type: Paper
Uncontrolled Keywords: Cell Line Cycloheximide/pharmacology HIV Enhancer HIV Long Terminal Repeat/*genetics HIV-1/*genetics Humans Kinetics Mutagenesis, Site-Directed Phytohemagglutinins/pharmacology TATA Box Tetradecanoylphorbol Acetate/pharmacology *Transcription, Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription
diseases & disorders > viral diseases > HIV
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene regulation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene regulation
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: April 1991
Date Deposited: 06 Jan 2016 20:52
Last Modified: 06 Jan 2016 20:52
PMCID: PMC359867
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32092

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving