Analysis of the human immunodeficiency virus long terminal repeat by in vitro transcription competition and linker scanning mutagenesis.
Previous studies designed to map the transcriptional regulatory sequences of the human immunodeficiency virus (HIV) long terminal repeat (LTR) have shown disparate results depending on the method of analysis. Experiments have shown that deletions 5' to -104 (relative to the transcription start site, +1) are not required for transcription in vitro, while other experiments have shown that various mutations in this 5' region of the HIV-1 LTR affect both reporter gene activity in transient expression systems and viral growth. To correlate in vitro and in vivo findings, we performed in vitro transcription competition studies to define minimal sequences necessary for competitive factor binding or competitive transcription complex formation. Using normal HeLa cell nuclear extracts, we found that transcription of a reporter gene run by the U3-R region was efficiently competed only by intact LTR DNA fragments representing virtually the entire U3-R region (-453 to +80). Smaller subfragments of the LTR were less effective competitors; these included fragments from -453 to -159, which had a modest competitive ability at higher competitor concentrations, -159 to +80, and -402 to -34, which were both relatively poor competitors. These findings indicate that although the U3-R region truncated to -104 is able to promote in vitro transcription, a more stable transcription complex appears to form on the entire U3-R region. Hence sequences between -453 and -104 appear to be significant in transcription complex formation. In vivo transfection competition studies confirmed these findings. Specific sequences between -453 and -104 which may affect expression or transcription complex formation were mapped using a set of linker-scanning mutants spanning the LTR.(ABSTRACT TRUNCATED AT 250 WORDS)
Zeichner
S L
SL
Division of Infectious Diseases, Children's Hospital of Philadelphia, Pennsylvania.
Kim
J Y
JY
Alwine
J C
JC
D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000502:physiology; D014158:Transcription, Genetic
D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases