RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator.
Tandem repeats of the transactivation response element (TAR) of the human immunodeficiency virus 1 (HIV-1) were generated using a specially constructed "tandemizing" plasmid, pGem-Tan. This plasmid exploits the rotational nonequivalence of Ava I restriction sites to generate multiple copies of an inserted sequence. Twelve tandem repeats of the TAR were then placed in sense and antisense orientations behind a strong human beta-actin gene promoter. The TAR constructs were transfected with an appropriate HIV-1-driven reporter and tat gene expression plasmids into NT2/D1 cells, a pluripotential human embryonic teratocarcinoma cell line. Twelve tandem TAR repeats in the sense orientation suppressed 85-90% of the transactivating function of the virus-encoded tat protein, whereas the antisense construct or constructs containing single copies of TAR in either sense or antisense orientations were relatively ineffective. The suppression was specific for reporter gene constructs containing an intact HIV-1 long terminal repeat: Reporter genes driven by other promoters or by an HIV-1 long terminal repeat lacking the TAR were not suppressed. Suppression of activation by tat required transcription into RNA: Similar constructs containing the TAR repeats but lacking a eukaryotic promoter failed to suppress tat activation. In the absence of tat, the TAR DNA stimulated 2- to 5-fold the expression of gene constructs driven not only by the HIV-1 long terminal repeat but also by the human beta-actin gene and the simian virus 40 promoters.
Graham
G J
GJ
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Maio
J J
JJ
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015696:Gene Products, tat / Q000235:genetics*; D015756:Genes, tat; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D012367:RNA, Viral / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D000199:Actins; D015696:Gene Products, tat; D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA