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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 T8-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 virusencoded 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 (3-actin gene and the simian virus 40 promoters.

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytesmacrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and SalmoneUla typhimurum histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-KB. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding 'y-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-KB pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

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