The human immunodeficiency virus (HIV) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The viral replication and high level gene expression of HIV virus require activation in trans by regulatory protein, TAT. The region of HIV needed for TAT mediated-regulation was termed the TAR (transacting responsive element), which is positioned between nucleotide - 1 7 and +80 relative to the transcription initiation site and the sequence from +14 to +44 is essential for the response to TAT protein. We developed a in vitro trancriptional assay and our previously results showed that the nuclear extract of HeLa/t2 cell, which contains TAT protein, and purified recombinant TAT protein from E. Coli specifically enhanced the transcription from HIV-LTR. In the past year, we also found that the DNA sequence of TAR region was needed in vitro transcription system by using competition assay. In order to be able to provide more directly evidence for the relations between the transactivating activity of TAT protein and the TAR sequence, we have set up a gel retardation assay. The result showed that when the HeLa/TAT nuclear extract (HeLa/TAT cells was transformed with TAT cDNA) was incubated with double strand DNA probe of TAR region, the gel retardation pattern appeared a clearly extra band compared with that of normal HeLa nuclear extract. The same result was obtained from the probes which either was between nucleotide +14 to +45 or between nucleotide +19 to +45, but this extra band was disappeared when the DNA probe was between nucleotide +25 to +45. This result indicated that the DNA sequence from +19 to +25 of TAR region is essential for this extra band. we also observed that the extra band can be competed out by specific DNA competitor (cold DNA TAR sequence) and anti-TAT antibody. These results imply that the formation of this specific DNA-protein complex was due to some factor(s) which was only contained in the cells transformed by TAT gene and it was relative to the TAT protein. These results give us some information about the transcriptional regulation by TAT protein and TAR sequence. We are now going to develop other assays to screen potential inhibitors of TAT activity.prevention or treatment of AIDS.
