Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps.

We have examined, using self-cleaving ribozymes, the intracellular trans-activation kinetics of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by viral protein Tat. Experiments were designed to effect a competition (during RNA chain elongation) between cleavage of a nascent RNA containing the Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-activation. We found that fast self-cleavage of nascent TAR-containing RNA abolished Tat trans-activation. Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate-limiting step in HIV-1 LTR trans-activation is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA polymerases that are tethered to functional TAR.     

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.     

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.