Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates.

Nucleic acid-based antiviral approaches have been tried against multiple HIV-1 genes with the purpose of down-regulating its replication. A unique stem-loop structure called TAR is present at the 5'-end of all HIV-1 transcripts; Tat and other cellular proteins bind to TAR and thus govern transcription. Therefore, HIV-1 TAR is an attractive target against which various antiviral approaches could be tried. We screened several DNA enzymes (Dz's) containing the 10-23 catalytic motif and a single Dz containing the 8-17 catalytic motif against the HIV-1 TAR RNA. Dz's directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. One of the two Dz's, namely Dz-475, showed moderate cleavage activity in complete absence of Mg(2+). Addition of unrelated sequences at the 5'-end of the HIV-1 TAR RNA rendered it susceptible to four additional Dz-mediated cleavages. Both Dz's (470 and 475) showed significant intracellular reduction of HIV-1 gene expression. Dz-475-treated cells showed significant protection against T-tropic and macrophage-tropic HIV-1 challenge. Dz-475-transfected T-lymphocytes, human PBMCs, or chronically infected cell lines showed marked viral resistance. Unique features of this antiviral strategy with respect to HIV-1 gene inhibition are discussed.