INTERACTION OF THE REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 WITH DNA

During DNA synthesis, the binding of human immunodeficiency virus (HIV ) reverse transcriptase (RT) to the template-primer precedes its bindi ng to nucleotide triphosphates. The interaction of oligonucleotide DNA with HIV-1 RT was investigated by using a gel retardation assay. Both homodimeric (p66/p66) and heterodimeric (p66/p51) isoforms of HIV-1 R T were capable of binding the DNA oligomers. Thus, ail further studies on the interaction of HIV-1 RT with DNA were done with heterodimeric RT. We have studied the conditions for optimal binding. The formation of the RT-DNA complex was primer-independent, and the extent of DNA bi nding was indistinguishable for both single-stranded and double-strand ed DNA (either blunt-ended or recessed). The DNA binding activity of t he RT was found to be dependent on oligonucleotide length. HIV-1 RT bi nds DNA with no apparent sequence specificity. Hence, this enzyme belo ngs to the sequence nonspecific DNA binding proteins. The interaction was found to be independent of DNA synthesis. The formation of the RT- DNA complex was not influenced by the presence of either template-comp lementary or noncomplementary dNTPs, indicating that neither DNA polym erization nor binding of the RT to the dNTP affects the stability of t he complex. The gel retardation assay was utilized to examine also the effect of various HIV-1 RT inhibitors (i.e., AZT-TP, ddTTP, TIBO, and 3,5,8-trihydroxy-4-quinolone) on the enzyme-DNA interaction. The resu lts indicate differences in the modes of action of these compounds. Wh ile there was a complete destabilization of the RT-DNB complex in the presence of 3,5,8-trihydroxy-4-quinolone, the addition of AZT-TP, ddTT P, or TIBO had no apparent effect on the stability of the complex. Mos t effective anti-HIV compounds are inhibitors of HIV RT; hence the int eraction of the enzyme with DNA might constitute a discrete step which can serve as a target for interference by novel specific anti-HIV RT drugs.