Regulation of HIV gene expression is crucially dependent on binding of the trans-activator protein, Tat, to the trans-activation response RNA element, TAR, found at the 5′ end of all HIV-1 transcripts. Tat-TAR interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. A new class of small-molecule peptidomimetic TAR RNA binders, conjugates of aminoglycosides and arginine, was recently designed [Litovchick, A., Evdokimov, A. G., and Lapidot, A. (1999) FEBS Lett. 445, 73-79]. Two of these compounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-arginine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transported and accumulated in cell nuclei. Here we present a detailed synthesis and chemical characterization of the aminoglycoside-arginine conjugates R3G and R4K as well as GB4K, the tetra-γ-guanidinobutyric derivative of kanamycin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitrate, and lead acetate footprinting. The conjugates interact with TAR RNA in the widened major groove, formed by the UCU bulge and the neighboring base pairs of the upper stem portion of TAR, the binding site of Tat protein, and Tat-derived peptides (e.g., R52). Our results suggest an additional binding site of R4K and R3G compounds, in the lower stem-bulge region of TAR. The antiviral activity of the conjugates in cultured equine dermal fibroblasts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.The trans-activation responsive RNA (TAR) 1 region of HIV long terminal repeat (LTR) regulates the viral gene expression via interaction with the HIV trans-activator protein, Tat, and thus is an attractive target for drug design strategies (1). TAR is found at the 5′ end of all HIV-1 transcripts. It adopts a hairpin secondary structure consisting of a highly conservative hexanucleotide loop and a threenucleotide bulge flanked by two double-stranded stems (2). TAR is a positive enhancer that stimulates the synthesis of productive transcripts. It is unique in terms of eukaryotic transcription control because it only functions as an RNA element. The activation by Tat is entirely dependent on the presence of the TAR RNA sequence. Tat activates expression by specific binding to TAR, which increases viral mRNA production several hundredfold by stimulation of the elongation capacity of RNA polymerase II (3). HIV Tat binds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1 LTR promoter. This process requires binding of Tat to the TAR bulge and of cyclin T to the TAR loop. The cyclin T-associated CDK9 kinase then induces phosphorylation of the C-terminal domain of RNA polymerase II, and of other polymerase II-associated proteins, leading to the transition from nonprocessive to processive transcription (4).Binding of Tat protein to TAR is mediated ...