SUNY at Albany
June 19-23, 2001
Drug binding to the Packaging Region of HIV-1
The packaging region or Psi-element of HIV-1 spans ~350 nt near the 5' end of the viral genome. The region contains the dimer initiation site (DIS) for formation of genomic-length RNA dimer, the primer binding site for reverse transcription, and unique structural elements to allow incorporation of HIV RNA into the budding virus particle. The importance of Psi-RNA in the life cycle of the virus, and in particular for viral infectivity, suggests that it may be a useful target for AIDS-directed drugs. However, the structures of its monomeric and dimeric forms, and the complex it forms with the nucleocapsid protein are unknown. Previously, we measured monomer-dimer equilibrium constants for various RNA sequences located in DIS and studied the binding of the nucleocapsid protein, NCp7, to stem-loop SL3 in Psi-RNA (1, 2).
Here we describe the results of quantitative footprinting and UV melting experiments using a 176 nt Psi-RNA from HIV-1 (LAI) and a group of aminoglycoside drugs and their analogues. The RNA was prepared by transcribing the appropriate DNA template using T7 RNA polymerase in the presence of the four ribonucleotide triphosphates followed by purification with PAGE. The first derivative of the melting curve revealed transitions at 24.6o, 31.2o, 45.5o, and 54.0o C, indicating that the 176 nt RNA has a complex secondary/tertiary structure. RNA folding programs indicated that the Psi-RNA fragment likely has four stem-loops, SL1-4, and a stem formed by Watson-Crick base pairing of the 5Õ and 3Õ ends of the molecule. Cleavage experiments with RNase I (essentially non-specific) and RNase T1 (specific for unpaired G) confirm this. Autoradiographic data from cleavage experiments in the presence and absence of drug were scanned and intensities for individual cleavage fragments were plotted as functions of drug concentration to yield footprinting plots. These reveal the sites of drug binding and relative values of the binding constants. Enhancements, or increases in cleavage rates with drug concentration, indicate regions of altered structure due to drug binding to the RNA.
For the aminoglycoside drug paromomycin, the highest affinity site on the 176-mer involves positions 235 and 236, in the main stem, and 266-269 and 274, located on one side of SL1 (3). The stem-loop SL1 contains the self complimentary sequence 5Õ-GCGCGC-3Õ which is critical for the formation of the HIV RNA dimer in the packaged form of the genome. Structural changes are induced by paromomycin mainly in the regions joining the stems of the various loops of the Psi-RNA. The conjugate ligand acridine-neomycin B, prepared by Tor and coworkers (4), possesses an intercalating acridine group appended via a disulfide linkage to one of the sugar residues of neomycin B. Analysis of the footprinting data revealed that this ligand binds similarly to paromomycin but sites 266-269 in the stem of SL1 are not involved. Comparison of the footprinting data for acridine-neomycin with that of neomycin alone revealed that the acridine moiety of the former may be bound (intercalated) to sites located in the base of the stem of SL1. We also studied the binding to the Psi-RNA of a disulfide-linked dimer of neomycin B (4). Since the dimer exhibits an initial protection pattern very similar to that of monomeric neomycin B, it appears that its interaction with the 176-mer RNA involves only one of the neomycin groups. The potential of Psi-RNA as a target for AIDS directed drugs will be presented and discussed.
The research was supported by NIH GM32691 to P. N. Borer.References and Footnotes
Mark P. McPike, Jerry Goodisman and James C. Dabrowiak,
Department of Chemistry, 111 College PL, CST 1-014,
Syracuse University, Syracuse, NY 13244-4100