Drug Targeting of HIV-1 RNA·DNA Hybrid Structures: Thermodynamics of Recognition and Impact on Reverse Transcriptase-Mediated Ribonuclease H Activity and Viral Replication †
ABSTRACT RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.
- SourceAvailable from: Andrew L Feig
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- "This chapter will lead the reader through the sample preparation, binding reaction, and data analysis required for the characterization of an RNA binding to another species. Examples of such experiment include two RNAs binding each other to form duplexes or tertiary structures (Mikulecky and Feig, 2006a; Mikulecky et al., 2004; Reymond et al., 2009; Takach et al., 2004; Vander Meulen et al., 2008), RNAs binding to proteins (McKenna et al., 2006; Niedzwiecka et al., 2004; Recht and Williamson, 2004; Recht et al., 2008), or small molecule ligands (Bernacchi et al., 2007; Gilbert and Batey, 2009; Li et al., 2004). Examples of all three classes of experiments have been measured successfully by ITC. "
ABSTRACT: Isothermal Titration Calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of RNA folding, RNA binding to small molecules, and RNA-protein interactions. The advent of extremely sensitive instrumentation and the increasing availability of ITC in shared facilities have made it increasingly valuable as a tool for RNA biochemistry. As an isothermal measurement, it allows analysis at a defined temperature, distinguishing it from thermal melting approaches (UV melting and differential scanning calorimetry, for instance) that provide thermodynamic information specific to the melting temperature. Residual structures at low temperature in the unfolded state and heat capacity changes lead to potential differences between thermodynamic values measured by ITC and those derived from melting studies. This article describes how ITC can be put to use in the study of RNA biochemistry.Methods in enzymology 01/2009; 468:409-22. DOI:10.1016/S0076-6879(09)68019-8 · 2.09 Impact Factor
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- "The RNA mediated synthesis of DNA was originally proposed  and substantiated  as an integral part of the retroviral life cycle. Expanded to eukaryotic cells , as much as 10% of the eukaryotic genome is a direct product of reverse transcription . Mechanistically similar to the transcription process, reverse transcription relies on the dual activity of reverse transcriptase (RT)  . "
ABSTRACT: Targeting nucleic acids using small molecules routinely uses the end products in the conversion pathway of "DNA to RNA, RNA to protein". However, the intermediate processes in this path have not always been targeted. The DNA-RNA interaction, specifically DNA:RNA hybrid formation, provides a unique target for controlling the transfer of genetic information through binding by small molecules. Not only do DNA:RNA hybrids differ in conformation from widely targeted DNA and RNA, the low occurrence within biological systems further validates their therapeutic potential. Surprisingly, a survey of the literature reveals only a handful of ligands that bind DNA:RNA hybrids; in comparison, the number of ligands designed to target DNA is in the thousands. DNA:RNA hybrids, from their scientific inception to current applications in ligand targeting, are discussed.Biochimie 08/2008; 90(7):1026-39. DOI:10.1016/j.biochi.2008.04.011 · 2.96 Impact Factor
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- "Considering that aminoglycoside ligands bind nucleic acid duplexes with an RNA:RNA > RNA:DNA >> DNA:DNA scale of binding affinities (56,57), the preferential interaction of neomycin B with the wild-type RNA:DNA hybrid provides important evidence regarding the determinants of PPT recognition. The fact that this non-covalent probe manifested greater affinity for the wild-type hybrid than for its duplex RNA analogue diminishes the significance of generic canonical A-form helical structure in determining binding specificity (56), and highlights the importance of helix plasticity in adapting favorable electronegative surfaces for ligand interactions (46). "
ABSTRACT: The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.Nucleic Acids Research 06/2008; 36(8):2799-810. DOI:10.1093/nar/gkn129 · 9.11 Impact Factor