The RT Biochemistry Section seeks to understand, through protein and nucleic acid mutagenesis, how HIV-1 reverse transcriptase (RT) interacts with the conformationally distinct nucleic acid duplexes encountered while converting the single-stranded RNA genome into an integration-competent double-stranded DNA. Using a combination of methods, we are investigating the synthetic (RNA- and DNA-dependent polymerase) and degradative [ribonuclease H (RNase H)] properties of this multifunctional retroviral enzyme, which continues to be a major target for development of therapeutic agents to inhibit HIV-1 replication and stem the progression of AIDS. (+) Strand DNA synthesis in retroviruses and LTR-retrotransposons initiates from the polyrpurine tract (PPT), the precision of which is critical to the integrity of the 5' LTR and its recognition by the viral integration machinery. Although PPT utilization has been studied with respect to alterations in RT or the sequence of the element, the molecular basis underlying this event remains obscure. Two recent studies from the RT Biochemistry Section have shed light on this processing, suggesting that the PPT actively participates in its processing by sequestering RT in an orientation placing the RNase H catalytic center over the biologically relevant processing site. Nucleoside analogs have been employed to understand the structural basis of PPT recognition in HIV-1 and the LTR-retrotransposon Ty3. These include: (a) thymine and cytosine isosteres, which locally remove hydrogen bonding (Rausch and Le Grice, 2004; Rausch et al., 2003; Lener et al., 2003); (b) locked nucleic acids, which locally decrease flexibility (Dash et al., J. Biol. Chem. 2004); (c) pyrrolo-dC, whose fluorescence is decreased in the double-stranded state (Dash et al., Nucleic Acids Res. 2004); and (d) abasic tetrahydrofuran lesions, which locally remove the base while preserving the sugar-phosphate backbone (Yi-Brunozzi & Le Grice, 2005). Collectively, these studies have highlighted important protein-nucleic acid interactions controlling PPT recognition. NMR studies have been initiated to understand the structural basis for PPT recognition in retroviruses and LTR-retrotransposons, suggesting that sugar pucker may play an important role in these events.
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