The RT Biochemistry Section combines its long-standing strengths in protein/nucleic acid complexes catalyzing specific steps in HIV replication with the development of new technologies to provide high-resolution information on their potential for therapeutic attack. Priority areas of research include: 1. Protein footprinting by mass spectrometry. 2. Protein and nucleic acid bioconjugate strategies. 3. Nucleic acid interference mapping. 4. Unnatural amino acids as probes of protein structure and function. 5. Drug discovery and efficacy. 1. Protein Footprinting by Mass Spectrometry Although nucleic acid footprinting has proven invaluable in understanding nucleoprotein complexes, an equivalent approach aimed at understanding changes in protein conformation has been lacking, due mainly to the paucity of probing reagents and sensitivity of detection. The former of these can be addressed by employing a variety of amino acid-modifying reagents, while improvements in mass spectrometry have allowed detection and sequencing of peptides in the nanomole to femptomole range. Current research exploits the sensitivity of lysine residues, of which HIV-1 reverse transcriptase (RT) contains 109, to modification with reagents such as pyridoxal phosphate or biotin, followed by resistance of modified residues by endoproteolytic digestion and their detection by mass spectrometry. As with nucleic acid footprinting, RT is treated in the absence and presence of ligand, after which its p66 and p51 subunits (sharing a common primary sequence) are resolved by denaturing polyacrylamide gel electrophoresis, digested in situ with endo-LysC, and examined by MALDI-TOF mass spectrometry. Using this approach, we have evaluated HIV-1 RT bound to the tRNA/vRNA initiation complex, in addition to HIV-1 integrase containing small-molecule antagonists. 2. Protein and Nucleic Acid Bioconjugate Strategies Bioconjugation describes the site-specific tethering of two molecules to generate a novel complex displaying the combined properties of its individual components. We have successfully used this approach to tether the photocrosslinking agent azidophenacyl to the RNase H domain of p66 RT to determine the location of its C terminus relative to nucleic acid duplexes. Current strategies extend these observations to site-specific attachment of chemical nucleases to RT and chemical proteases to structurally diverse nucleic acids encountered during replication. These complementary approaches can be evaluated by high-resolution gel electrophoresis and mass spectrometry, respectively, and provide precise information on the interaction of particular RT domains with nucleic acid in solution. We are planning to use several novel nucleic acids containing chemical proteases/nucleases (e.g., the HIV replication primer tRNALys,3, containing Fe-EDTA or ortho-phenanthroline-Cu at its 5' terminus) to investigate the dynamics of initiation of reverse transcription. In conjunction, a variety of photoactive, fluorescent and nucleolytic agents will be attached at specific sites of the retroviral polymerase to understand the contribution of its subdomains. 3. Nucleic Acid Interference Mapping Using synthetic chemistry, it is now possible to introduce a variety of modified nucleosides into the RNA or DNA component of nucleic acid duplexes to probe regions critical to their interaction with the retroviral replication machinery. We have successfully initiated this approach and will extend it to understand the structural features of the (+) strand polypurine tract (PPT) RNA primer governing its processing from (+) RNA or (+) DNA sequences. As an example, A-tract-induced distortions at two regions of the HIV-1 PPT are formed in the absence of RT, serving to "guide" the RNase H domain to cleave at the appropriate site. By introducing 2,4-difluorotoluene into the DNA template as a thymine isostere, hydrogen bonding is weakened, after which processing of the modified PPT-containing duplex can be followed. With respect to the RNA primer, the adenine analogs 2-aminopurine and 2,6-diaminopurine alter and strengthen the hydrogen bonding pattern, respectively, while the guanine analog inosine weakens this. These analogs and additional modified nucleosides will be introduced into the PPT to better understand the structural deformations underlying its processing. 4. Unnatural Amino Acids as Probes of Protein Structure and Function In contrast to the 20 natural amino acids, unnatural amino acids represent a rich and diverse source of reagents to probe protein structure by analyzing the individual regions of a particular amino acid. An example of this approach, which combines the use of chemically misacylated suppressor tRNAs with efficient cell-free protein-synthesizing systems, is the introduction of aspartic acid analogs (e.g., allo-aspartic acid, dimethyl aspartic acid) into HIV-1 protease to understand the role of the counterpart active site residues. Our laboratory has now successfully introduced the first analogs into HIV-1 RT (2-naphthylalanine) and purified a heterodimer whose p66 subunit is selectively modified. We will continue this approach using newly synthesized bis-aminoacylated suppressor tRNAs. Current work focuses on the introduction of tyrosine analogs (nor-tyrosine, ortho-tyrosine and meta-tyrosine) for p66 tyrosine 501, a critical residue of the "RNase H primer grip" and also involved in resistance to the RNase H antagonist BBNH. 5. Drug Discovery and Efficacy Although disruption of HIV-1 RNase H function destroys virus infectivity, there is still only a handful of RNase H inhibitors in the literature. Nonradioactive approaches are being developed for use in high-throughput screening for potential RNase H antagonists. Two fluorescent substrates have been developed, one of which is now being introduced to screen a library of 100,000 compounds, after which a variety of secondary and tertiary assays are planned. While expanding our search for potent and selective RNase H inhibitors, this project will also provide a rich source of reagents with which we can investigate RNase H function in greater detail. The drug discovery program has been extended to include HIV-1 integrase and combined with high-resolution protein footprinting (discussed above) to determine the site of drug interaction. Isothermal titration calorimetry is being applied to a study of first- and second-generation nonnucleoside-based HIV-1 RT inhibitors.