HIV-1 reverse transcriptase (RT) is a multifunctional enzyme responsible for the synthesis of the HIV genome and is essential for the replication of HIV, the causative agent of AIDS. RT is of great medical importance due to its role in the HIV life cycle and as a source of the high mutation rate of HIV. Rapid generation of HIV variants presents one of the greatest obstacles to universally effective treatments for AIDS. PMPA, also known as Tenofovir, is a dNTP substrate analog that inhibits DNA synthesis by RT via chain termination. PMPA is highly specific for RT, resulting in low toxicity to the host. Notably, the emergence of HIV variants resistant to PMPA occurs with much lower frequency and severity than with existing treatments for AIDS. Through the studies outlined in this proposal I hope to obtain basic mechanistic information about polymerase catalysis by capturing """"""""snapshots"""""""" of key polymerization intermediates, in part, by using covalent trapping methods. Specifically X-ray crystallography will be used to obtain three- dimensional structures of HIV RT bound with:1) DNA and PMPA- diphosphate (ternary complex), using wild-type and K65R mutant RT; 2) DNA terminated with PMPA in a pre-translocated complex, and 3) DNA terminated with PMPA in a post-translocation, dead-end complex. The composite picture obtained from these structures will aid in understanding the specificity by which PMPA inhibits RT in particular and the general principles of RT-nucleotide analog interactions that should facilitate the design of more effective treatments for AIDS.
| Tuske, Steve; Sarafianos, Stefan G; Clark Jr, Arthur D et al. (2004) Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir. Nat Struct Mol Biol 11:469-74 |
