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18th International HIV Drug Resistance WorkshopBasic Principles & Clinical ImplicationsJune 9–13 2009, Fort Myers, Florida, USA |
HIV-1 REVERSE TRANSCRIPTASE FIDELITY VARIANTS ALTER LEVELS OF MUTATION DURING REPLICATION
Antivir Ther 2009; 14 Suppl 1:A24 (abstract no. 22)
DV Nissley1,2, O Alptürk3, Z Ambrose3, K Eaton2, JN Strathern2 and N Sluis-Cremer3
1BSP, SAIC-Frederick, Inc., NCI-Fredrick, Frederick, MD, USA; 2GRCBL, NCI-Fredrick, Frederick, MD, USA; 3University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
BACKGROUND: HIV-1 generates genetic diversity through error prone reverse transcription at sufficient levels to respond to environmental challenges while maintaining efficient replication. Advantageous mutations acquired via replication make it possible for HIV-1 to evade host defense systems, avoid immune response and develop antiviral drug resistance. Viral diversity may be generated by cellular RNA polymerase during transcription of the provirus or by the RNA- and DNA-dependent polymerase activities of HIV-1 reverse transcriptase (RT) during cDNA synthesis. Because HIV-1 RT lacks a conventional proofreading activity and has been reported to replicate nucleic acids with low fidelity, it has been assumed that most diversity is generated during reverse transcription.
METHODS: Previously, we identified error prone HIV-1 RT variants by mutating HIV-1 RT and screening for fidelity variants in a genetic reverse transcription/fidelity assay. In this system, RT activity is uncoupled from viral replication and is monitored in a retro-element with a reverse transcription indicator gene while fidelity is measured by reversion of an inactive fidelity indicator gene. Fidelity variants were characterized using purified enzymes and viral replication assays.
RESULTS: RT variants that affect the fidelity of reverse transcription map to both expected and unexpected regions of HIV-1 RT. Fidelity variants that cluster in the fingers region (V60A, A62T, I63K and F77S) most likely alter nucleotide binding and specificity. p51 subunit β7/β8 loop variants (T131I and I132L) may be exerting a long-range effect on nucleotide discrimination at the active site as has been demonstrated previously for I132M, which confers hypersensitivity to nucleoside analogs. Variants present in the αA helix (C38S and E42K) may alter the interaction between RT and template nucleic acid as it feeds into the active site. Several of the fidelity variants have been shown to misincorporate nucleotides using biochemical and retrotransposition assays, and some compromise viral replication.
CONCLUSION: We characterized HIV-1 RT variants that increase mutation frequency, alter the mutational spectrum and affect viral replication. These fidelity variants map to multiple regions in RT and expand the repertoire of enzyme:nucleic acid interactions that determine the quality of replication. These variants may be useful to compare the evolution of drug resistance in low fidelity RT viruses with that of wild-type and to determine whether fidelity variants play a role in generating drug resistance.
ACKNOWLEDGEMENTS: Funded in part by NCI Contract N01-CO-12400.
2009-06-09
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