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Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir

This study explores the structural basis for HIV-1 reverse transcriptase (RT) drug resistance to zidovudine (AZT) and tenofovir. It investigates NRTI inhibition and resistance, excision mechanisms, and the impact of specific mutations on drug discrimination and viral fitness.

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Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir

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  1. Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir Kalyan Das CABM & Rutgers University, NJ, USA

  2. NRTI - Inhibition and Resistance • NRTI Inhibition • Nucleoside/nucleotide analog • Gets incorporated at the DNA-primer terminus by RT and acts as a chain terminator • NRTI Resistance • Different RT mutations or sets of mutations emerge in response to different NRTIs • A mutant RT has the ability to discriminate the drug from normal nucleotides • Discrimination can occur @ • Binding • Incorporation • Excision

  3. AZTTP ATP’ AZT-resistance Mutations • AZT-MP gets incorporated • RT removes AZT-MP by excision • Excision is reverse of polymerization • ATP is the primary excision substrate in vivo • ATP excises AZT-MP to form AZTppppA Meyer et al. 1998, PNAS 95:1347 Meyer et al. 1999, Mol. Cell 4:35 Boyer et al. 2001. J. Virol. 75:4832

  4. Methods Five crystal structures were determined • wt RT/dsDNA/AZTppppA (3.1 Å resolution) • AZTr RT/dsDNA/AZTppppA (3.2 Å) • AZTr RT/dsDNA terminated with AZTMP at N-site (3.6 Å) • AZTr RT/dsDNA terminated with AZTMP at P-site (2.9 Å) • apo AZTr RT (2.6 Å)

  5. primer primer primer primer template template template template dTTP dTTP D67N D67N T215Y T215Y K70R K70R M41L M41L K219Q K219Q palm palm palm palm fingers fingers fingers fingers AZTppppA dNTP Incorporation and AZT-Resistance Mutations

  6. T215Y K70R Binding of AZTppppA to AZTr RT/dsDNA Complex Primer AppppAZT R72 K65 T215Y K70R K70R and T215Y are Excision Enhancing Mutations (EEMs)

  7. DNA primer YMDD AZTppppA (I)  template  ' R72  ' T/Y215 K65 AZTppppA (II) K/R70 Site I b3  ATP’ R70 Y215 Site II ATP binds differently to wild-type and EEM RT The ATP as an excision substrate binds differently to wild-type RT and EEM/TAM RT Wild-type RT does not have high specificity for ATP binding The mutations create a new ATP-specific binding site

  8. K65R Background • K65R is an NRTI resistance mutation in HIV-1 RT: • Selected by TDF, ABC, ddI, and occasionally d4T • Observed in 2-5% of antiretroviral-experienced patients • Low-level resistance to all NRTIs, with the exception of AZT which remains susceptible

  9. K65R Background • K65R biochemical functions: • Decreases incorporation rate (kpol) of dNTPs and NRTIs • Decreases NRTI excision • Increases fidelity • Decreases viral replication capacity

  10. Structures of K65R RT/dsDNA/TFV-DP (3.0 Å; R 0.251; R-free 0.284) K65R RT/dsDNA/dATP (3.3 Å; R 0.254; R-free 0.286) thumb RNase H fingers TFV-DP/ dATP DNA template DNA primer palm p51 p66

  11. dNTP Binding Site primer : template dNTP K65 b3     palm fingers

  12. Y115 Y115 Q151 Q151 R72 R72 R65 R65 K65R and R72 form a Molecular Platform Like K65 in wt RT structures, R65 also interacts with the -phosphate. The guanidinium planes of arginines at positions 65 and 72 stack to form a Molecular Platform. R72 is highly conserved; mutations at R72 impair RT polymerization. How does the platform discriminate TFV-DP from dATP?

  13. Binding of dATP and TFV-DP to K65R RT Q151 Y115  b3 R72 1.7 R65 TFV-DP dATP

  14. Y115 R72 N N   N N R65  dATP dATP and TFV-DP show alternate R65/R72 rotameric conformations Y115 R72 N N   N R65  TFV-DP

  15. K65R mutation: • Does not significantly alter interaction of residue 65 with dNTP • Forms a Molecular Platform with R72 that may work as a “Check Point” • Reduces dNTP incorporation • Reduces NRTI excision • Increases fidelity 3. The platform has alternate rotameric conformations when TFV-DP vs. dATP binds - Causes discrimination of TFV-DP from dATP

  16. K65R and Excision Enhancing Mutations • K65R: • Decreases excision • Increases AZT susceptibility • K65R and EEMs (TAMs): • Antagonistic for mutation development

  17. K65R and M184V • M184V is a primary mutation emerges against 3TC and FTC • M184V with K65R • Increases resistance to ABC and ddI • Partial re-sensitization to TFV

  18. 3TC/FTC resistance site TFV resistance site Excision Enhancing Mutations dATP/AZT Y115 V184 AZT- TP R72  Mg2+ ' ATP’ R65 ' Y215 ATP’ ' R70

  19. Conclusions • EEMs create a site for binding ATP as excision substrate • K70R and T215Y help ATP binding • K65R forms a molecular platform that is responsible for • selective NRTI resistance, reduced dNTP incorporation, reduced excision and reduced viral fitness • The K65R/R72 platform cross-talks with other NRTI resistance mutations • With M184V across the substrate ribose ring • Negatively with L74V through the templating base • Negatively with EEMs (K70R and T215Y)

  20. Acknowledgements Rutgers University CABM Xiongying Tu Rajiv Bandwar Arthur D. Clark, Jr. Joseph Bauman Stefan Sarafianos Steve Tuske Eddy Arnold Chemistry Qianwei Han Barbara L. Gaffney Roger A. Jones Gilead Sciences, Inc. Kirsten White Joy Feng Michael Miller HIV DRP Paul L. Boyer Stephen H. Hughes NIH funded

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