1. Kalyan Das CABM & Rutgers University, NJ, USA
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
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
Binding
Incorporation
Excision 1.

3. AZT-resistance Mutations
ATP’
AZTTP
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 J. Virol. 75:4832
There was no clue of the role of AZT-resistance mutations for almost 10 years till Mayer et al discovered the excision characteristic of RT.

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) 1.

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

6. Binding of AZTppppA to AZTr RT/dsDNA Complex
Primer
AppppAZT
R72
T215Y
K70R
K65
T215Y
K70E mutation reduces excision whereas, K70R enhances excision.
K70R
K70R and T215Y are Excision Enhancing Mutations (EEMs)

7. ATP binds differently to wild-type and EEM RT
T/Y215
K/R70
K65
R72
YMDD    '
DNA primer
template
AZTppppA (I)
AZTppppA (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
Y215
R70
Site I
Site II
ATP’
Wild-type RT does not have high specificity for ATP binding
Superposition of wt HIV-1 RT (green ribbon)/AZTppppA (gray) on G5-mutant RT (cyan/gray ribbon)/AZTppppA (yellow) complexes. The mutated amino acid residues R70 and Y215 apparently enhance binding of ATP( the preferred substrate for AZT excision) at the site II.
The molecular surface of G5-mutant RT shows both the sites for ATP-binding. The G5-mutant does not alter the conformation of Site I, rather, generates Site II, which has stronger affinity for ATP than Site I.
Summary of the interactions of AZTppppA in the wild-type (A) and AZTr (B) HIV-1 RT excision product complexes. Residues are not shown; these residues lie on the bottom of the AZTppppA-binding site in both wild-type and AZTr excision product complexes. The main-chain amide nitrogens of these amino acids are positioned to interact with the negatively charged phosphates of AZTppp-, and may help AZTppppA to bind to RT.
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 1.

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
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 primer
DNA template
palm
p51
p66

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

12. 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?
Y115
Q151
R72
R65

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

14. dATP and TFV-DP show alternate R65/R72 rotameric conformations
Y115
dATP   
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
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. dATP/AZT Y115 V184 R72 R65 Y215 ATP’ R70 3TC/FTC resistance site TFV
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 Xiongying Tu Rajiv Bandwar NIH funded
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