Dimitrios Coutsinos, Cedric F' Invernizzi, Hongtao Xu, Bluma G' Brenner and Mark A' Wainberg

1
In vitro Molecular Characterization of the
Development of the K65R and M184V Mutations in
Subtype C HIV-1
Dimitrios Coutsinos, Cedric F. Invernizzi,
Hongtao Xu, Bluma G. Brenner and Mark A.
Wainberg McGill University AIDS Centre Jewish
General Hospital Montreal, Canada
XVI International HIV Drug Resistance
Workshop Barbados, June 12-16 2007
2
Signature Drug Resistance Patterns in Subtype C
HIV-1

• Subtype C viruses are more prone to develop
  V106M
• - 40 of EFV-experienced patients with subtype C
• - 2 of EFV-experienced patients with subtype B
• K65R is selected more rapidly in subtype C than
  B isolates in CBMC cell culture.
• - 6 to 8 weeks for Subtype C
• - 43 weeks for Subtype B
• Clinical data show a higher frequency of K65R in
  ddI-experienced patients from Botswana.

(Brenner, B et al. AIDS. 2003 17 F1-F5)
(Morris, L et al. AIDS. 2003 17
1698-1700) (Grossman, Z et al. AIDS. 2004 18
909-915)
(Brenner, B et al. AIDS. 2006 20 F9-F13)
(Doualla-Bell, F et al. Antimicrob Agents
Chemother. 2006 50 4182-4185)
3
K65R Drug Resistance Pathway in Subtype C HIV-1

• There is no obvious reason to anticipate subtype
  differences in treatment responses with
  combination ART
• - Studies have shown no evidence of higher rates
  of K65R development among patients with non-B
  subtypes or with subtype C HIV-1 receiving ART.
• - Subtype C RT enzymes are very similar to
  subtype B RT enzymes with respect to initiation
  and synthesis of (-)ssDNA, TFV incorporation and
  ATP-mediated excision.

(Miller, MD et al. AIDS. 2007 21 265-266)
(DART Virology Group and Trial Team. AIDS. 2006
20 13911399)
(Coutsinos, D et al. 14th CROI, Los Angeles, 2007
Poster no.585)
4
Rationale
Subtype B HIV
64 65 66
183 184 185

• 5- AAG AAA AAA TAC ATG GAT -3

Wild-Type
5- AAG AGA AAA TAC GTG GAT -3
Resistant
Subtype C HIV
5- AAA AAG AAA TAT ATG GAT -3
Wild-Type
5- AAA AGG AAA TAT GTG GAT -3
Resistant
To elucidate the mechanistic propensity for
acquisition of K65R and M184V in B and C subtypes
using templates derived from the pol nucleotide
sequence
5
Reverse Transcription Leading to K65R in Subtype
C and B
(Subtype C) 5- GCU AUA AAA AAG AAA GAC AGC
-3 (Subtype B) 5- GCC AUA AAG AAA AAA GAC
AGC -3
64 65 66
()RNA
(Subtype C) 5- ACT GTC TTT CTT TTT TAT AGC
-3 (Subtype B) 5- ACT GTC TTT TTT CTT TAT
GGC -3
66 65 64
(-)ssDNA
64 65 66
(Subtype C) 5- GCC ATA AAA AAG AAA GAC AGT
-3 (Subtype B) 5- GCC ATA AAG AAA AAA GAC
AGT -3
()dsDNA
6
K65R Acquisition (-)ssDNA Synthesis from ()RNA
Subtype B RT/Template
Subtype C RT/Template

• There are no differences that explain the
  distinct patterns of K65R acquisition between
  both subtypes.

7
K65R Acquisition ()dsDNA Synthesis from (-)ssDNA
Subtype B RT/Template
Subtype C RT/Template

• In subtype C, pausing is seen at the exact
  nucleotide responsible for the AAG to AGG
  mutation which gives rise to K65R.

8
K65R Acquisition ()dsDNA Synthesis from (-)ssDNA
Subtype C RT Subtype B Template
Subtype B RT Subtype C Template

• The differential patterns of DNA synthesis
  between subtypes are defined by the template
  sequence rather than the enzyme used.

9
M184V Acquisition (-)ssDNA Synthesis from ()RNA
Subtype B RT/Template
Subtype C RT/Template

• There are no differences that explain the
  distinct patterns of M184V acquisition between
  both subtypes.

10
M184V Acquisition ()dsDNA Synthesis from
(-)ssDNA
Subtype B RT/Template
Subtype C RT/Template

• There are no differences that explain the
  distinct patterns of M184V acquisition between
  both subtypes.

11
Conclusions

• Subtype-specific differences exist
• Pausing is seen at the 65 region with subtype C
  but not subtype B template.
• - Rapid emergence of K65R in subtype C RT appears
  to be based on the pol nucleotide sequence.
• Emergence of M184V is not favored by the pol
  gene nucleotide sequence in both subtype B and C.
• Clinical relevance of these observations
  requires long-term follow-up of patients on
  antiretroviral nucleosides in subtype-C endemic
  areas.

12
Acknowledgements

• Mark A. Wainberg
• Bluma Brenner
• Cédric Invernizzi
• Hongtao Xu
• Daniela Moisi
• Maureen Oliveira
• All the members of
• the Wainberg lab
• Matthias Götte