Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance

doi:10.1073/pnas.0609642104

Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance

*Viral Mutation Section and
^†Host-Virus Interaction Unit, HIV Drug Resistance Program, and
^‡Data Management Services, Inc., National Cancer Institute, Frederick, MD 21702

Contributed by John M. Coffin, October 30, 2006 (received for review September 24, 2006)

Abstract

We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3′-azido-3′-deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.

Footnotes

^§To whom correspondence may be addressed. E-mail: john.coffin{at}tufts.edu or vpathak{at}ncifcrf.gov
Author contributions: G.N.N., K.A.D.-F. contributed equally to this work; G.N.N., K.A.D.-F., and V.K.P. designed research; G.N.N. and K.A.D.-F. performed research; S.P. contributed new reagents/analytic tools; G.N.N., K.A.D.-F., S.P., F.M., M.J.F., J.M.C., and V.K.P. analyzed data; and G.N.N., K.A.D.-F., and V.K.P. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609642104/DC1.
Abbreviations:

AZT,

3′-azido-3′-deoxythymidine;

cn,

connection domain;

d4T,

2,3-didehydro-2,3-dideoxythymidine;

NRTI,

nucleoside reverse transcriptase inhibitor;

pol,

polymerase;

rh,

RNase H;

RT,

reverse transcriptase;

TAM,

thymidine analog resistance mutation.