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Mechanism by which N348I in HIV-1 Reverse Transcriptase Confers Dual Zidovudine/Nevirapine Resistance
Soo-Huey Yap*1,2, J Radzio3,4, N Sluis-Cremer3, and G Tachedjian1,5
1Burnet Inst, Melbourne, Australia; 2Monash Univ, Churchill, Australia; 3Univ of Pittsburgh Sch of Med, PA, US; 4Univ of Pittsburgh Grad Sch of Publ Hlth, PA, US; and 5Monash Univ, Clayton, Australia
Background: The efficacy of combination therapies
that include zidovudine (AZT) and nevirapine (NVP) for the treatment of HIV-1
infection can be explained, in part, by the observed synergy between these
drugs and the antagonism between their respective resistance mutations (e.g.,
K70R for AZT and Y181C for NVP). The molecular mechanism of synergy between
these 2 drugs is complex and involves a direct effect of NVP on the chemistry
of AZT-MP excision, and also a NVP-induced stimulation of reverse transcriptase
(RT) RNase H activity which indirectly affects AZT-MP excision by prematurely
destroying the RNA/DNA duplex. We recently described the identification of
N348I, which occurs early in AZT/NVP based therapies and confers resistance to
both drugs. The objective of this study was to determine the biochemical
mechanisms responsible for this phenotype.
Methods: The AZT-MP excision activity of wild type and
mutant (N348I, K70R, Y181C) HIV-1 RT was assessed in the absence and presence
of NVP using different, but complementary, biochemical assay systems. The
effects of the resistance mutations, alone and in combination with NVP, on
RNase H activity of the RT were also investigated.
Results: N348I confers dual AZT/NVP resistance via 2
inter-related mechanisms. In the first, N348I decreases the ability of NVP to
inhibit HIV-1 RT. In the second, N348I significantly decreases the rate of RT
RNase H cleavage, which provides RT with more time to efficiently excise AZT-MP
from an RNA/DNA template/primer. Furthermore, the ability of NVP to stimulate
RNase H is also significantly reduced compared with the wild type enzyme.
Therefore, when both AZT-TP and NVP are combined in RNA-dependent DNA
polymerization reactions, the combined effects of N348I on NVP binding, RNase H
activity, and AZT-MP excision activity allows the enzyme to replicate
relatively efficiently. In comparison, the K70R mutations confer AZT resistance
but remains sensitive to NEV inhibition, and Y181C confers NVP resistance, but
is hyper-sensitive to inhibition by AZT.
Conclusions: This study defines the mechanism by
which N348I confers dual AZT/NVP resistance and explains why this mutation may
be selected in response to therapies containing these drugs. Furthermore, it
expands our knowledge of how mutations, distal from the polymerase active site
and NNRTI-binding pocket, can confer drug resistance.
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