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N348I in HIV-1 Reverse Transcriptase Counteracts the Antagonism between Thymidine Analogue Mutations and Y181C
Jessica Radzio*1,2, S-H Yapp3, R Harrigan4, G Tachedjian3, and N Sluis-Cremer2
1Univ of Pittsburgh Grad Sch of Publ Hlth, PA, US; 2Univ of Pittsburgh Sch of Med, PA, US; 3Macfarlane Burnet Inst, Melbourne, Australia and Monash Univ, Churchill, Australia; and 4BC Ctr for Excellence in HIV/AIDS, Vancouver, Canada
Background: The efficacy of combination antiretroviral
therapies that include both zidovudine (AZT) and nevirapine (NVP) is due, in
part, to the antagonistic interactions between thymidine analog mutations (TAM)
and Y181C. In this regard, we recently reported that patients
treated with AZT and NVP were 2.6 times more likely to acquire an N348I
mutation in the connection domain of HIV-1 reverse transcriptase (RT) than
patients not treated with these drugs, and furthermore, that this mutation was
highly associated with both TAM and Y181C. The goal of this study was to
determine whether N348I counteracted the antagonism between TAM and Y181C.
Methods: The AZT and NVP susceptibilities of wild-type,
N348I, K70R, Y181C, K70R/N348I, Y181C/N348I, K70R/Y181C, K70R/Y181C/N348I,
M41L/T215Y, M41L/T215Y/N348I, M41L/T215Y/Y181C, and M41L/T215Y/Y181C/N348I
HIV-1 virus or RT were investigated using antiviral or biochemical assays.
Results: As expected, the addition of Y181C to molecular
clones of HIV-1 that included the K70R or M41L/T215Y mutations restored viral
sensitivity to AZT. However, viruses that contained Y181C, TAM, and N348I
displayed significant levels of AZT resistance. The antagonism between Y181C and
TAM was also observed in biochemical assays using purified, recombinant RT on
both DNA/DNA and RNA/DNA template/primers. The N348I mutation in RT
counteracted this antagonism on RNA/DNA, but not DNA/DNA, template/primers, a
phenotype that was due to N348I decreasing the enzyme’s RNase H activity. In addition,
we demonstrate that RT-containing N348I can synthesize DNA more efficiently
when both AZT and NVP are included in the assay as compared to RT that contain
TAM, Y181C, or TAM and Y181C.
Conclusions: These studies demonstrate the ability of N348I
to counteract the antagonism between TAM and Y181C in both biochemical and
cell-based assays. These data represent experimental evidence of the
restoration of the AZT-resistance phenotype in the presence of an antagonistic
mutation, suggesting that N348I may represent a novel pathway to dual AZT-NNRTI
resistance.
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