Mutational Patterns Associated with Reduced and Increased Susceptibility to NcRTI in >6000 Clinical HIV-1 Isolates
Dirk Jochmans*1, H Van Marck1, M Van Ginderen1, I De Baere1, P Dehertogh1, A Peeters1, B Kesteleyn1, T Pattery2, P Mc Kenna2, and K Hertogs1
1Tibotec, Mechelen, Belgium and 2Virco, Mechelen, Belgium
Background: Nucleotide-competing reverse transcriptase
inhibitors (NcRTI) are potent inhibitors of HIV replication with a unique
mechanism of action. They bind the active site of RT and compete with the next
incoming nucleotide. To further investigate the effect of RTI-resistance-associated
mutations on the activity of NcRTI, the susceptibility of >6000 recent
clinical isolates for a prototype compound (NcRTI-1, EC50 for wild
type HIV-1 = 30 nM) was determined.
Methods: Virological methods included phenotyping,
genotyping, constructing site-directed-mutant (SDM) strains and in vitro resistant virus selection.
Results: More than 80% of the profiled clinical
isolates remained susceptible for NcRTI-1 (fold change in EC50 [FC]
<4). No cross-resistance was observed between NcRTI-1 and efavirenz (EFV),
nevirapine (NVP), tenofovir (TDF), zidovudine (AZT), stavudine (d4T), abacavir
(ABC), zalcitabine (ddC), or didanosine (ddI). Some cross-resistance with
lamivudine (3TC) and emtricitabine (FTC) could be detected (Pearson Correlation
Coefficient = 0.59). Analysis of the genotype of >1700 of these viruses
showed that the combination of M184V + Y115F correlated most with reduced
susceptibility to NcRTI-1. This was confirmed in SDM strains: single mutations M184V and Y115F showed a FC
of 5.0 and 7.9, respectively. The combination of both resulted in a FC of 39.
Analysis of the dataset also indicated that the K65R mutation is associated
with hypersusceptibility to NcRTI-1 and that it reverses M184V-induced
resistance. This was confirmed using a M184V + K65R SDM strain (FC = 0.63). The
interplay between K65R and M184V was also explored by replicating wild type
HIV-1 in the presence of both NcRTI-1 and TDF. This showed that, in contrast to
3TC or ABC, NcRTI-1 prevents the selection of K65R. Further breakdown of the
dataset showed that NcRTI-1 activity is not influenced by the presence of
thymidine analog mutations (TAM), the 69ins-MDR complex, the Q151M-MDR complex,
or mutations associated with NNRTI-resistance.
Conclusions: Analysis of HIV-1 clinical isolates and
SDM strains showed that the combination of RT mutations M184V and Y115F is
associated with a decreased susceptibility to NcRTI. In addition, amino acid
change K65R was found to restore the susceptibility of M184V carrying viruses
to NcRTI. These data, together with the observation that neither TAM, nor the
69ins-MDR complex, nor the Q151M-MDR complex influences susceptibility to NcRTI,
warrant further exploration of this novel class of HIV inhibitors.