Background:  Nucleotide-competing reverse transcriptase inhibitors (NcRTI) are a novel class of HIV-1 reverse transcriptase inhibitors. Despite their non-nucleotidic chemical structure, they inhibit reverse transcriptase by binding its active site in competition with the next incoming nucleotide. This novel mechanism of action translates in a unique resistance profile. In earlier selection experiments using a low multiplicity of infection, mutation M184V was most often selected. Here we investigate the mutational pattern that appears when a high multiplicity of infection is used for in vitro selection in the presence of the prototype compound NcRTI-1 (EC₅₀ = 30 nM).

Methods:  In 4 independent experiments, a high multiplicity of infection (>1 CCID₅₀/cell) of wild type HIV-1 was replicated in the presence of constant concentrations of NcRTI-1.

Results:  At NcRTI-1 concentrations of 170xEC₅₀, a virus with mutation L234F in the HIV reverse transcriptase gene was selected. Antiviral testing showed that L234F is associated with a high (>100-fold) change in NcRTI-1 susceptibility and a moderate change in nevirapine susceptibility (<10-fold). The efficacy of other nucleoside reverse transcriptase inhibitors (NRTI) and non-NRTI (NNRTI) remained unaffected. The change in susceptibility for NcRTI-1 was confirmed using purified enzyme. Interestingly, recombinant L234F RT showed a defect in dimerization.

When L234F virus was grown in the presence of increasing concentrations of NNRTI, known NNRTI-resistance-associated mutations were selected. Importantly in 3 of 4 experiments the L234F mutation reverted to wild-type.

Conclusions:  In the presence of a high viral input, a novel mutation in the HIV reverse transcriptase gene was selected by NcRTI-1. Presence of L234F resulted in a considerable reduction in NcRTI-susceptibility, and a less pronounced change in susceptibility for nevirapine. This is not unexpected, since position 234 is located in the primer grip of the reverse transcriptase p66 subunit, close to the NNRTI pocket. No cross-resistance with other reverse transcriptase inhibitors was observed. Replication of the selected virus in the presence of NNRTI showed the reversal of the L234F mutation to wild type. This could indicate that L234F interferes with the replication capacity of the virus and/or that it is incompatible with canonical NNRTI mutations. The biochemical mechanism of NcRTI-resistance by L234F and its relevance in the clinical setting remains to be clarified. But the fact that this mutation is only selected by NcRTI further underscores the unique features of this novel class of HIV reverse transcriptase inhibitors.