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Background:  Next to the diketo acids (DKA), selective inhibitors of the strand transfer step of the HIV-1 integration process (integrase strand transfer inhibitors, INSTI), the pyranodipyrimidines (PDP) were identified as a second class of authentic integrase (IN) inhibitors. V-165 is the most potent congener. PDP interact with the binding of IN to the DNA and are thus referred to as integase binding inhibitors (INBI).

Methods:  We have now studied the development of antiviral resistance to V-165 by growing wild type HIV-1 strains in the presence of increasing concentrations of this compound. The selected strains were analysed genotypically and phenotypically. Mutant integrase enzymes were generated and evaluated in an enzymatic oligonucleotide based assay and by fluorescence correlation spectroscopy for their activity, affinity for DNA and susceptibility to the different IN inhibitors. In addition, the replication kinetics of the different selected strains have been investigated.

Results:  The IN mutation V165I was identified in a selected strain starting from HIV-1(III_B), whereas the double mutation T206S/S230N emerged in a selected strain starting from HIV-1(NL4.3). The mutant viruses showed a > 9-fold reduction in susceptibility to V-165. Interestingly, a minor phenotypic cross-resistance to DKA was observed. The enzymatic activity of the V165I and T206S/S230N mutants was reduced by 2-fold as compared to wild type IN activity. Although the mutant viruses were resistant to inhibition by V-165, the corresponding enzyme displayed only a 2- to 3-fold reduction in sensitivity to V-165. The replication kinetics of the selected strains pointed to a hampered replication due to the IN mutations.

Conclusions:  Passaging of HIV-1 in the presence of V-165 resulted in V165I and T206S/S230N mutations in HIV IN. These mutations were associated with reduced replication capacities of the selected virus strains. We confirmed that these mutations are important for the interaction of PDP with HIV-1 IN. Apparently, mutations in the PDP binding site interfere with the enzymatic activity of HIV-1 IN. This may explain the difficulty in selecting PDP-resistant strains in cell culture.

Keywords: Resistance; Pyranodipyrimidines; integrase