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5th International Workshop on HIV Drug Resistance & Treatment Strategies4-8 June 2001, Scottsdale, Arizona |
Diketo acids are potent and selective inhibitors of HIV- 1 integrase-mediated strand transfer in vitro and in infected cells and thus are effective as inhibitors of HIV-1 replication. We previously reported on HIV-1 resistance selection using two related compounds. These studies demonstrated that loss of susceptibility required the acquisition of integrase active site mutations, T66I, S153 and M154I, and that combinations of 66 and either 153 or 154 were required. Related analogues with significantly improved potency have now been identified and one of these analogues was used to follow the evolution of integrase in response to increasing selective pressure with time. Active site mutations in integrase were acquired sequentially as follows: (i) S153Y; (ii) T66I and S153Y; and (iii) S153Y and S155N with concomitant loss of T66I. Viruses containing these mutations were constructed and susceptibility to a panel of related inhibitors and relative infectivity compared. As anticipated, each successive mutation resulted in an increased loss of susceptibility to the selecting agent: WT = S153Y (1×) <T66I/S153Y (6×) <S153Y/N155S (70×). Increased resistance, however, was accompanied by a loss of replicative capacity as measured in a single-cycle infection assay: WT = S153Y (1×) >T66I/S153Y (0.14×) >S153Y/N155S (0.05×). Viruses constructed with all three active site mutations were not viable, thus explaining the observed absence of T66I in the context of the S153Y/N155S variant. In addition, the decrease in fitness observed in a single-cycle assay translated into a more profound defect in a multiple cycle infection and in the absence of continued selective pressure, the resistant genotype was not stable and revertants were rapidly obtained. The parallel between increasing resistance and decreasing fitness observed in the HIV-1 viral constructs was recapitulated in studies of integrase enzymatic activity, but only when Mg (and not Mn) was provided as the divalent cation cofactor. These studies provide the first evidence that Mg is the relevant divalent cation for integration in vivo, provide additional evidence validating integrase as an important and viable therapeutic target for HIV-1 and suggest fitness is an important factor influencing the acquisition of resistance to these novel antiretroviral agents.
PRESENTING AUTHOR: D Hazuda
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