Selection and characterization of HIV-1 showing reduced susceptibility to the non-peptidic protease inhibitor tipranavir

Tipranavir is a novel, non-peptidic protease inhibitor, which possesses broad antiviral activity against multiple protease inhibitor-resistant HIV-1. Resistance to this inhibitor however has not yet been well described. HIV was passaged for 9 months in culture in the presence of tipranavir to select HIV with a drug-resistant phenotype. Characterization of the selected variants revealed that the first mutations to be selected were L33F and I84V in the viral protease, mutations which together conferred less than two-fold resistance to tipranavir. At the end of the selection experiments, viruses harbouring 10 mutations in the protease (L10F, I13V, V32I, L33F, M36I, K45I, I54V, A71V, V82L, I84V) as well as a mutation in the CA/SP1 gag cleavage site were selected and showed 87-fold decreased susceptibility to tipranavir. In vitro, tipranavir-resistant viruses had a reduced replicative capacity which could not be improved by the introduction of the CA/SP1 cleavage site mutation. Tipranavir resistant viruses showed cross-resistance to other currently approved protease inhibitors with the exception of saquinavir. These results demonstrate that the tipranavir resistance phenotype is associated with complex genotypic changes in the protease. Resistance necessitates the sequential accumulation of multiple mutations.