Background:  Atazanavir (ATV) is a potent, once daily HIV protease inhibitor (PI) recently approved for the treatment of HIV-1 infections, which has a distinct resistance profile relative to other PI. A unique I50L signature resistance substitution was identified in both PI-naïve and treatment-experienced patients failing ATV-containing regimens. Viruses containing I50L display ATV-specific resistance and increased susceptibilities to other PI. Studies focusing on the phenotype of HIV-1 protease molecule containing an I50L were undertaken to better understand this distinctive resistance profile.

Methods:  Expression and processing of p55 by recombinant HIV gag-pol proteins was evaluated in vitro using a transient cell-based assay. Protease susceptibilities to PI were determined using Western blots to monitor p24 accumulation. Proteases with an I50L and/or A71V substitutions inserted into a variety of protease backbones were evaluated.

Results:  Transient expression of the HIV gag-pol gene resulted in the time-dependent accumulation of p24, and processing could be blocked by the inclusion of HIV PI in a dose-dependent manner. Western blot-derived EC50 values showed that the I50L substitution caused reduced protease susceptibility to ATV and increased susceptibility to the other marketed PI. A clear correlation was observed between changes in protease susceptibility and in vitro antiviral activity in cell culture. The degree of increased susceptibility among the other PI appeared to vary. Combining I50L with other primary PI resistance substitutions caused, in each case, reduced susceptibility to ATV and increased susceptibility to the other PI. Increased susceptibility to IDV, LPV, RTV, and SQV was observed consistently, whereas NFV susceptibility changes were somewhat dependent on backbone sequence. As observed in clinical studies, APV susceptibility increases appear to be only modestly impacted by I50L. Similar to the reduced replicative capacity observed for I50L-containing viruses, cleavage efficiency of the gag-pol constructs was also markedly reduced.

Conclusions:  The increased susceptibility of I50L-containing viruses to other PI appears to stem from the intrinsic susceptibility of the I50L protease. The I50L substitution can counteract the reduced susceptibility of HIV protease to other PI caused by other primary mutations, both in laboratory and clinical isolates.

Keywords: atazanavir; resistance; protease inhibitor