Viral evolution in response to the broad-based retroviral protease inhibitor TL-3

TL-3 is a protease inhibitor developed using the feline immunodeficiency vi rus protease as a model. It has been shown to efficiently inhibit replicati on of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to kno wn protease inhibitors. To dissect the spectrum of molecular changes in pro tease and viral properties associated with resistance to TL-3, a panel of c hronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M461/F53L/V 82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and st ructurally modeled the protease mutant containing six changes. Virus contai ning six changes was found to be 17-fold more resistant to TL-3 in cell cul ture than was wild-type virus but maintained similar in vitro replication k inetics compared to the wild-type virus. Analyses of enzyme activity of pro tease variants with one, three, and six changes indicated that these enzyme s, compared to mild-type protease, retained 40, 47, and 61% activity, respe ctively. These results suggest that deficient protease enzymatic activity i s sufficient for function, and the observed protease restoration might impl y a selective advantage, at least in vitro, for increased protease activity .