Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3

Abstract

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication 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 known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

“…Next, we consecutively chose 15 substrates predicted to be noncleavable by the CLM by HXB2 HIV-1 protease, allowing at most four amino acids to be identical at any same positions among all the substrates already selected (including the cleavable substrates already chosen above). If none of the remaining substrates met the requirements, a five-amino-acid similarity was allowed (Table 3, numbers [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]. We then used the CLM to predict cleavability of the chosen 30 substrates by mutant HIV-1 proteases I84V, L90M, and I84V þ L90M.…”

Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

“…One of our goals is to understand the molecular basis of human immunodeficiency virus type 1 (HIV-1) and FIV protease (PR) substrate and inhibitor specificity in order to develop broadspectrum PR inhibitors that will inhibit wild-type and drugresistant PRs. This approach has led to the development of TL-3, an inhibitor that is capable of inhibiting FIV, simian immunodeficiency virus, HIV-1, and several drug-resistant HIV-1 strains ex vivo (5,25,26), as well as other potential inhibitors with broad efficacy (24,33,34). FIV PR, like HIV-1 PR, is a homodimer, but each monomer is composed of 116 amino acids, as opposed to 99 amino acids for HIV-1 PR (Fig.…”

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

“…Comparisons of the 3 dimensional structures of the two PRs led to the rational design of TL-3, a broad-based PR inhibitor capable of blocking infection by FIV, simian immunodeficiency virus (SIV), and HIV-1, as well as many drug-resistant HIV-1 variants (10,12,21,23,31,32). Similar approaches comparing the structures of FIV and drug-resistant HIV-1 PRs to that of WT HIV-1 PR have led to the development of additional PR-inhibiting compounds with broadened efficacy (8,9,19,29,41,42).…”