HIV-2 Protease resistance defined in yeast cells

Abstract

Background: Inhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast.

Results: Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC50 values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC50 values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain.

Conclusion: This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.

Figure 1

HIV-2 Protease expression in yeast induces cell growtharrest. A) BY4741 cells transformed either with pRS316Gal1/10 (1), pRS316Gal1/10-HIV2PR (2) or pRS316Gal1/10-HIV2PR-D25A were plated on minimal selective media containing glucose and replica-plated on galactose containing media in the presence or the absence of 200 μM Saquinavir. Yeast patches were observed after 2 days incubation at 30°C. B) 0.25 OD₆₀₀ of yeast cells transformed either with HIV-2_ROD Protease (wt), or with a genetically inactivated version of HIV-2_ROD Protease (D25A), were incubated in 5 ml of liquid SGalC-Ura for 60 hours. At defined time points cell growth was measured (OD₆₀₀). C) Soluble yeast cell extracts obtained from 1OD₆₀₀ of growing cells were run on a SDS 17% PAGE, and subjected to Western blot analysis. 1: BY4741 [pRS316Gal1/10-HIV2PR] grown in glucose, 2: 25 ng of purified recombinant HIV-2 PR [34], 3: BY4741 [pRS316Gal1/10-HIV2PR(D25A)] grown in galactose, 4 : BY4741 [pRS316Gal1/10-HIV2PR] grown in galactose in the presence of 60 μM LPV.

Figure 2

Susceptibility of HIV-2_ROD Protease to Protease Inhibitors in transformed yeast 0.02 OD₆₀₀/well of BY4741 [pRS316Gal1/10-HIV2PR] cells were either incubated in Galactose (A) or Glucose (B) containing synthetic media in the presence of increasing amounts of LPV and SQV (from 1.5 μM to 200 μM) in a 96 well plate. After 48 h at 30°C cellular growth was determined by measuring cell density at 600nm and plotted against PI concentrations. Results are presented as the percentage of cell growth; [(OD₆₀₀ cells grew in Galactose with PI – OD₆₀₀ cells grew in Galactose)/OD₆₀₀cells grew in Glucose] × 100.

Figure 3

Amino acid sequences of Proteases from HIV-2 infecting isolates. PCR amplified HIV-2 Protease coding regions from 7 infected individuals were sequenced as described in Material and Methods and translated to amino acid sequence. Conservatives amino acid substitutions are in green while non-conservatives are in red.