Impact of human immunodeficiency virus type 1 resistance to protease inhibitors on evolution of resistance to the maturation inhibitor bevirimat (PA-457)

Abstract

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

FIG. 1.

Representation of the BVM resistance and PIR PR mutations examined in this study. HIV-1 Gag and PR are represented at the top. The MA, CA, NC, and p6 domains and the SP1 and SP2 spacer peptides in Gag are indicated. The alignment shows each of the BVM resistance and PR mutations. (Adapted from reference .)

FIG. 2.

PIR mutations induce general defects in Gag processing and a modest delay in virus replication. (A) Biochemical characterization of Gag expression and proteolytic processing. Transfected HeLa cells were cultured with 0 or 1 μg/ml BVM and were metabolically labeled with [³⁵S]Met/Cys. Released virions were pelleted by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. SU, surface; TM, transmembrane. (B) Phosphorimager analysis of radioimmunoprecipitation assays to quantify the percentage of proteolytic processing intermediate CA-SP1 relative to total CA-SP1 plus CA: % CA-SP1 = [CA-SP1/(CA-SP1 + CA)] × 100. Error bars indicate standard deviations (n ≥ 8). (C) Replication kinetics in Jurkat T cells of WT and PIR molecular clones. Cultures were maintained in 0 or 1 μg/ml BVM. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. The experiment shown is representative of eight independent experiments in which the delay between the replication of the WT and PIR clones cultured without BVM ranged from 0 to 12 days, with an average delay of 5 days. Detectable virus replication was not observed in the presence of 1 μg/ml BVM. (D) Phosphorimager analysis of radioimmunoprecipitation assays to quantify the percentage of proteolytic processing intermediate MA-CA relative to total Gag: cellular % MA-CA = [MA-CA/(Pr55 Gag + MA-CA + CA-SP1 + CA)] × 100, and virion % MA-CA = [MA-CA/(MA-CA + CA-SP1 + CA)] × 100. Error bars indicate standard deviations (n ≥ 8). One-way ANOVA was performed using jmp software to test differences between means. Statistically significant differences between pairs of means are indicated with a solid line for BVM-treated versus non-BVM-treated samples with the same PR and a dashed line for samples encoding WT or PIR PR but with the same inhibitor treatment. **, P < 0.01; *, P < 0.05.

FIG. 3.

Biochemical quantification of virion-associated CA-SP1 in BVM-resistant clones expressing either WT or PIR PR. Viral lysates were prepared, and the percent CA-SP1 was calculated as indicated in the Fig. 2 legend. Error bars indicate standard deviations (n ≥ 3). ANOVA was performed as described in the Fig. 2 legend. **, P < 0.01; *, P < 0.05.

FIG. 4.

PIR and BVM resistance mutations synergize to delay virus replication. Jurkat T cells were transfected with the indicated BVM-resistant CA (A) or SP1 (B) mutants expressing either WT PR (closed symbols) or PIR PR (open symbols) and were cultured without BVM (blue circle) or with 50 ng/ml (red triangle) or 1 μg/ml (green square) BVM. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. Results shown are representative of four independent experiments.

FIG. 5.

Replication kinetics of the SP1-A3V mutant coupled with the PIR changes. Jurkat T cells were transfected with WT pNL4-3 (closed symbols), pNL4-3/PIR, or pNL4-3/A3V PIR. (A) Cultures were maintained either without BVM (blue symbols) or with 1 μg/ml BVM (green symbols). (B) Cultures were maintained in no BVM (blue circle), 50 ng/ml (red triangle) BVM, or 1 μg/ml (green square) BVM. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. Virus from the SP1-A3V PIR flask (A) was harvested on day 44 and used to infect fresh Jurkat T cells (B). DNA was extracted at peak RT activity, and the Gag-PR coding region was PCR amplified and sequenced.

FIG. 6.

PIR changes in PR delay the development of BVM resistance. Ten Jurkat flasks each were transfected with WT pNL4-3 (red) or pNL4-3/PIR (blue) and cultured in parallel in the presence of 50 ng/ml BVM. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. DNA was extracted at peak RT activity, and the Gag-PR coding region was PCR amplified and sequenced.