In vitro resistance to the human immunodeficiency virus type 1 maturation inhibitor PA-457 (Bevirimat)

Abstract

3-O-(3',3'-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.

FIG. 1.

Selection for PA-457 resistance. (A) Ten flasks of pNL4-3-transfected Jurkat cells were cultured in parallel in the presence of 50 ng/ml PA-457. As controls, two flasks transfected with either pUC19 or pNL4-3 were cultured without PA-457. Genomic DNA was extracted at peak RT activity and analyzed by PCR amplification and DNA sequencing. (B) Mutations identified in the selection experiments shown in panel A. Pr55^Gag is represented at the top, with the MA, CA, NC, and p6 domains and the SP1 and SP2 spacer peptides indicated. The alignment shows each of the six potential PA-457 resistance mutations identified.

FIG.2.

CA-SP1 processing of PA-457-resistant mutants displays insensitivity to PA-457. HeLa cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations and were cultured without PA-457 or in the presence of either 1.0 or 2.0 μg/ml PA-457. Cells were metabolically labeled for 2 h with [³⁵S]Met/Cys, and released virions were pelleted by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig, and processing of CA-SP1 to CA was analyzed by SDS-PAGE and fluorography (A) followed by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (B). Error bars indicate standard deviations (n = 2).

FIG.2.

CA-SP1 processing of PA-457-resistant mutants displays insensitivity to PA-457. HeLa cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations and were cultured without PA-457 or in the presence of either 1.0 or 2.0 μg/ml PA-457. Cells were metabolically labeled for 2 h with [³⁵S]Met/Cys, and released virions were pelleted by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig, and processing of CA-SP1 to CA was analyzed by SDS-PAGE and fluorography (A) followed by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (B). Error bars indicate standard deviations (n = 2).

FIG. 3.

The extent of CA-SP1 processing does not correlate with PA-457 resistance. HeLa cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations and pulse-labeled for 15 min with [³⁵S]Met/Cys. Cells were chased for the indicated times in unlabeled medium, and cell lysates were immunoprecipitated with HIV-Ig. The extent of processing of CA-SP1 to CA was analyzed by SDS-PAGE and fluorography (A and B) followed by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (C and D). Error bars indicate standard errors of the means (n = 4 [C] or 3 [D]).

FIG. 4.

Biochemical characterization of PA-457-resistant mutants in an in vitro assembly system. [³⁵S]Met-labeled assembled Gag was used as substrate for proteolytic processing by 3 h of incubation with purified PR in the presence or absence of PA-457 at the indicated concentrations. (A) Portions of gels from representative experiments showing CA-SP1 and CA. The concentration of PA-457 is given at tops of gel lanes (0.6 μg/ml ∼ 1 μM; 6 μg/ml ∼ 10 μM). (B) Quantitative representation of the gel data from panel A normalized to the maximum amount of processing as seen in the no-drug control. (C) Extent of cleavage achieved under standard reaction conditions in the absence of drug, presented as percentage of CA-SP1 relative to total CA-SP1 plus CA. For panels B and C, error bars indicate the standard deviations for six replicate experiments. Note that the data for SP1-A1V were reported in a previous study (38) and are recapitulated here for comparison.

FIG. 5.

Replication kinetics of PA-457-resistant viruses. Jurkat T cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations. Cultures were maintained either without PA-457 or with 50 ng/ml or 1.0 μg/ml PA-457. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis.

FIG. 6.

CA-G225S compensates for the replication defect imposed by SP1-A3V. (A) Jurkat T cells were transfected with the indicated WT or mutant molecular clones. Cultures were maintained either without or with 1.0 μg/ml PA-457. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. (B and C) Effect of the CA-G225S mutation on the extent of cleavage of CA-SP1 to CA. HeLa cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations and pulse-labeled for 15 min with [³⁵S]Met/Cys. Cells were chased for the indicated times, cell lysates were immunoprecipitated with HIV-Ig, and the extent of processing of CA-SP1 to CA was analyzed by SDS-PAGE and fluorography (B) followed by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (C). Error bars indicate standard errors of the means (n = 3).

FIG. 7.

Replication of SP1-A3V at various concentrations of PA-457. Ten flasks of Jurkat T cells transfected with pNL4-3-SP1-A3V were cultured in parallel in the presence of 50 ng/ml or 1.0 μg/ml PA-457. Cells transfected with pUC19, WT pNL4-3, or pNL4-3-SP1-A3V, cultured without PA-457, served as controls. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis.

FIG. 8.

SP1-A3T exhibits inefficient virus release and Gag processing. HeLa cells were transfected with WT pNL4-3 or derivatives containing the indicated mutations and were cultured without PA-457 or in the presence of 1.0 μg/ml PA-457. Cells were metabolically labeled with [³⁵S]Met/Cys, and released virions were pelleted by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE and fluorography (A) followed by phosphorimager analysis to quantify virus release efficiency, calculated as the amount of particle-associated Gag as a fraction of total (cell plus virion) Gag (B) and the cellular ratio of Pr55^Gag to total CA-SP1 plus CA (C). Error bars indicate standard errors of the means (n = 6 [B] or 5 [C]).

FIG. 9.

Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h posttransfection and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.

FIG. 10.

Amino acid positions to which PA-457 resistance maps are highly conserved between HIV-1 isolates. An amino acid sequence alignment of the CA-SP1 boundary region of Gag is shown. The alignment was constructed from the 2004 Los Alamos HIV-1 sequence database group M consensus sequences and SIVmac239. Arrows indicate amino acid positions to which PA-457 resistance maps (; http://hiv-web.lanl.gov/content/hivdb/CONSENSUS/M_GROUP/Consensus.html).