Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat

Abstract

Background: The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8.

Results: In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance.

Conclusions: Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

Figure 1

Mutagenesis at SP1 residues 6-8 results in varying degrees of CA-SP1 processing in the presence of BVM. (A) Mutagenesis of SP1 residues 6-8. HIV-1 Gag is represented at the top. The MA, CA, NC and p6 domains and the SP1 and SP2 spacer peptides are indicated. The alignment shows the pNL4-3 wild type (WT) amino acid sequence at the CA-SP1 junction in Gag and the panel of SP1 mutant derivatives examined in this study. The residues to which BVM resistance was previously mapped in vitro are shaded in grey. (B and C) Quantitative CA-SP1 processing assay. HeLa cells were transfected with WT pNL4-3 and the panel of SP1 mutant derivatives and cultured either without BVM or in 1 μg/ml BVM. 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 (B) and by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (C). Error bars indicate standard deviations (n = 3-5).

Figure 2

Residue 6-8 mutations result in varying levels of resistance to BVM. (A) Virus stocks produced from HeLa cells either in the absence of BVM or in the presence of 1 μg/ml BVM were used to infect the TZM-bl indicator cell line. Infectivity was measured 48 h postinfection by determining levels of luciferase activity. Relative infectivity was calculated by normalization of the untreated WT virus at the 12.5 μl viral input to 100%. Paired t tests were performed to evaluate differences between means. Statistically significant differences between pairs of means are indicated (*** P = 0.0001, ** P = 0.001, * = 0.01). (B) Virus inputs were verified by confirming that virus stocks contained comparable RT activities. All data shown are means and standard deviations from three independent experiments, performed in duplicate.

Figure 3

Replication kinetics of viruses containing mutations in SP1 residues 6-8. Jurkat T cells were transfected with WT pNL4-3 and the panel of SP1 mutant derivatives and cultured either without BVM or in 50 ng/ml or 1 μg/ml BVM. Cells were split every 2 days, and supernatants were reserved at each time point for RT analysis. All originally introduced mutations were maintained. The grey boxes indicate those cultures in which an additional mutation is acquired; both the introduced and the acquired mutations are indicated. The results shown are representative of at least 2 independent experiments. Results from the repeat experiment are described in the text.