Schematic molecular model of PF-46396 and BVM binding sites.

<p>The ribbon diagrams illustrate two adjacent CA-CTD/SP1 monomers in the hexagonal complex of the immature capsid. The atomic coordinates of the CA-CTD and SP1 domains were obtained from PDB 3H4E <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Pornillos1" target="_blank">[66]</a> and PDB 1U57 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Morellet1" target="_blank">[27]</a>, respectively. Residues 220–223 that link the CA-CTD and SP1 domains of each monomer were not modeled due to a lack of experimental data. The relative orientations of the adjacent CA-CTD domains were taken from the models of Bharat et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Bharat1" target="_blank">[61]</a>. The sites of resistance mutations are labeled and color-coded for location: Green, blue and red for the MHR, CA-CTD/SP1 boundary, and individual I201 residue, respectively. Yellow indicates the sites of secondary substitutions that rescue the G156E and P157S mutants. Also shown are stick figures of the PF-46396 and BVM compounds at the heights of their respective binding sites predicted from the locations of the resistance mutations (color-code: C – brown, N - blue, O - red, H – white, and Fl – green). The rotational orientation of PF-46396 is arbitrary, but that of BVM is taken from the photoafinity data of Nguyen et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Nguyen1" target="_blank">[45]</a>. The proximity of the MHR of the left monomer to the CA-CTD/SP1 region of the right one suggests that the binding sites of the two compounds straddle adjacent monomers. This would explain the observed necessity of Gag assembly into the immature capsid structure for BVM cleavage inhibition (see text).</p

The SP1-V7A polymorphism has a lesser effect on sensitivity to PF-46396 than to BVM.

<p>293T cells were transfected with WT pNL4-3 or pNL4-3 derivatives bearing SP1-V7A or SP1-T8A polymorphisms and treated with 1 µM BVM or PF-46396. CA-SP1 processing efficiency was examined in virions by radioimmunoprecipitation analysis. P values: **, p<0.01; dashed line, no significant difference. Error bars indicate SD; N = 3.</p

Second-site compensatory changes for PF-46396-dependent CA mutants.

<p>Second-site compensatory changes for PF-46396-dependent CA mutants.</p

PF-46396-dependent mutants are not rescued by BVM.

<p>[A] Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence (−) and presence (+) of 2 µM BVM. Positions of Pr55^Gag (PrGag), Pr41^Gag (p41), CA-SP1 and CA are indicated. Lower panels show phosphorimager-based quantification of relative virus release efficiency (VRE) and % virion CA-SP1, calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 4. [B] Virus replication kinetics in the absence and presence of BVM. The Jurkat T-cell line was transfected with WT or mutant pNL4-3 and propagated in the presence of 0, 0.1, or 1.0 µM BVM. Virus replication was monitored by RT activity, shown in cpm/µl.</p

PF-46396 blocks CA-SP1 processing. [A] Chemical structures of BVM and PF-46396.

<p>[B] Radioimmunoprecipitation analysis of virion-associated CA and CA-SP1 in the presence of increasing concentrations of BVM or PF-46396. [C] Quantification of the % CA-SP1 relative to total CA+ CA-SP1 in virion fraction at the indicated concentrations of BVM or PF-46396. Error bars indicate SD; N>3.</p

Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket

<div><p>Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for <em>in vitro</em>. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.</p> </div

Summary of PF-46396-resistance mutations selected.

#<p>From ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Adamson5" target="_blank">[51]</a>.</p>*<p>In parentheses is the number of times the indicated mutation was selected.</p

PF-46396 can rescue the assembly/release defect of some but not all MHR mutants.

<p>Top panel: Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence and presence of 5 µM PF-46396. Positions of Pr55^Gag (PrGag), Pr41^Gag (p41), CA-SP1, and CA are indicated. Lower panel shows phosphorimager-based quantification of relative virus release efficiency (VRE), calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 3. Note that PF-46396 significantly enhances the release of virus particles for E159Q (and G156E, as shown earlier) but not for Q155N, G156V, or E159D.</p

Virus replication kinetics in the absence (top) and presence (bottom) of PF-46396.

<p>The Jurkat T-cell line was transfected with WT or mutant pNL4-3 and propagated in the presence of 0 or 5 µM PF-46396. Virus replication was monitored by RT activity, shown in cpm/µl.</p