Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1

Abstract

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.

Fig. 1.

FRET-based HIV expression system. Plasmids encoding full-length molecular infectious HIV (HIV_NL4-3) clones that produce CFP- or YFP-tagged PR were prepared using the PCR-mediated recombination method as described in Materials and Methods. A linker consisting of five alanines was inserted between PR and the fluorescent protein. A phenylalanine-proline site (F/P) that HIV PR cleaves was introduced between the fluorescent protein and RT. Shown are structural representations of PR monomers and dimer in association with the linker atoms and fluorescent proteins. FRET occurs when the two fluorescent proteins become 1 to 10 nm apart. If an agent that is capable of inhibiting the dimerization of PR monomer subunits is present when the CFP- and YFP-tagged PR monomers are produced within the cell upon cotransfection, no FRET occurs. If certain amino acid substitutions (AA) such as D29N (shown below) are introduced, PR subunits do not get dimerized and no FRET occurs.

Fig. 2.

DRV blocks the dimerization of both pHIV-PR_WT-encoded PR and pPR_WT-encoded PR. (A) COS7 cells were cotransfected with pHIV-PR_WT^CFP plus pHIV-PR_WT^YFP in the absence or presence of 1 μM DRV or APV. On day 3 after transfection, CFP^A/B ratios were determined using an FV500 confocal laser microscope. When the average value of CFP^A/B ratios was greater than 1.0, it was judged that the dimerization of PR occurred, whereas when it was less than 1.0, it was judged that the dimerization did not occur. (B) COS7 cells were cotransfected with a pair of wild-type PR-expressing plasmids (pPR_WT^CFP plus pPR_WT^YFP) in the absence or presence of 1 μM DRV or APV, and CFP^A/B ratios were determined as described above. Note that DRV inhibited the dimerization of PR when it was expressed as HIV virions and virion-free PR. The results of statistical evaluation of the changes in the CFP^A/B ratios, determined in the presence or absence of DRV or APV, using the nonparametric Mann-Whitney U test, are as follows. (A) For the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) versus the CFP^A/B ratios in the presence of 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}), P = 0.00001, and for CFP^{A/B_{No Drug}} versus CFP^{A/B_{1.0 APV}}, P = 0.42. (B) For CFP^{A/B_{No Drug}} versus CFP^{A/B_{1.0 DRV}}, P = 0.000003, and for CFP^{A/B_{No Drug}} versus CFP^{A/B_{1.0 APV}}, P = 0.60.

Fig. 3.

Dimerization profiles of single PR mutants in the presence of DRV. (A) COS-7 cells were cotransfected with pHIV-PR_WT^CFP plus pHIV-PR_WT^YFP (shown as WT^CFP/WT^YFP) or mutated pairs such as pHIV-PR_P1A^CFP plus pHIV-PR_P1A^YFP (shown as P1A^CFP/P1A^YFP) in the absence or presence of 1 μM DRV. On day 3 after transfection, CFP^A/B ratios were determined. (B) COS7 cells were cotransfected with plasmid pair pHIV-PR_A28S^CFP and pHIV-PR_A28S^YFP in the absence or presence of an agent (1 μM GRL-0216, DRV, GRL-98065, TPV, or TMC126), and CFP^A/B ratios were determined as described above. (A) The statistical evaluation of all the changes in the CFP^A/B ratios determined in the presence or absence of DRV using the nonparametric Mann-Whitney U test, gave P values ranging 0.000037 to 0.044, except for the P value for the pair A28S^CFP and A28S^YFP, which was 0.57. (B) The differences between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) and the CFP^A/B ratios in the presence of 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}) were statistically insignificant, indicating that all of the agents examined failed to block the dimerization of A28S^CFP/A28S^YFP.

Fig. 4.

Replication kinetics of HIV-PR^YFP with wild-type or mutated PR. (A) 293T cells were transfected with pHIV-PR_WT^YFP or mutated pHIV-PR^YFP (if pHIV-PR_P1A^YFP was used, it is shown as P1A^YFP), and the amounts of p24 Gag in the culture supernatants were determined 48 h after transfection. (B) MT-4 cells (10⁵) were exposed to the harvested supernatant of each infectious HIV-PR^YFP clone shown in panel A (100 ng of p24 Gag protein/ml) for 6 h, washed twice with phosphate-buffered saline (PBS), and further cultured in 7 ml of complete medium. Culture supernatants (50 μl) were harvested every other day, and virus replication was monitored by the amounts of p24 Gag produced in the culture supernatants. Replication kinetics of various HIV-PR^YFP mutants are shown over 11 days. In subpanels a, b, and c, the replication kinetics of infectious clones carrying mutations in the active site, N ternimus, and C terminus, respectively, are shown. Note that recombinant HIV clones, whose replication rates were relatively poor, are illustrated in subpanel a. The experiments that generated data in subpanels a and b were performed on the same occasion. Thus, two controls (HIV-1_NL4-3sma and HIV-PR_WT^YFP) in subpanel b serve as controls in subpanel a as well.

Fig. 5.

Dimerization inhibition profiles of selected HIV-1 PR mutants. COS7 cells were cotransfected with a pair of HIV-PR^CFP and HIV-PR^YFP strains either wild type or carrying single amino acid (AA) substitutions, such as the R8A, R8Q, or L24A, in the absence of drug. The CFP^A/B ratios were determined at the conclusion of the 3-day period of culture. The differences between the CFP^A/B ratios of the WT and the CFP^A/B ratios of the mutant had P values of 0.000099 for R8A, 0.000084 for R8Q, and 0.0000014 for L24A.

Fig. 6.

DRV fails to inhibit the dimerization of the protease of a highly DRV-resistant HIV_8MIX^P51 variant. COS7 cells were transfected with a pair of plasmids encoding a full-length molecular infectious HIV-1 clone (HIV_8MIX^P51) containing CFP- or YFP-tagged PR with 14 amino acid substitutions (L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82I, I84V, and L89M) in the presence or absence of 0.1, 1, or 10 μM DRV. On day 3 after transfection, CFP^A/B ratios were determined as described in the legend to Fig. 2. HIV_NL4-3 served as a reference. Note that 0.1 and 1 μM DRV failed to block the dimerization of the protease of HIV_8MIX^P51, while the same concentration of DRV blocked protease dimerization in HIV_NL4-3. The differences between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) and the CFP^A/B ratios in the presence of 0.01 μM DRV (CFP^{A/B_{0.01 DRV}}), between the CFP^A/B ratios in the presence of 0.01 μM DRV (CFP^{A/B_{0.01 DRV}}) and 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}), and between the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) and 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}) had P values of 0.32, 0.0025, and 0.34 for HIV_NL4-3, respectively. The differences between the CFP^{A/B_{No Drug}} and the CFP^{A/B_{0.1 DRV}}, between the CFP^{A/B_{0.1 DRV}} and CFP^{A/B_{1.0 DRV}}, and between the CFP^{A/B_{1.0 DRV}} and the CFP^{A/B_{10.0 DRV}} had P values of 0.42, 0.022, and 0.26, respectively, for HIV_8MIX^P51.

Fig. 7.

Amino acid changes conferring DRV resistance on HIV. (A) Locations of amino acid substitutions V32I, L33A/F, I54M, and I84V associated with HIV's DRV resistance. The location of Asp29 (D29), which is known to be an essential amino acid for dimerization, is also shown. (B) Profiles of DRV's dimerization inhibition of PR carrying a single amino acid substitution. COS7 cells were cotransfected with a pair of HIV-PR^CFP and HIV-PR^YFP variants carrying wild-type PR or a single amino acid substitution such as V32I, L33F, I54M, or I84V, each of which was found to be associated with the development of HIV resistance to DRV, in the presence of 1 μM DRV, further cultured, and the CFP^A/B ratios were determined. Note that none of the amino acid substitutions introduced blocked the dimerization of PR. The statistical evaluation of all the changes in the CFP^A/B ratios determined in the presence or absence of DRV, conducted using the nonparametric Mann-Whitney U test, showed P values ranging from 0.00000034 (3.4E−07) to 0.0026. (C) Profiles of DRV's dimerization inhibition of PR carrying combined amino acid substitutions. COS7 cells were cotransfected with a pair of HIV-PR^CFP and HIV-PR^YFP variants carrying combined amino acid substitutions such as V32I and I84V, V32I, L33F, and I84V, V32I, L33F, and I54M, or V32I, L33F, I54M, and I84V. The COS7 cells were further cultured in the continuous presence of 0, 0.1, and 1 μM DRV, and the CFP^A/B ratios were determined at the conclusion of the 3-day period of culture. The differences between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) and the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) and between the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) and 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}) had P values of 0.0015 and 0.42 for V32I and I84V, 0.0047 and 0.15 for V32I, L33F, and I84V, 0.033 and 0.07 for V32I, L33F, and I54M, and 0.3 and 0.0000073 for V32I, L33F, I54M, and I84V, respectively.

Fig. 8.

Effects of V82A and V82I substitutions on DRV's activity to inhibit PR dimerization. COS7 cells were cotransfected with the pHIV-PR^CFP and pHIV-PR^YFP pair of plasmids carrying a single V82A or V82I substitution or four combined mutations (V32I, L33F, and I54M plus V82A or V82I). The COS7 cells were further cultured in the continuous presence of 0.1 and 1 μM DRV, and the CFP^A/B ratios were determined at the conclusion of the 3-day period of culture. Note that combined with other three substitutions V32I, L33F, and I54M, V82A did not have a significant effect on DRV's dimerization inhibition activity, while V82I compromised the dimerization inhibition of DRV at 0.1 and 1.0 μM. The differences between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) and the CFP^A/B ratios in the presence of 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}) had P values of 0.00017 for V82A and 0.0027 for V82I. The differences between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{No Drug}}) and the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) and between the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) and 1.0 μM DRV (CFP^{A/B_{1.0 DRV}}) were 0.000055 and 0.38 for V32I, L33F, I54M, and V82A and 0.026 and 0.91 for V32I, L33F, I54M, and V82I, respectively.