Mechanism of Darunavir (DRV)'s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance

Abstract

Darunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIV_DRV^R_P51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIV_DRV^R_P51 are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIV^I54M and rHIV^I84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIV^V32I also developed high-level DRV resistance. However, wild-type HIV_NL4-3 (rHIV^WT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIV^WT, rHIV^V32I was highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIV^WT with DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1's DRV resistance development and elucidate, at least in part, a mechanism of DRV's high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.IMPORTANCE Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1's drug resistance. However, the mechanism(s) of the DRV's high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIV^WT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIV^WT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1's resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

Keywords: HIV-1; V32I; darunavir; drug resistance; dual mechanism; genetic barrier; protease inhibitors.

FIG 1

In vitro selection of highly DRV-resistant variants using various infectious HIV-1 clones. The impact of four amino acid substitutions, V32I, L33F, I54M, and I84V, on the development of DRV resistance was examined. (A and B) rHIV^WT, rHIV^V32I/I54M, rHIV^L33F/I84V, and rHIV^{V32I/L33F/I54M/I84V} (A) and rHIV^V32I, rHIV^L33F, rHIV^I54M, and rHIV^I84V (B) were propagated in the presence of increasing concentrations of DRV in MT-4 cells. At the conclusion of each passage, cell-free supernatant was harvested from the culture and subsequently added to a following culture replenished with the same number of uninfected target cells, and the virus was further propagated. This passage was repeated every 1 to 3 weeks for a total of 17 to 50 weeks.

FIG 2

Emergence of the V32I substitution during the selection of highly DRV-resistant HIV-1 variants but not in HIV^WT and HIV^L33F. (A) Amino acid sequences deduced from the nucleotide sequence of the protease-encoding region (direct sequencing) were determined using proviral DNA. In the selection with DRV, proviral DNA was extracted at week 50 for HIV^WT and HIV^L33F, at week 36 for HIV^V32I, at weeks 7, 24, and 36 for HIV^I54M, at weeks 8, 17 and 29 for HIV^I84V, at week 22 for HIV^V32I/I54M, at week 26 for HIV^L33F/I84V, and at week 17 for HIV^{V32I/L33F/I54M/I84V}. (B) Absence of the V32I substitution in two infectious clones, rHIV^WT and rHIV^L33F, which were selected with DRV over 50 weeks. Two clones, rHIV^WT and rHIV^L33F, selected with DRV over 50 weeks (generating HIV^WT-WK50 and HIV^L33F-WK50, respectively), apparently failed to develop DRV resistance as shown in Fig. 1. To confirm the absence of V32I, HIV^WT-WK50 and HIV^L33F-WK50 were further cloned (20 clones), and each clone generated was sequenced. Note that the V32I substitution did not emerge in either of the virus populations. The consensus sequence of pNL4-3 is illustrated at the top of panels A and B as a reference. Amino acids that are identical to those in the consensus sequence at individual amino acid positions are indicated by dots. The fractions of the virus which each clone is presumed to have originated from over the total number of clones examined are shown to the right of the sequences.

FIG 3

rHIV^A71V rapidly develops DRV resistance, acquiring the V32I substitution. rHIV^M46I and rHIV^A71V were propagated in the presence of increasing concentrations of DRV in MT-4 cells. The selection was carried out in a cell-free manner over a total of 23 or 50 weeks. Amino acid substitutions identified in the protease of rHIV^A71V at 14 and 23 weeks and that of rHIV^M46I at 50 weeks are shown, indicated by arrows. Note that rHIV^M46I apparently failed to develop DRV resistance, while rHIV^A71V rapidly developed DRV resistance, acquiring the V32I substitution.

FIG 4

DRV’s protease dimerization inhibition activity also increases against HIV^V32I compared to HIV^WT. (A, top) Plasmids encoding full-length molecular infectious HIV_NL4-3 clones, which produce CFP- or YFP-tagged protease (PR), were generated using the PCR-mediated recombination method as described in Materials and Methods. A linker consisting of five alanines was inserted between PR and each fluorescent protein. The phenylalanine-proline site (F/P) that PR cleaves was introduced between the fluorescent protein and reverse transcriptase (RT). AA, amino acids. (Bottom) Structural representations of PR monomers and dimer in association with the linker atoms and fluorescent proteins are shown below the schematic representation of the plasmids. FRET occurs when the two fluorescent proteins come close in the proximity of 1 to 10 nm. If an agent that inhibits the dimerization of 2-PR monomer subunits is present when the CFP- and YFP-tagged PR monomers are produced within the cell upon cotransfection, FRET does not occur. (B) COS-7 cells were cotransfected with plasmids encoding either of full-length molecular infectious HIV_NL4-3 clones producing CFP- or YFP-tagged wild-type protease (HIV^WT) and cultured in the presence or absence of various concentrations of DRV. COS-7 cells were also cotransfected with plasmids encoding either of HIV_NL4-3 clones producing CFP- or YFP-tagged V32I-carrying protease (HIV^V32I). After 72 h, cultured cells were examined in the FRET-HIV-1 assay system and the CFP^A/B ratios (y axis) were determined. The mean values of the ratios obtained are shown as bars. A CFP^A/B ratio that is greater than 1 signifies that protease dimerization occurred, whereas a ratio that is less than 1 signifies the disruption of protease dimerization. All the experiments were conducted in a blind fashion. For rHIV^WT, the P values were 0.3816 for the CFP^A/B ratio in the absence of drug (CFP^A/B_{No Drug}) versus the CFP^A/B ratio in the presence of 10 nM DRV (CFP^A/B_10-DRV), 0.004 for CFP^A/B_{No Drug} versus CFP^A/B_100-DRV. For rHIV^V32I, the P values were 0.1111 for CFP^A/B_{No Drug} versus CFP^A/B_0.1-DRV, 0.001 for CFP^A/B_{No Drug} versus CFP^A/B_1-DRV, and 0.0086 for CFP^A/B_{No Drug} versus CFP^A/B_10-DRV.

FIG 5

V32I reduces the viral fitness of HIV-1, while A71V recovers the compromised fitness. Replication profiles of rHIV^WT, rHIV^V32I, and rHIV^V32I/A71V were examined. (A and B) Replication kinetics of the three clones were examined. MT-4 cells (10⁵) were exposed to each infectious clone (calibrated to have 10 ng of p24 Gag of each preparation) and cultured in the absence (A) or presence (B) of 3 nM DRV. Virus replication was monitored by the amounts of p24 Gag produced in the culture supernatants. The results shown are representative of results from three independent experiments. The failure of rHIV^V32I replication in the presence of 3 nM DRV suggests that rHIV^V32I is more susceptible to DRV than rHIV^WT and rHIV^V32I/A71V are. (C and D) The replication profiles of HIV^WT versus HIV^V32I and HIV^V32I versus HIV^V32I/A71V in the absence (solid line) or presence (dashed line) of 3 nM DRV were examined using the competitive HIV replication assay (19). MT-4 cells (10⁵) were exposed to an equal amount of two infectious clones to be compared and cultured for 7 days. The cell-free supernatant harvested at the conclusion of each culture passage (7 days) in the absence (solid line) or presence (dashed line) of 3 nM DRV was transferred to fresh MT-4 cell culture. High-molecular-weight DNA extracted from infected cells at the end of each passage was subjected to nucleotide sequencing, and percent populations of the mixture were estimated by the heights of the electropherogram obtained through the sequencing. The proportions of Val and Ile at position 32 in protease (panel C) and of Ala and Val at position 71 in protease (panel D) were determined.

FIG 6

Comparison of binding fashion of DRV or APV to PR^Val32 or PR^Ile32. (A) In the structure, DRV contains a bis-tetrahydrofuranyl urethane (bis-THF) at the P2 site instead of APV’s tetrahydrofuranyl urethane (THF) moiety. (B) The Connolly surface interactions of DRV or APV with residue 32 are shown in stereo. Surface colors are as follows: gray for DRV or APV, green for V32, and magenta for I32. DRV is shown with green carbons, and APV is shown with gray carbons. The figure shows that DRV has better interaction with substituted I32 than with wild-type V32. The interactions of APV with V32 or I32 are similar.

FIG 7

The mature dimerized HIV-1 protease in complex with DRV and proposed pathway of development of DRV resistance. (A) Proposed pathway of development of DRV resistance of HIV-1. Upon DRV selection, rHIV^WT, rHIV^L33F, and rHIV^M46I clones did not develop DRV resistance. rHIV^V32I and rHIV^I54M relatively rapidly acquired A71V, and rHIV^I54M subsequently acquired V32I. These clones continued to acquire multiple amino acid substitutions and became highly resistant to DRV. Upon DRV selection, rHIV^V32I/I54M quickly acquired A71V and became highly resistant to DRV. rHIV^A71V acquired V32I and eventually became resistant to DRV. rHIV^I84V quickly acquired A71V, subsequently acquired V32I, and became resistant to DRV. rHIV^L33F/I84V continued to propagate in the presence of DRV, acquired V32I but without acquiring A71V, and became resistant to DRV. rHIV^L33F/I84V’s acquisition of high-level DRV resistance despite the absence of A71V strongly suggest that its DRV resistance acquisition involved alternate albeit unidentified pathway of DRV resistance development. (B) The locations of V32, L33F, M46, I54, A71, and I84 are shown in the dimerized protease. V32, I54, and I84 are located in the active site of the dimerized protease; however, A71 is distant from the active site.