In Vitro Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors

Abstract

We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1_MIX) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1_MIX became highly resistant to DRV, with a 50% effective concentration (EC₅₀) ∼333-fold greater than that against HIV-1_NL4-3. HIV-1_MIX at passage 51 (HIV-1_MIX^P51) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1_MIX^P51 was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1_MIX^P51 had a resemblance with HIV-1_C of the HIV-1_MIX population, and selection using HIV-1_C was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1_C^P51, likely contributed to conferring a replication advantage on HIV-1_MIX^P51 by reducing intravirion cyclophilin A content. HIV-1_MIX^P51 apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1_MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1_MIX^P51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

Successful antiviral drugs, in theory, exert their virus-specific effects by interacting with viral receptors, virally encoded enzymes, viral structural components, viral genes, or their transcripts without disturbing cellular metabolism or function. We have designed and synthesized a series of nonpeptidyl protease inhibitors (PIs) that are potent against HIV-1 variants resistant to a number of PIs. One such anti-HIV-1 agent, darunavir (DRV), containing a structure-based designed privileged nonpeptidic P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl-urethane (bis-THF) (9, 10, 16), has been used worldwide as a first-line drug for the treatment of drug-naive patients with HIV-1 infection and those who harbor multidrug-resistant HIV-1 (HIV-1_MDR) variants and do not respond to previously existing highly active antiretroviral therapy (HAART) regimens. It has been reported that DRV has a high genetic barrier to development of HIV-1 resistance (4, 5) and that most patients with HIV-1 infection treated with other PIs respond favorably to DRV-based salvage therapy (19), while a variety of amino acid substitutions potentially related to HIV-1 resistance to DRV have been reported (4, 19). For elucidation of the mechanism of development of resistance to DRV by HIV-1, it is critical to acquire highly DRV-resistant HIV-1 variants, which should be of high utility in further designing more efficacious and resistance-deferring anti-HIV-1 drugs.

In the present work, we attempted to select DRV-resistant variants by propagating wild-type HIV-1_NL4-3, a mixture (HIV-1_MIX) of 8 highly PI-resistant HIV-1 clinical isolates, and each of the isolates separately. In vitro selection of HIV-1 variants highly resistant to DRV was unsuccessful when HIV-1_NL4-3 was used, in agreement with previous findings of De Meyer et al. (3). In contrast, when HIV-1_MIX was propagated, a highly DRV-resistant viral population was selected at relatively early passages. The population that replicated in the presence of 5 μM DRV at passage 51 (HIV-1_MIX^P51) contained as many as 14-amino-acid substitutions in the protease-encoding region. HIV-1_MIX^P51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

MATERIALS AND METHODS

Cells and viruses.

MT-4 cells were grown in RPMI 1640-based culture medium supplemented with 10% fetal calf serum (FCS) (PAA Laboratories GmbH, Linz, Austria) plus 50 U of penicillin and 100 μg of kanamycin per ml. COS7 and 293T cells were grown in Dulbecco's modified Eagle medium (DMEM)-based culture medium supplemented with 10% FCS plus 50 U penicillin and 100 μg kanamycin per ml. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat of HIV-1-seronegative individuals using Ficoll-Hypaque density gradient centrifugation and cultured in RPMI 1640-based culture medium containing 10% FCS and antibiotics with 10 μg of phytohemagglutinin (PHA-PBMC) for 3 days prior to drug susceptibility assays. The following HIV strains were used for the drug susceptibility assay and selection experiments: HIV-1_NL4-3, clinical HIV-1 strains from drug-naive patients with AIDS (HIV-1_ERS104pre) (22), and 8 HIV-1 clinical isolates, HIV-1_A, HIV-1_B, HIV-1_C, HIV-1_G, HIV-1_TM, HIV-1_MM, HIV-1_JSL, and HIV-1_SS, which were originally isolated from patients with AIDS who had failed existing anti-HIV regimens after receiving 9 to 11 anti-HIV-1 drugs over the previous 32 to 83 months in the late 1990s and which contained 9 to 14 amino acid substitutions corresponding to the protease-encoding region which have reportedly been associated with HIV-1 resistance to various PIs; these were genotypically and phenotypically characterized as multi-PI-resistant HIV-1 variants (25). All of the variants employed were susceptible to DRV, with 50% effective concentrations (EC₅₀s) less than 0.029 μM (10-fold increase in EC₅₀) (16; also unpublished data).

Antiviral agents.

Darunavir (DRV) (previously designated TMC114), a novel nonpeptidic PI containing bis-THF, was designed and synthesized by A. K. Ghosh as described previously (9, 11). TMC126 is the prototype of DRV. Both TMC126 and DRV contain the bis-THF moiety (24), while TMC126 and DRV have 4-methoxybenzenesulfonamide and sulfonamide isostere, respectively (see Fig. S1 in the supplemental material). GRL-02031 and GRL-03021, both of which are structurally related to DRV and highly potent against multi-PI resistant HIV-1 in vitro (14), were newly designed and were synthesized by A. K. Ghosh and S. Leshchenko-Yashchuk (see Fig. S1 in the supplemental material). These two compounds were used as control drugs in the in vitro drug selection experiments. Detailed synthetic methods for GRL-02031 and GRL-03021 will be described elsewhere. Saquinavir (SQV) and ritonavir (RTV) were kindly provided by Roche Products Ltd. (Welwyn Garden City, United Kingdom) and Abbott Laboratories (Abbott Park, IL), respectively. Amprenavir (APV) was a kind gift from GlaxoSmithKline (Research Triangle Park, NC). Nelfinavir (NFV), indinavir (IDV), and lopinavir (LPV) were kindly provided by Japan Energy Inc., Tokyo, Japan. Atazanavir (ATV) was a kind gift from Bristol Myers Squibb (New York, NY). Tipranavir (TPV) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health.

Generation of PI-resistant HIV-1 using HIV-1_NL4-3 in vitro.

MT-4 cells (10⁵/ml) were exposed to HIV-1_NL4-3 (500 50% tissue culture infected doses [TCID₅₀s]) and cultured in the presence of various PIs at an initial EC₅₀. Viral replication was monitored by determining the amount of p24 Gag produced by MT-4 cells. The culture supernatants were harvested on day 7 and used to infect fresh MT-4 cells for the next round of culture in the presence of increasing concentrations of each drug. When the virus began to propagate in the presence of the drug, the drug concentration was generally increased 2- to 3-fold. Proviral DNA samples obtained from the lysates of infected cells were subjected to nucleotide sequencing.

Generation of highly DRV-resistant HIV-1 using HIV-1_MDR in vitro.

Eight highly multi-PI-resistant primary HIV-1 strains (HIV-1_A, HIV-1_B, HIV-1_C, HIV-1_G, HIV-1_TM, HIV-1_MM, HIV-1_JSL, and HIV-1_SS) were isolated from patients with AIDS who had failed existing anti-HIV regimens after receiving 9 to 11 anti-HIV-1 drugs over the previous 32 to 83 months. These strains, which contained 9 to 14 amino acid substitutions corresponding to the protease-encoding region that have reportedly been associated with HIV-1 resistance to various PIs, were mixed and propagated in both MT-4 cells and PHA-PBMCs as previously described (25). The mixture on day 7 of culture was propagated in fresh MT-4 cells. The culture supernatant was harvested and used to infect fresh MT-4 cells for the selection experiment. To determine the existence of 8 clinical isolates, viral RNA was purified from each indicated supernatant using the QIAamp viral RNA minikit (Qiagen Inc., Valencia, CA), and reverse transcription-PCR (RT-PCR) was carried out using the Superscript First-Strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Each primary strain was also harvested, and the culture supernatant was used for the selection experiment. The culture supernatants were harvested on day 7 and used to infect fresh MT-4 cells for the next round of culture in the presence of increasing concentrations of DRV. When the virus began to propagate in the presence of the drug, the drug concentration was generally increased 2- to 3-fold. Proviral DNA samples obtained from the lysates of infected cells were subjected to nucleotide sequencing. This DRV selection procedure was carried out until the DRV concentration reached 1 or 5 μM.

Replication kinetics of DRV-resistant HIV-1 variant and wild-type HIV-1_NL4-3.

MT-4 cells (2.4 × 10⁵) were exposed to the DRV-selected HIV-1 variant at passage 39 (HIV-1_MIX^P39) or a wild-type HIV-1_NL4-3 preparation containing 30 ng p24 in 6-well culture plates for 3 h, and these MT-4 cells were divided into three fractions, each cultured with or without DRV (final concentration of MT-4 cells, 10⁴/ml; drug concentrations, 0, 0.1, and 1.0 μM). The amounts of p24 were measured every 2 days for up to 9 days in culture.

Drug susceptibility assay.

To determine the sensitivity of HIV-1_ERS104pre and clinical multidrug-resistant HIV-1 isolates, PHA-PBMC (10⁶/ml) were exposed to 50 TCID₅₀s of each HIV-1 isolate and cultured in the presence or absence of various concentrations of drugs in 10-fold serial dilutions in 96-well microtiter culture plates. PHA-PBMCs were derived from a single donor in each independent experiment. Thus, to obtain the data, three different donors were recruited. To determine the drug susceptibilities of a laboratory HIV-1 strain (HIV-1_NL4-3) and DRV-selected HIV-1 variants, MT-4 cells were used as target cells. MT-4 cells (10⁵/ml) were exposed to 100 TCID₅₀s of wild-type HIV-1_NL4-3 and DRV-selected HIV-1 variants in the presence or absence of various concentrations of drugs and were incubated at 37°C. On day 7 of culture, the supernatant was harvested and the amount of p24 Gag protein was determined by using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse F; Fujirebio Inc., Tokyo, Japan) (18). The drug concentrations that suppressed the production of the p24 Gag protein by 50% (EC₅₀) were determined by comparison with the level of p24 production in drug-free control cell cultures. All assays were performed in duplicate or triplicate.

RESULTS

In vitro selection of HIV-1 variants resistant to DRV using wild-type HIV-1_NL4-3.

We attempted to select HIV-1 variants with DRV by propagating a wild-type laboratory HIV-1 strain, HIV-1_NL4-3, in MT-4 cells in the presence of increasing concentrations of DRV as previously described (24). HIV-1_NL4-3 was initially exposed to 0.003 μM DRV and underwent 90 passages in the presence of DRV at concentrations up to 0.1 μM. We simultaneously and independently selected HIV-1 variants in the presence of RTV, APV, LPV, or ATV. As shown in Fig. S2A in the supplemental material, HIV-1 variants that replicated in the presence of 1 μM RTV, APV, LPV, and ATV emerged by passages 13, 21, 30, and 39, respectively, while HIV-1 exposed to DRV continued to replicate poorly and failed to further replicate in the presence of ∼0.1 μM DRV, indicating that the emergence of a DRV-resistant HIV-1 variant was substantially delayed compared to results with other PIs examined and HIV-1 failed to acquire significant resistance to DRV. We also determined the nucleic acid sequence of the protease-encoding region of the proviral DNA isolated from infected MT-4 cells at passages 1, 30, 60, and 90 in DRV selection. As shown in of Fig. S3 (Exp. 1) in the supplemental material, the virus contained R41I, L63P, and V82I substitutions at passage 30 and beyond.

Selection of DRV-resistant HIV-1 using HIV-1_NL4-3 preselected against TMC126.

Since we failed to obtain HIV-1 variants highly resistant to DRV as described above, we next used as a starting strain an HIV-1_NL4-3 variant that was selected over 9 passages against TMC126 (HIV-1_TMC126^P9), a bis-THF-containing PI prototype of DRV, which was potent against a wide spectrum of PI-resistant HIV-1 variants as previously described (24). This HIV-1_TMC126^P9, at a later passage (by passage 15), developed the A28S substitution, located at the active site of the enzyme, and acquired a high level of resistance to TMC126 and DRV (24). HIV-1_TMC126^P9 was initially exposed to 0.003 μM DRV, representing an EC₅₀ of DRV for the virus, and underwent 85 passages in the presence of DRV. In these selection experiments, we also selected HIV-1 variants against two bis-THF-containing or bis-THF-related ligands, cyclopentanyl-THF-containing-PIs GRL-02031 and GRL-03021 (see Fig. S1). It took only 16 and 36 passages for the concentrations of GRL-02031 and GRL-03021 under which HIV-1_TMC126^P9 replicated to reach 1 μM, respectively. However, HIV-1_TMC126^P9 selected against DRV gradually lost its replication capability, and when DRV went beyond ∼0.1 μM, the virus again failed to replicate, and the maintenance of the viral culture became highly difficult (see Fig. S2B in the supplemental material). HIV-1_TMC126^P9 replicating in the presence of 5 μM GRL-02031 contained M46I, I47V, V82I, I84V, and N98I encoded in the protease-encoding region of the gene at passage 23, while the virus replicating in the presence of 5 μM GRL-03021 contained L10I, G16E, V32I, M46I, I47V, I54L, V82A, and I84V at passage 53 (see Table S1 in the supplemental material).

The protease-encoding region of the proviral DNA isolated from HIV-1_TMC126^P9-infected MT-4 cells was cloned and sequenced at passages 1, 25, 55, and 85 upon DRV selection. The nucleic acid sequences of the protease-encoding region at various passages are depicted in of Fig. S3 (Exp. 2) in the supplemental material. By passage 25, the virus had acquired the V82I substitution. By passage 55, the virus had additionally acquired the R41S substitution, and by passage 85, the virus had acquired the K70E and V82M substitutions. The A28S substitution, which was seen when HIV-1 was selected against bis-THF-containing TMC126 (24) or brecanavir/GW640385 (BCV) (23), did not emerge.

It is of note that the multitude of amino acid substitutions in protease observed when HIV-1_NL4-3 or HIV-1_TMC126^P9 was used as a starting virus was moderate. Importantly, the two independent selection experiments described above strongly suggested that the emergence of a DRV-resistant HIV-1 variant is substantially delayed compared to the emergence of HIV-1 variants resistant to other PIs, as De Meyers et al. previously described (3), and that HIV-1 does not acquire significant levels of resistance to DRV (see Fig. S2 and S3 in the supplemental material).

Selection of DRV-resistant HIV-1 using a mixture of multi-PI-resistant HIV-1 isolates.

As described above, when a single HIV-1 strain was used as a starting virus for selection against DRV, highly DRV-resistant HIV-1 variants were not obtained. Thus, we employed a mixture of 8 HIV-1 clinical isolates resistant to multiple PIs, expecting that homologous recombination from one isolate to another among them takes place in the presence of escalating doses of DRV and can expedite the emergence of highly DRV-resistant HIV-1 variants. The 8 primary HIV-1 strains were isolated from patients with AIDS who had failed various antiviral regimens after receiving 9 to 11 anti-HIV-1 drugs over 32 to 83 months, and these strains contained 9 to 14 amino acid substitutions corresponding to the protease-encoding region, which are associated with HIV-1 resistance to various PIs (25).

There were a few reportedly DRV resistance-associated mutations (V11I, L33F, and G73S) in the 8 multi-PI-resistant primary isolates (Fig. 1). The mixture of these 8 isolates was obtained by propagation in PHA-PBMC as previously described (25). Each of the 8 isolates was then propagated in a mixture of an equal number of PHA-PBMC and MT-4 cells in an attempt to adapt them to replication in MT-4 cells. The cell supernatant was harvested on day 7 of coculture (PHA-PBMC and MT-4 cells), and the viruses were further propagated in fresh MT-4 cells. When a portion of culture medium was subjected to RT-PCR, molecular cloning, and nucleic acid determination immediately after a mixture of the 8 isolates was added to fresh MT-4 cells, the presence of all 8 isolates was confirmed with the presence of amino acid substitutions corresponding to the protease-encoding gene (day 0 in Fig. 1). However, by day 7, the virus population was comprised of two isolates, HIV-1_C and HIV-1_G. When examined on days 14 and 21, the predominant virus population seemed to have been derived from HIV-1_C (Fig. 1).

FIG. 1.

Sequence analysis of the protease-encoding regions in the mixture of 8 HIV_MDR isolates. Viral RNA was purified from each indicated supernatant using the QIAamp viral RNA minikit (Qiagen Inc., Valencia, CA), and RT-PCR was carried out using the Superscript First-Strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The amino acid sequences of protease deduced from nucleotide sequences of the protease-encoding region of HIV-1 clones determined are shown. The fraction of clones examined is indicated on the right. The amino acid sequence of protease of a wild-type pNL4-3 clone is shown as a reference. Identity with this sequence at individual amino acid positions is indicated (dots).

When the mixed isolates were further propagated in a cell-free transmission manner in fresh MT-4 cells in the presence of increasing concentrations of DRV (Fig. 2), the DRV concentration with which the virus continued to replicate relatively quickly reached 0.1 μM by passage 21. The virus continued to replicate at 1 μM DRV at passage 39 and further propagated eventually in the presence of a 5 μM concentration (passage 51) (Fig. 2). The protease-encoding region of the proviral DNA isolated from infected MT-4 cells was cloned and sequenced at passages 1, 10, 30, and 51 during the DRV selection. Individual protease sequences at each passage are depicted in Fig. 3A. At passage 1, the virus had a variety of amino acid substitutions, L10I, I15V, K20R, L24I, M36I, M46L, F53S, I54V, I62V, L63P, K70Q, V82A, and L89M, compared to wild-type HIV-1_NL4-3. By passage 10, the virus acquired V32I substitutions, and by passage 30, the virus acquired the I84V substitution. By passage 51, the virus had also acquired the L33F, I54M, and V82I substitutions.

FIG. 2.

In vitro selection of HIV-1_MIX and HIV-1_C resistant to DRV. A mixture of 8 HIV-1 isolates resistant to multiple PIs (triangles) or an HIV-1_C strain (circles) was passaged in the presence of increasing concentrations of DRV in MT-4 cells. The selection was carried out in a cell-free manner for a total of 60 passages, with drug concentrations escalating from 0.006 to 5.0 μM. Nucleotide sequences of proviral DNA were determined using cell lysates of HIV-1-infected MT-4 cells at the termination of each indicated passage.

FIG. 3.

Amino acid sequences of HIV-1_MIX and HIV-1_C passaged in the presence of DRV. (A) The amino acid sequences of the Gag-protease-encoding region from 4 different passages of each strain derived from HIV-1_MIX passaged in the presence of DRV are indicated. The top line shows the Gag-protease sequence of the wild-type pNL4-3 clone. Identity of each amino acid with that from pNL4-3 (top) at an individual amino acid position is indicated by a dot. (B) The amino acid sequences of the Gag-protease-encoding region from 4 different passages of each strain derived from HIV-1_C passaged in the presence of DRV are indicated.

Since the predominant virus population seemed to have been derived from HIV-1_C when examined on day 14 (Fig. 1), another selection experiment was conducted using HIV-1_C as a starting isolate. The concentration of DRV with which HIV-1_C grew reached 0.1 μM by passage 25 and 1 μM by passage 51 (Fig. 2). It is of note that after HIV-1_C achieved its replication at a DRV concentration of 0.1 μM, the DRV concentration curve for HIV-1_C substantially diverged from that for HIV-1_MIX (Fig. 2). Also, HIV-1_C replicated poorly and failed to further replicate in the presence of more than 1.8 μM DRV. These data suggest that HIV-1_MIX had some advantages in acquiring DRV resistance.

Failure of selection of DRV-resistant variants using single HIV-1 isolates.

Since it was suspected that genetic homologous recombination was mechanically involved in the emergence of HIV-1_MIX^R, we conducted further selection experiments using each single HIV-1 variant of HIV-1_MIX under the same conditions as the ones we used to obtain HIV-1_MIX. In the process of culture, only HIV-1_C could replicate, and 7 other HIV-1 strains were lost during cell-free transmission. When HIV-1_C was propagated in the presence of increasing concentrations of DRV (Fig. 2), HIV-1 variants resistant to DRV, which replicated at 1.0 μM, emerged by passages 51. The protease-encoding region of the proviral DNA isolated from infected MT-4 cells was cloned and sequenced at passages 1, 10, 31, and 50 upon DRV selection. An individual protease sequence at each passage is depicted in Fig. 3B. At passage 1, the virus had the L10I, I15V, K20R, L24I, M36I, M46L, F53S, I54V, I62V, L63P, K70Q, V82A, and L89M substitutions compared to the HIV-1_NL4-3 sequence. At passage 10, HIV-1_C had the V32I substitution. By passage 31, the virus had acquired the I84V substitution. By passage 50, the virus had acquired the A71V and L89I substitutions.

We also examined whether the virus had acquired mutations in the Gag region on passages 1, 10, 31, and 50 of the selection with DRV. At passage 1, HIV-1_C had the K15R, Q28R, R76K, I82V, V84T, D93E, T122A, N124D, N125S, Q127T, V159I, S176A, H252N, T280V, S368C (the p24/p2 cleavage site substitution), P373S, I376V, I378V, K380R, T389I, K403R, R406K, D425E, A431V (the p7/p1 cleavage site substitution), L449F (the p1/p6 cleavage site substitution), P478T, L483M, A487S, S488A, R490K, and S495N substitutions. By passage 10, the V35I substitution emerged, and it persisted thereafter. By passage 50, the V128I and Q199H substitutions had additionally emerged (Fig. 3B).

HIV-1_MIX that was selected to be highly DRV resistant (HIV-1_MIX^R) likely developed DRV resistance through possible homologous recombination.

Since HIV-1_MIX^R apparently had some advantages in acquiring DRV resistance without significantly compromising viral fitness compared to HIV-1_C, as described above, we examined whether HIV-1_MIX^R acquired amino acid substitutions in the Gag region at passages 1, 10, 30, and 51 of DRV selection (Fig. 3A). On passage 1, the virus had the K15R, Q28R, R76K, I82V, V84T, D93E, T122A, N124D, N125S, Q127T, V159I, S176A, H252N, T280V, S368C (the p24/p2 cleavage site substitution), P373S, I376V, I378V, K380R, T389I, K403R, R406K, D425E, A431V (the p7/p1 cleavage site substitution), L449F (the p1/p6 cleavage site substitution), P478T, L483M, A487S, S488A, R490K, and S495N substitutions. By passage 10 and beyond, the H219Q (within the CypA-binding loop), I223V (within the CypA-binding loop), and I247V substitutions emerged and persisted. By passage 30 and beyond, the V35L and A118V substitutions emerged. By passage 51, the G11E and L21M substitutions emerged.

It is of note that HIV-1_MIX had acquired the two amino acid substitutions H219Q and I223V, located in the CypA binding loop of the Gag protein, as early as by passage 10 (Fig. 3A). Since certain amino acid substitutions are known to affect the viral fitness of HIV-1 (7) and it was possible that HIV-1_MIX might have acquired these two substitutions through homologous recombination, we examined the amino acid sequences of each isolate of HIV-1_MIX. Although HIV-1_MIX^R was thought to have derived predominantly from HIV-1_C, HIV-1_C was devoid of H219Q and I223V (see Fig. S4 in the supplemental material). Since HIV-1_C selected to be DRV resistant (HIV-1_C^R) was still devoid of the two substitutions, it was thought more likely that HIV-1_MIX^R acquired H219Q and I223V from other HIV-1 variants within HIV-1_MIX through homologous recombination rather than spontaneously acquiring the substitutions during the DRV selection (Fig. 2). Indeed, HIV-1_A and HIV-1_B had I223V; HIV-1_G carried both H219Q and I223V (see Fig. S4). Thus, it was thought that although all three strains (HIV-1_A, HIV-1_B, and HIV-1_G) apparently disappeared during selection with DRV, either or all of the three strains served as a donor(s) to provide the two substitutions to HIV-1_MIX^R.

The role of another amino acid substitution, I247V, which has been identified in HIV-1 isolates (13 of 156 different HIV-1 isolates in the HIV Sequence Compendium 2008/Los Alamos HIV Sequence Database) remains to be elucidated, although it is possible that I247V was incorporated into HIV-1_MIX^R alongside H219Q and I223V through homologous recombination. These data suggest that at least these two substitutions contributed the favorable replicative ability of HIV-1_MIX^R compared to that of HIV-1_C^R. We generated four different clones that contained a variety of amino acid substitutions identified in the protease- and Gag-encoding genes of the DRV-selected mixture population (see Table S2 in the supplemental material); however, all such recombinant HIV-1 clones we generated failed to replicate (data not shown).

H219Q and I223V substitutions reduce the virion content of CypA.

One salient difference between HIV-1_MIX^R and HIV-1_C^R was the presence of the H219Q and I223V substitutions as described above. These two substitutions are located in the CypA binding loop of the Gag protein, which regulates the intravirion content of CypA, which is believed to play an essential role early in the HIV-1 replication cycle. CypA perhaps destabilizes the capsid (p24 Gag protein) shell during viral entry and uncoats (6) and/or performs an additional chaperon function, facilitating correct capsid condensation during viral maturation (12).

Also, based on the data from crystal structure analyses by Gamble et al. (6) of the p24 Gag protein complexed with CypA, showing that His²¹⁹ binds to Asn⁷¹ and Gln¹¹¹ of CypA through a hydrogen bond and a hydrophobic contact(s), respectively, we postulated that the H219Q and I223V substitutions cancel or weaken such interactions, resulting in the reduction of binding of p24 to CypA and of CypA incorporation into daughter virions in CypA-rich MT-4 cells. Thus, we determined the CypA content of the cells where HIV-1 was propagated and of virions used in the present study. As shown in Fig. S5A and B in the supplemental material, the CypA contents in 10⁵ MT-2 cells (relative density, 100%), 10⁵ H9 cells (91.0%), and 10⁵ MT-4 cells (66.6%) appeared to be greater than those in 10⁵ PHA-PBMC (21.6%), suggesting that MT-2 and H9 cells contained 3 to 5 times as much CypA per cell as PHA-PBM, in agreement with our previous findings (7). We subsequently determined the virion-associated CypA amounts in HIV-1_NL4-3, HIV-1_C^P51 (replicative at 1 μM DRV), HIV-1_MIX^P39 (replicative at 1 μM DRV), and HIV-1_MIX^P51 (replicative at 5 μM DRV), employing Western blotting using anti-p24 Gag and anti-CypA antisera. The virions in each supernatant were pelleted by ultracentrifugation and subsequently subjected to SDS-PAGE.

As shown in Fig. S5B in the supplemental material, p24 signal densities in HIV-1_NL4-3, HIV-1_C^P51, HIV-1_MIX^P39, and HIV-1_MIX^P51 were 100, 75.4, 101.2, and 83.3%, respectively, compared with that in HIV-1_NL4-3 (serving as a standard at 100%). CypA signal densities in HIV-1_NL4-3, HIV-1_C^P51, HIV-1_MIX^P39, and HIV-1_MIX^P51 were 100, 103.4, 68.4, and 61.9%, respectively, compared with that in HIV-1_NL4-3 (serving as a standard at 100%) (see Fig. S5C). Ratios of densities of the CypA signal relative to each p24 Gag signal were 1.0, 1.37, 0.68, and 0.74 for HIV-1_NL4-3, HIV-1_C^P51, HIV-1_MIX^P39, and HIV-1_MIX^P51, respectively. These data suggest that both the H219Q and I223V substitutions improved HIV-1_MIX^R replication in CypA-rich MT4 cells by reducing CypA incorporation into daughter virions. The impact of the H219Q and I223V substitutions on the structure of the CypA-binding loop of the Gag protein was also examined using molecular dynamic simulation (see Fig. S6).

Significantly reduced susceptibilities of HIV-1_MIX^R to DRV and other PIs.

We also examined the susceptibilities of HIV-1_MIX^R to a variety of FDA-approved PIs, including DRV in MT-4 cells (Table 1). HIV-1_MIX harvested on passage 1 (HIV-1_MIX^P1) was already resistant to APV with an EC₅₀ (0.28 μM) 28-fold greater than the EC₅₀ with the wild-type clinical isolate HIV-1_ERS104pre, to NFV with a 41-fold greater EC₅₀ (0.66 μM), and to LPV with a 17-fold greater EC₅₀ (0.26 μM). The EC₅₀s of RTV and IDV against HIV-1_MIX^R were both >1 μM. In contrast, SQV, ATV, and TPV relatively maintained their antiviral activity against HIV-1_MIX at early stages of DRV selection. However, HIV-1_MIX^P39 was found to be highly resistant to DRV (EC₅₀ >333-fold greater than that against HIV-1_ERS104pre). HIV-1_MIX^P51 was highly resistant to APV, IDV, NFV, RTV, LPV, and ATV (all with EC₅₀s of >1 μM) and also had significant resistance against SQV (33-fold increases in the EC₅₀) and TPV (18-fold increases in the EC₅₀) (Table 1). We also determined replication kinetics of HIV-1_NL4-3 along with that of HIV-1_MIX^P39, which turned out to be capable of replicating in the presence of 1 μM DRV. As shown in Fig. 4, when HIV-1_MIX^P39 was propagated in MT-4 cells in the presence or absence of 0.1 or 1 μM DRV, there was no discernible difference observed in the replication kinetics of HIV-1_MIX^P39 compared to that of HIV-1_NL4-3 in the absence of DRV.

TABLE 1.

High levels of HIV-1_MIX^R resistance to DRV and other PIs

Virus	EC50 (μM) of druga
	SQV	APV	IDV	NFV	RTV	LPV	ATV	TPV	DRV
HIV-1ERS104pre (wild type)	0.009	0.025	0.021	0.016	0.030	0.015	0.005	0.10	0.003
HIV-1MIXP1	0.034 (4)	0.28 (11)	>1 (>48)	0.66 (41)	>1 (>33)	0.26 (17)	0.021 (4)	0.060 (0.6)	0.005 (2)
HIV-1MIXP10	0.026 (3)	0.45 (18)	>1 (>48)	>1 (>63)	>1 (>33)	0.22 (14)	0.035 (7)	0.023 (0.2)	0.013 (4)
HIV-1MIXP20	0.27 (30)	>1 (>40)	>1 (>48)	>1 (>63)	>1 (>33)	0.39 (26)	0.39 (78)	0.18 (2)	0.12 (40)
HIV-1MIXP30	0.30 (33)	>1 (>40)	>1 (>48)	>1 (>63)	>1 (>33)	>1 (>67)	>1 (>200)	0.33 (3)	0.31 (100)
HIV-1MIXP39	0.35 (39)	>1 (>40)	>1 (>48)	>1 (>63)	>1 (>33)	>1 (>67)	>1 (>200)	0.41 (4)	>1 (>333)
HIV-1MIXP51	0.30 (33)	>1 (>40)	>1 (>48)	>1 (>63)	>1 (>33)	>1 (>67)	>1 (>200)	1.79 (18)	>1 (>333)

^aHIV-1_MIX was propagated in the presence of increasing concentrations of DRV, harvested at passages 1, 10, 20, 30, 39, and 51(HIV-1_MIX^P1, HIV-1_MIX^P10, HIV-1_MIX^P20, HIV-1_MIX^P30, HIV-1_MIX^P39, and HIV-1_MIX^P51, respectively), and examined for susceptibilities to DRV and other PIs listed. In the assay, MT-4 cells (10⁴) were exposed to each HIV-1 preparation (100 TCID₅₀s), and to determine EC₅₀s of each drug against HIV-1, the inhibition of p24 Gag protein production by each drug was used as an endpoint. The numbers in parentheses represent fold changes of EC₅₀s against each HIV-1 preparation compared to EC₅₀s against a wild-type clinical strain, HIV-1_ERS104pre. All assays were conducted in triplicate, and the mean values are shown.

FIG. 4.

Replication kinetics of HIV-1_MIX^P39 in the presence of DRV. MT-4 cells were exposed to HIV-1_NL4-3 or HIV-1_MIX^P39 and cultured in the presence or absence of 0.1 or 1.0 μM DRV. Viral replication was monitored by measuring p24 Gag protein in the culture supernatant.

DISCUSSION

Darunavir (DRV) potently inhibits the enzymatic activity and dimerization of HIV-1 protease (15, 16) and exhibits a high genetic barrier to HIV-1 development of resistance to DRV (4, 5). In a relatively small percentage of heavily drug-experienced patients, a variety of amino acid substitutions potentially related to HIV-1 resistance to DRV have been reported (4, 19); however, the mechanism of development of HIV-1 resistance to DRV still remains to be elucidated. For determining the mechanism of development of HIV-1 resistance to DRV, it is critical to acquire highly DRV-resistant HIV-1 variants, which should be of great utility.

In the present study, we attempted to select DRV-resistant HIV-1 variants by propagating a mixture of HIV-1 variants (HIV-1_MIX) isolated from 8 patients with AIDS who had received antiretroviral therapy over 32 to 83 months and were not responding to antiretroviral regimens in the presence of DRV. DRV-resistant HIV-1 at passage 51 (HIV-1_MIX^P51) replicated in the presence of 5 μM DRV and contained 14 mutations (L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82I, I84V, and L89M) encoded in the protease-encoding region.

A set of mutations (V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V, and L89V) was identified in HIV-1 isolated from those failing DRV-containing regimens that were associated with a diminished virological response to DRV boosted with a low dose of ritonavir at week 24 in the POWER studies (4). The most common mutations identified were V32I, L33F, I54M/L, I84V, and L89V (17, 21). In the present study, HIV-1 variants resistant to DRV, which replicated in the presence of 1 and 5 μM DRV, emerged by passages 39 and 51, respectively. The protease-encoding region of the proviral DNA isolated from infected MT-4 cells was cloned and sequenced at passages 1, 10, 30, and 51 upon DRV selection. Individual protease sequences at each passage are depicted in Fig. 3A. On passage 1, the virus had L10I, I15V, K20R, L24I, M36I, M46L, F53S, I54V, I62V, L63P, K70Q, V82A, and L89M substitutions compared to wild-type HIV-1_NL4-3. By passage 10 and beyond, the virus additionally acquired a V32I substitution. By passage 30 and beyond, the virus contained an I84V substitution. By passage 51, the virus had acquired L33F, I54M, and V82I substitutions and was found to contain 14 mutations, L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82I, I84V, and L89M, corresponding to the protease-encoding region. It is of note that the four mutations (V32I, L33F, I54M, and I84V) HIV-1 acquired in the present study were the ones identified in highly DRV-resistant HIV-1 variants.

With respect to HIV-1's acquisition of cross-resistance to TPV and DRV, our recent results showed both compounds blocked the dimerization of HIV-1 protease in the fluorescence resonance energy transfer (FRET)-based HIV-1 expression assay (15). Since HIV-1_MIX^P51 can propagate in the presence of 5 μM DRV, it is likely that DRV is no longer capable of inhibiting the dimerization of the protease with the mutations seen in HIV-1_MIX^P51. Considering that conventional protease inhibitors, such as SQV, RTV, NFV, APV, and LPV, failed to block the dimerization of HIV-1 protease (15), it appears that the activity inhibiting the proteolytic function of protease is independent from that inhibiting the dimerization of HIV-1 protease, although it should be determined what mutations (a single mutation or combined mutations) seen in HIV-1_MIX^P51 are responsible for the viral acquisition of the ability to escape from DRV's protease dimerization inhibition. It is also yet to be determined whether and what mutations seen in HIV-1_MIX^P51 can confer resistance to TPV on HIV-1.

During reverse transcription, reverse transcriptase is known to frequently switch a template from one genomic RNA strand to another, yielding recombinant proviral DNA, which represents a mosaic consisting of multiple parent genomic components. Indeed, there is firm evidence that a single CD4⁺ target cell can be infected with multiple HIV-1 virions both in vitro and in vivo (2, 13). If resultant proviral DNA acquires mutations in one strand that confer HIV-1 resistance to one drug and mutations in the other strand that are associated with HIV-1 resistance to the other drug, such proviral DNA-containing daughter virions will be resistant to both drugs, a process called homologous recombination (1, 20, 26). Homologous recombination is thus highly likely to accelerate the emergence of multidrug and multiclass drug resistance in infected individuals. In the present work, the nucleic acid sequence of the protease-encoding region of HIV-1_MIX^P51 was virtually identical to that of HIV-1_C^R, suggesting that HIV-1_C had resistance to DRV, immediately predominated over other 7 HIV-1 isolates, and continued to propagate in the presence of DRV, suggesting a lack of involvement of homologous recombination in the emergence of highly DRV-resistant HIV-1 variants. Thus, we further conducted a selection experiment using HIV-1_C as a starting HIV-1 isolate. However, HIV-1_C's DRV resistance acquisition was substantially delayed compared to DRV resistance acquisition of HIV-1_MIX^P51 in two independent selection experiments (Fig. 2 and data not shown). Therefore, we determined the nucleic acid sequence of the gag gene in both HIV-1_MIX^P51 and HIV-1_C^R and readily found that the former had two mutations (H219Q and I223V) in the CypA-binding loop and I247V by passage 10 while the latter lacked all three mutations (Fig. 3A and B). Among the 8 isolates used, only the original HIV-1_G contained these three mutations (see Fig. S4 in the supplemental material). The H219Q substitution in the viral CypA binding loop, a polymorphic amino acid change, has been shown to confer a replication advantage on HIV-1 in CypA-rich target cells (8). As expected, both HIV-1_MIX^P39 and HIV-1_MIX^P51 had substantially less CypA content within virions (see Fig. S5).

When we generated four different clones that contained a variety of amino acid substitutions identified in the protease- and Gag-encoding genes of the DRV-selected mixture population (see Table S2 in the supplemental material), only recombinant HIV_p1 (rHIV_p1) replicated, while other recombinant HIV-1 clones failed to replicate, although all the amino acid substitutions identified in the protease- and Gag-encoding genes of the replicative mixture HIV-1 population were introduced to such recombinant clones. When we generated recombinant HIV-1 virions with two amino acid substitutions mutated back (H219Q/I223V to H219/I223; designated rHIV_p530-MB), these virions also failed to replicate. Suspecting that other amino acid substitutions residing in a minor HIV-1 population helped the mixture population replicate through homologous recombination in the mixture population, we added such an amino acid substitution, A196T or A196S, to rHIV_p530-MB (designated rHIV_p530-MB196T and rHIV_p530-MB196T). However, such recombinant clones also failed to replicate. It is unclear at this time how these recombinant HIV-1 virions failed to replicate; however, it is possible that other unidentified yet critical amino acid substitutions are required for the replication of the DRV-selected mixture population. It should be noted that in general, the population size of HIV-1 in patients is relatively larger, the magnitude of replication is greater, and the duration of HIV-1 replication is longer than in a test tube. It is also of note that the appearance of mutations can be largely affected by stochastic phenomena, i.e., rates and orders of appearance of mutations in vitro compared to the in vivo situation.

In summary, the present results demonstrated the first successful in vitro selection of highly DRV-resistant HIV-1 variants and a new method for efficiently selecting drug-resistant HIV-1 variants in a test tube when such variants are hardly generated in vitro and in vivo. The present data also suggest that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance with robust viral fitness comparable to the fitness of wild-type HIV-1 when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination occurs.