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Human immunodeficiency virus type 1 (HIV-1) entry is an attractive target for therapeutic intervention. Two drugs that inhibit this process have been approved: the fusion inhibitor T20 (enfuvirtide [Fuzeon]) and, more recently, the CCR5 blocker maraviroc (Selzentry). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared to the first-generation peptide T20. We selected T1249-resistant HIV-1 variants in vitro by serial virus passage in the presence of increasing T1249 doses after passage with wild-type and T20-resistant variants. Sequence analysis revealed the acquisition of substitutions within the HR1 region of the gp41 ectodomain. The virus acquired mutations of residue V38 to either E or R in 10 of 19 cultures. Both E and R at position 38 were confirmed to cause resistance to T1249, as well as cross-resistance to T20 and C34, but not to the third-generation fusion inhibitor T2635. We also observed substitutions at residues 79 and 90 (Q79E and K90E), which provide modest resistance to T1249 and, interestingly, T2635. Thus, the gp41 amino acid position implicated in T20 resistance (V38 replaced by A, G, or W) is also responsible for T1249 resistance (V38 replaced by E, R, or K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that call for similar but not identical resistance mutations. All T1249-resistant viruses with changes at position 38 are cross resistant to T20, but not vice versa. Furthermore, substitutions at position 38 do not provide resistance to the third-generation inhibitor T2635, while substitution at positions 79 and 90 do, suggesting different resistance mechanisms.
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the envelope (Env) glycoproteins gp120 and gp41. Env is arranged on the virus particle as trimeric spikes, comprising three gp120 molecules and three gp41 molecules, anchored within the viral membrane via the gp41 transmembrane domain. The first step in HIV-1 entry is the binding of gp120 to CD4 followed by binding to a coreceptor on the T-cell surface that triggers conformational changes in Env, resulting in the insertion of the hydrophobic N-terminal fusion peptide of gp41 into the target cell membrane (reviewed in reference 18). Subsequent changes within gp41 involve two leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), assembling into a highly stable six-helix bundle structure which juxtaposes the viral and cellular membranes for the fusion event (9, 49, 52). The change in free energy associated with this structural transition is predicted to be sufficient to cause lipid mixing and membrane fusion (25, 39). Peptide fusion inhibitors that bind to one of the HR motifs can block this conformational switch and thus inhibit viral entry (4, 10, 34, 54).
T20, the first approved fusion inhibitor for HIV-1, is a 36-amino-acid peptide that mimics HR2 and acts by binding to HR1, thus preventing the HR1-HR2 interaction (Fig. (Fig.1)1) (29, 32, 53, 54). In vitro passaging of HIV-1 in the presence of increasing T20 concentrations resulted in the selection of resistant virus variants with mutations within a stretch of three HR1 amino acids, glycine-isoleucine-valine (the GIV motif, HXB2 amino acid positions 36 to 38 of gp41) (16, 22, 43, 44, 55, 56). Resistance mutations have also been identified within the viral quasispecies of patients on T20 therapy, specifically at positions 36 to 45 (1, 5, 12, 40, 42, 51).
The fusion inhibitor C34 also corresponds to part of HR2 (Fig. (Fig.1;1; see Fig. Fig.5b)5b) (35). The inhibitory activity of C34 depends on its ability to bind to a prominent hydrophobic pocket in HR1, which may make it less susceptible to the evolution of drug-resistant viruses (36, 43). In vitro passaging of HIV-1 in the presence of increasing C34 concentrations resulted in the selection of resistant virus with the V38E substitution (2). An second-generation fusion inhibitor, T1249, which has greater inhibitory potency than C34 and is also effective against T20-resistant variants in vitro and in vivo, was developed (20, 21). T1249 is composed of sequences from HIV-1, HIV-2, and simian immunodeficiency virus (20, 47). The C-terminal region of T1249 is almost identical to T20, but the peptide differs in the N-terminal sequence, which extends a further three residues (total length, 39 amino acids) and bears little similarity to T20 in its amino acid composition (Fig. (Fig.1).1). In vitro resistance has been described in a study using a randomized mutagenesis strategy in HR1 (13). Changes at positions 37, 38, and 40 were found to cause T1249 resistance. In vivo resistance development during treatment with T1249 was linked to mutations G36D, V38E, Q40K, N43K, and A50V in HR1 and N126K and S138A in HR2 (20, 38). Recently, third-generation fusion inhibitor peptides were described as having increased helical structure and high HR1/HR2 bundle stability (T2635 and variants thereof) (17). These peptides are active against T20- and T1249-resistant viruses, and no T2635 resistance has been reported so far.
In this study, we selected T1249 drug-resistant HIV-1 variants in vitro. Our results demonstrate that the same amino acid position, position 38, that is implicated in T20 and C34 resistance is also involved in T1249 resistance, although different substitutions are required. These findings indicate that T20 and T1249 exhibit very similar inhibition modes that trigger similar but not identical escape routes. To evaluate the clinical significance of the described mutations, the cross-resistance to T20 and T2635 was determined. Interestingly, substitutions at positions 38 caused increased susceptibility to T2635. In contrast, T1249 resistance mutations at positions 79 and 90 confer cross-resistance to T2635.
Amino acids are indicated by the single-letter code. Peptides were synthesized by solid-phase peptide synthesis by using a 4-(2,4-dimethoxyphenyl-Fmoc (Rink-Amide) resin (BACHEM Biochemica, Heidelberg, Germany) on a Syro synthesizer (MultiSynTech, Witten, Germany). All amino acids were purchased from BACHEM Biochemica and used as N-α-(Fmoc) protected with side chain functionalities protected as N-tert-butoxycarbonyl (KW), O-tert-Butyl (DESTY), N-Trityl (HNQ), S-Trityl (C), S-2,2,4,6,7 (C), or N-pentamethyl dihydrobenzofurane-5-sulfonyl (R) groups. A coupling protocol using a 6.5-fold excess of HBTU-HOBt-amino acid-DIPEA (1:1:1:2) in NMP with a 30-min activation time using double couplings was employed. Peptides were cleaved from the resin by reaction with trifluoroacetic acid (TFA; 15 ml g−1 resin) containing 13.3% (by weight) phenol, 5% (by volume) thioanisole, 2.5% (by volume) 1,2-ethanedithiol, and 5% (by volume) milliQ-purified H2O for 2 to 4 h at room temperature. The crude peptides were purified by reversed-phase high-performance liquid chromatography (HPLC), either on a DeltaPack (25 mm or 40 mm [inner diameter] by 100 mm [length], 15-μm particle size, 100-Å pore size; Waters, Milford, MA) or on an XTerra (50 mm by 4.6 mm [inner diameter], 2.5-μm particle size; Waters, Milford, MA) RP-18 preparative C18 column with a linear AB gradient of 1 to 2% solvent B min−1 where solvent A was 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile. The correct primary ion molecular weights of the peptides was confirmed by electron-spray ionization mass spectrometry on a ZQ (Micromass, Almere, The Netherlands) or Quattro II (VG Organic, Cheshire, United Kingdom) mass spectrometer.
For the selection of T1249-resistant viruses, SupT1 cells were transfected with 1 μg DNA of either the wild-type HIV-1LAI molecular clone or several T20-resistant variants: V38A, V38G, V38W, and V38W/N126K mutants (5). Transfected cells were split 1 day posttransfection into three to six separate culture flasks, and 0.5 × 106 fresh SupT1 cells were added to initiate the evolution cultures. We started the selection with a concentration of 5 ng/ml T1249 for the T20-sensitive wild type and 20 ng/ml for T20-resistant variants, which is sufficient to reduce replication by >90%. We initially split 100 μl culture (cells and supernatant) when required onto uninfected SupT1 cells. At each passage, the T1249 drug concentration was increased on average 1.5 times. When HIV-induced cytopathic effects and increased CA-p24 production were apparent, virus replication was maintained by passage of cell-free culture supernatant onto uninfected SupT1 cells. We used escalating volumes of cell-free culture supernatant to infect 5 ml fresh SupT1 cells (0.5 × 106 cells). Initially, we started by passaging 100 μl cell-free supernatant onto fresh cells. We used less supernatant in subsequent passages, from 100 μl in the second passage to a minimum of 10 μl. Cells and supernatant samples were taken at regular time points and stored at −70°C. Cell culturing, transfections, and CA-p24 determination were performed as previously reported (5, 24).
HIV-1-infected cells (1 ml culture) were pelleted by centrifugation at 4,000 rpm for 4 min and the supernatant was analyzed for CA-p24 content and stored at −70°C. The cell pellet was lysed in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.5% Tween 20 and incubated with 500 μg proteinase K/ml at 56°C for 60 min and heat inactivated at 95°C for 10 min. The complete env genes from proviral DNA sequences were PCR amplified from solubilized cellular DNA by using the Expand high-fidelity PCR system according to the manufacturer's instructions (Roche, Mannheim, Germany). Briefly, after incubation for 5 min at 94°C, the reaction mixture was subjected to 35 PCR cycles in a type 9700 DNA thermal cycler (Perkin Elmer, Waltham, MA), with each cycle including a denaturation step for 30 s at 94°C, an annealing step for 30 s at 60°C, and an extension step for 3 min at 68°C. This was followed by a final extension step of 7 min at 68°C. The PCR was performed with 50 ng sense and antisense primers (WS1 [5′-ATAAGCTTAGCAGAAGACA-3′] and 3′envMD4 [5′-GCAAAATCCTTTCCAAGCCC-3′]) in a 50-μl PCR. DNA products were analyzed on a 1% agarose gel that was prestained with ethidium bromide. PCR products were sequenced directly using the DNA BigDye Terminator sequencing kit (ABI, Foster City, CA) and an ABI 377 automated sequencer.
The full-length molecular clone of HIV-1LAI (pLAI) was used to produce wild-type and mutant viruses (41). We already described the wild-type variant with the GIV-SNY sequence (V38) as observed in a patient isolate (different from the GIV-NNY sequence that is present in the HIV-1LAI molecular clone) and the T20-resistant V38A, V38G, and V38W variants (5). The GIV-SNY (N125S) variant shows a slight decrease in fitness compared to the GIV-NNY variant (data not shown). The plasmid pRS1, designed to subclone mutant env genes, was described previously (45). Mutations were introduced into pRS1 by using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and the entire env gene was verified by DNA sequencing. Mutant env genes (corresponding to V38E, V38R, V38K, N43K, Q79E, and K90E mutations) in pRS1 were cloned back into pLAI as SalI-BamHI fragments. Because of the common appearance of charged amino acids at position 38, we also made a V38K mutant, even though we did not observe this mutant in the evolution experiments.
The SupT1 T-cell line was maintained in RPMI 1640 supplemented with 10% fetal calf serum and penicillin and streptomycin (both at 100 U/ml) and incubated at 37°C with 5% CO2. SupT1 cells were transfected with HIV-1 molecular clones by electroporation. Briefly, 5 × 106 cells were washed in RPMI 1640 with 20% fetal calf serum, mixed with 1 μg of DNA in 0.4-cm cuvettes, and electroporated at 250 V and 975 μF, followed by resuspension of cells in RPMI 1640 with 10% fetal calf serum. The transfected cells were split at day 1 posttransfection and cultured with 100 ng/ml T20, 100 ng/ml C34, and 25, 100, or 200 ng/ml T1249. CA-p24 production was determined from culture supernatant taken at various days posttransfection.
The TZM-bl reporter cell line (15, 51) stably expresses high levels of CD4 and HIV-1 coreceptors CCR5 and CXCR4 and contains the luciferase and β-galactosidase genes under the control of the HIV-1 long-terminal-repeat promoter. The TZM-bl cell line was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (TZM-bl from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. [Durham, NC]). One day prior to infection, TZM-bl cells were plated on a 96-well plate in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1× minimum essential medium nonessential amino acids, and penicillin-streptomycin (both at 100 units/ml) and incubated at 37°C with 5% CO2. Each virus variant was produced in C33A cells by calcium phosphate precipitation as previously described (14). A fixed amount of virus (1 ng CA-p24) was preincubated for 30 min at room temperature with 0, 0.15, 0.46, 1.37, 4.12, 12.35, 37.04, 111.1, 333.3, 1,000 or 3,000 ng/ml of T20 or T1249 or 0, 0.051, 0.15, 0.46, 1.37, 4.12, 12.35, 37.04, 111.1, 333.3, or 1,000 ng/ml of T2635. This mixture was added to the cells in the presence of 400 nM saquinavir (Roche, Mannheim, Germany) and 40 μg/ml DEAE in a total volume of 200 μl. Two days postinfection, the medium was removed and cells were washed once with phosphate-buffered saline (PBS) and lysed in reporter lysis buffer (Promega, Madison, WI). Luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI) and a Glomax luminometer according to the manufacturer's instructions (Turner BioSystems, Sunnyvale, CA). All infections were performed in duplicate, and luciferase measurements were also performed in duplicate. Uninfected cells were used to correct for background luciferase activity. The infectivity of each mutant without inhibitor was set at 100%. Nonlinear regression curves were determined and 50% inhibitory concentrations (IC50s) were calculated using Prism software version 4.0c. The relative infectivities of molecular clones compared to HIV-1LAI were calculated for all T1249 escape mutants. Luciferase activity without inhibitor of quadruple infections was measured in duplicate and corrected for background luciferase activity. Infectivity of HIV-1LAI wild type was normalized to 100% and relative infectivity for the other mutants was calculated.
The recombinant N36(L6)C34 model peptide and its variants were expressed in the Escherichia coli strain BL21(DE3)/pLysS by using a modified pET3a vector (Novagen, San Diego, CA). The sequence of N36(L6)C34 is SGIVQQQSNL LRAIEAQQHL LQLTVWGIKQ LQARVLSGGR GGWMDWEREI SNYTKQIYTL IEESQNQQEK NEQELL (with the six-residue linker underlined). Substitutions were introduced into the pN36/34 plasmid (5) by the method used by Kunkel (30) and were verified by DNA sequencing. The cells were grown at 37°C in LB medium to an optical density of 0.7 at 600 nm and harvested by centrifugation 4 h postinduction with 0.5 mM isopropylthio-β-d-galactoside. Cells were lysed by glacial acetic acid and centrifuged to separate the soluble fraction from inclusion bodies. The soluble fraction containing peptide was subsequently dialyzed into 5% acetic acid overnight at 4°C. Peptides were purified from the soluble fraction to homogeneity by reverse-phase HPLC (Waters, Milford, MA) on a C18 preparative column (Vydac, Hesperia, CA) by using a water-acetonitrile gradient in the presence of 0.1% TFA and lyophilized. Peptide identities were confirmed by electrospray mass spectrometry (Voyager Elite; PerSeptive Biosystems, Framingham, MA). Protein concentrations were determined by the method of Edelhoch (19). Proteinase K digestion was performed with protease/protein ratios of 1:100 (wt/wt) at room temperature in PBS (pH 7.0). Proteolysis was quenched by addition of phenylmethylsulfonyl fluoride to a final concentration of 2 mM. Proteolytic fragments were analyzed by reverse-phase HPLC as described above and identified by N-terminal sequencing and mass spectrometry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on 16.5% polyacrylamide gels using a Tris-Tricine buffer system (46).
Circular dichroism (CD) experiments were performed on an 62A/DS (Aviv Associates, Lakewood, NJ) spectropolarimeter equipped with a thermoelectric temperature control at 10 μM peptide concentration in PBS (50 mM sodium phosphate, pH 7.0, 150 mM NaCl). CD spectra were collected from 260 to 200 nm at 4°C, using an average time of 5 s, a cell path length of 0.1 cm, and a bandwidth of 1 nm. An ellipticity value at 222 nm ([θ]222) value of −33,000 degrees cm2 dmol−1 was taken to correspond to 100% helix (11). Thermal stability was determined by monitoring [θ]222 as a function of temperature. Thermal melts were performed in two-degree intervals with a 2-min equilibration at the desired temperature and an integration time of 30 s. All melts were reversible. Superimposable folding and unfolding curves were observed, and >90% of the signal was regained upon cooling. Temperatures of midpoint unfolding transitions (Tm) were estimated by evaluating the maximum of the first derivative of [θ]222 in relation to the temperature data (8). Equilibrium ultracentrifugation measurements were carried out on an XL-A analytical ultracentrifuge equipped with an An-60 Ti rotor (Beckman Coulter, Fullerton, CA) at 20°C as described previously (48). Protein solutions were dialyzed overnight against PBS (pH 7.0), loaded at initial concentrations of 10, 30, and 100 μM, and analyzed at rotor speeds of 20,000 and 23,000 rpm. Data were acquired at two wavelengths per rotor speed setting and processed simultaneously with a nonlinear least-squares fitting routine (26). Solvent density and protein partial specific volume were calculated according to solvent and protein composition, respectively (31). The apparent molecular masses of all N36(L6)C34 variants were within 10% of that calculated for an ideal trimer, with no systematic deviation of the residuals.
T1249-resistant HIV-1 isolates were selected by repeated passage of HIV-1LAI on the SupT1 T-cell line in the presence of increasing concentrations of T1249. We multiple independent cultures with different HIV-1 variants (V38 wild-type virus and T20-resistant variants V38A, V38G, V38W, and V38W/N126K). Cultures were started by transfection of the HIV-1LAI molecular clones into SupT1 cells. T1249 was added at concentrations of 5 ng/ml for the T20-sensitive wild-type virus (V38) and 20 ng/ml for all T20-resistant variants, a drug level that is sufficient to reduce virus replication by >90%. Drug pressure was increased on average 1.5-fold at each passage, and virus and cell samples were stored at several time points. Viral replication was monitored via CA-p24 measurements and the appearance of virus-induced syncytia. Sequence analysis was performed on day 46 for cultures started with the wild-type virus and on day 33 for cultures started with the T20-resistant viruses (Table (Table1).1). The wild-type and T20-resistant viruses replicated efficiently with 40 and 55 ng/ml T1249, respectively.
We PCR amplified the gp41 ectodomain from proviral DNA and analyzed the sequence for acquired mutations. Sequence analysis of the viral population revealed the acquisition of mutations within the HR1 region, but no changes in HR2 were detected. Ten of the 19 cultures acquired mutations at position 38, which is also implicated in T20 and C34 resistance (one V38E mutant, two A38E mutants, three G38R mutants, and four W38R mutants). Interestingly, V38 and V38A evolve to E (observed three times), whereas V38G and V38W (alone and combined with N126K) evolve to R (observed seven times). This pattern is governed by the underlying codon changes (Table (Table1;1; see Discussion for details). We also observed other gp41 changes: Q79E (culture V5), K90E (culture V1), and N43K (in combination with V38A; culture A1). A double mutation, A38E/A96T, was also observed in culture A2. Cultures V2, V4, V6, W3, WK3, and WK4 did not reveal any mutational changes within the gp41 ectodomain, suggesting that T1249 resistance in these variants is caused by changes outside the gp41 ectodomain, for example in gp120. We constructed the HIV-1LAI V38E, V38R, Q79E, K90E, and N43K mutants. The N43K mutant was generated in the wild-type context, thus, without the V38A substitution. Because of the common appearance of charged amino acids at position 38, we also constructed a V38K mutant, although we did not observe this mutant during the evolution experiments.
We tested the impact of the observed gp41 mutations on in vitro virus replication and resistance to T1249, T20, and C34. Viral DNA constructs were transfected into the SupT1 T-cell line and cultured in the presence or absence of inhibitor (Fig. (Fig.2).2). Delayed virus spread in the absence of drug indicated that some variants have a reduced replication capacity (e.g., V38K, V38R, Q79E, and K90E variants) (Fig. (Fig.2a).2a). However, the V38E and N43K variants replicated at levels similar to those of the wild type.
As expected, replication of the wild-type virus (V38) was strongly inhibited by all inhibitors. In contrast, all HR1 variants with substitutions at amino acid position 38 (V38E, V38R, and V38K) showed resistance to T1249 and cross-resistance to T20 and C34. The V38E variant could replicate efficiently without drug and at T1249 concentrations up to 25 ng/ml but was inhibited at 100 ng/ml T1249. This mutant was fully resistant to T20 and C34 at concentrations of 100 ng/ml. The V38R and V38K variants were more resistant to T1249 than V38E and fully resistant to 100 ng/ml of T20 and C34. The HR1 mutant Q79E exhibited reduced replication capacity and showed modest resistance to T1249 with slight cross-resistance to T20 and C34. The K90E mutant with a substitution outside the HR1 region in the loop of gp41 displayed very low replication capacity but was able to replicate at low levels in the presence of 25 ng/ml T1249. In fact, levels of replication of the Q79E and K90E variants were improved in the presence of drug, suggesting a drug-stimulatory effect as described previously (3, 5). N43K mutant virus has been observed in T20- and T1249-treated patients (33, 38) and indeed had a low level of T20 resistance but no apparent T1249 or C34 resistance, although we tested this mutant without V38A, the context in which it was selected in the evolution experiment.
To quantitate the infectivity of the T1249 escape variants, we performed single-cycle infection assays (Fig. (Fig.3).3). We also included our previously described T20-resistant V38A, V38W, and V38G mutants (5) for comparison. Luciferase activity in TZM-bl reporter cells was measured 2 days postinfection. Infectivity of HIV-1LAI wild-type virus was normalized to 100%, and the relative infectivities of the HIV-1LAI mutants were calculated. All mutants showed diminished infectivity, with V38R, V38K, K90E, and Q79E mutants showing the most profound infectivity defects. These results are generally in concert with the replication capacities (Fig. (Fig.22).
We tested the susceptibility of wild-type virus to three spectra of peptide inhibitors (Fig. (Fig.4a4a and Table Table2).2). T20 inhibited wild-type virus with an IC50 of ~45 ng/ml. The IC50 of T1249 was fourfold lower (~12 ng/ml), and the IC50 of T2635 was again fourfold lower (~3.3 ng/ml). We next established the levels of T1249 resistance and cross-resistance to the first- and third-generation fusion inhibitors T20 and T2635 of the in vitro selected virus variants (Fig. 4b, c, and d and Table Table2).2). All mutants with an amino acid substitution at position 38 of gp41 showed high levels of resistance to T20, but only the T1249 escape mutants were resistant to T1249, with the V38E mutant showing the highest level of resistance (24-fold) (Table (Table2).2). The V38R mutant, which was selected in several evolution cultures, and the newly constructed V38K mutant showed moderate resistance to T1249 (6.7-fold and 2.9-fold, respectively). Again, the N43K mutant did not confer T1249 resistance (in the absence of V38A). The T20- and T1249-resistant V38 variants showed no cross-resistance to T2635, and some (V38A, V38W, V38G, and V38E mutants) appeared to be even more sensitive to inhibition by T2635. Interestingly, the Q79E and K90E variants, which were somewhat resistant to T20 and T1249 (1.6- and 4.0-fold to T20 and 3.4- and 3.0-fold to T1249, respectively), were modestly resistant to T2635 (4.1-fold and 6.8-fold, respectively).
During HIV-1 entry, the HR1 and HR2 segments in gp41 associate to form a highly thermostable six-helix bundle (Fig. 5b and c) (9, 34, 49, 52). The formation of this six-helix bundle is thought to be mechanistically and thermodynamically coupled to HIV-1 membrane fusion (18). Prior to bundle formation, a prehairpin intermediate containing the HR1 coiled-coil trimer which is the target for the HR2-based inhibitors T20, C34, T1249, and T2635 is present (18). T20 resistance mutations in HR1 destabilize the six-helix bundle, suggesting that a decreased T20-HR1 association is underlying the resistance phenotype (5). To determine the effects of the T1249 resistance mutations on the folding, stability, and conformation of the gp41 core, we introduced each of the V38E, V38R, V38K, and N43K substitutions into the soluble N36(L6)C34 six-helix bundle model peptide that is formed by covalent attachment of the N36 (HR1) and C34 (HR2) peptides by a short flexible linker (Fig. (Fig.5a5a).
CD spectroscopy analysis was used to measure the extent of α-helical structure. Monitoring of the typical ellipticity at 222 nm indicated that the wild-type and T1249-resistant variants contain >90% helical structure (Table (Table3).3). Sedimentation equilibrium experiments suggest that all peptides exist in a discretely trimeric state over a 10-fold protein concentration range (10 to 100 μM) (shown for V38E in Fig. Fig.5d;5d; Table Table3).3). We conclude that the introduction of T1249 resistance mutations into the HR1 region of gp41 does not perturb the overall folding and structure of the six-helix bundle, in agreement with the essential role of bundle formation during fusion.
The thermal unfolding of each variant at 10 μM protein concentration was also monitored by CD. The sigmoidal transitions observed at 222 nm (Fig. (Fig.5e)5e) indicate a cooperative disruption of the helical structure with increasing temperature. The midpoints (Tm values) of the transition of V38K, V38R, V38E, and N43K are 74, 74, 72, and 78°C, respectively, compared to a Tm of 80°C for the wild-type molecule, indicating that the substitutions cause a slight destabilization of the six-helix bundle (Table (Table3).3). Intriguingly, the pretransitional slopes and the levels of steepness of the main transition differ greatly for the wild-type bundle and its variants, indicating that the introduction of the charged side chains at positions 38 and 43 alter the mechanism of thermal unfolding. We have not observed this phenomenon with the neutral T20 resistance mutations at position 38 (5).
To probe structural consequences of the introduction of a charged amino acid in HR1, we compared the sensitivities of the N36(L6)C34 variants to proteolytic degradation by proteinase K (Fig. (Fig.6).6). Consistent with a lower thermal stability, the V38K, V38R, and V38E variants exhibited increased sensitivity to proteolysis compared to the wild-type peptide, as indicated by the rapid disappearance of the undigested peptide band and proteolytic products. In contrast, the N43K mutant shows a slight decrease in protease sensitivity (Fig. (Fig.6).6). Moreover, the proteolytic fragmentation patterns of the V38K, V38R, and V38E peptides differs greatly from those of the wild-type and N43K peptides. Digestion of these two peptides yields N36/HR1 (residues 34 to 70) (observed mass, 4,083 Da; expected mass, 4,082 Da) and C34/HR2 (residues 117 to 150) including the N-terminal linker Ser-Gly-Gly-Arg-Gly-Gly (observed mass, 4,788 Da; expected mass, 4,789 Da). Limited proteolysis of V38K, V38R, and V38E variants generates N7(L6)C22, spanning residues 64 to 70 (N7) and 117 to 138 (C22) connected by the linker (observed mass, 4,115 Da; expected mass, 4,116 Da). These results indicate that the N-terminal end of N36/HR1, which contains the charged residue at position 38, is not properly folded and more susceptible to proteolysis. We conclude that the T1249 resistance mutations V38K, V38R, and V38E do not affect the overall formation of the gp41 core structure but destabilize the six-helix bundle conformation.
In this study, we selected HIV-1 variants resistant to T1249 in vitro. Interestingly, gp41 amino acid position 38, implicated in T20 resistance, is also involved in T1249 resistance. Although the interface of the peptide inhibitor with HR2 is quite large, the resistance mutations primarily appear at or near position 38. Because of the large contact surface, the virus can perhaps easily compensate for point mutations, and therefore mutations at the docking site of the peptide inhibitor will have a more dramatic impact on peptide binding. The LLSGIV stretch has been shown to be a critical docking site for T20 (50), and this may explain the critical role of position 38 in resistance development. Whereas T20 resistance is mediated by hydrophobic and noncharged amino acid substitutions at position 38 in HR1 (V38A, V38G, and V38W), T1249 resistance appears to require charged amino acids (V38E, V38R, and V38K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that call for similar but not identical mechanisms of resistance. T1249 is a more potent inhibitor than T20, likely due to its higher HR1 binding affinity. A different gp41 amino acid substitution (involving charged residues) is apparently needed to prevent T1249 binding as opposed to T20 binding. A randomized mutagenesis study that focused on gp41 residues 37 and 38 previously showed the importance of residue 38 in T1249 resistance (3, 5, 5a). Our viral escape study, without an a priori bias for any specific residue, confirms the importance of residue 38 but also demonstrates that other changes can confer T1249 resistance. Besides mutations at position 38, substitutions at the C terminus of the HR1 domain (Q79E) and in the loop (K90E) were found to cause resistance to T1249 as well.
In single-cycle infection experiments, we measured up to 24-fold T1249 resistance for the V38E mutant and lower levels of resistance (3.0- to 6.7-fold) for the V38R, V38K, Q79E, and K90E variants. The previously described T20-resistant V38A, V38G, and V38W variants provided only a low level of T1249 resistance (2.1- to 2.7-fold) (Table (Table2).2). Interestingly, none of the V38 variants provide cross-resistance to the third-generation fusion inhibitor T2635. In fact, some position 38 variants were found to be more susceptible to T2635 than the wild type was. In contrast, the Q79E and K90E mutants exhibited modest levels of resistance to all three spectra of peptide inhibitors. These observations suggest that resistance to T2635 differs mechanistically from T20 and T1249 resistance. While the position 38 substitutions directly affect the HR1-peptide interaction, this is probably not the case for the Q79E and K90E substitutions because they are located outside the actual peptide binding site. Possibly they accelerate the HR1-HR2 association and thereby restrict the time frame in which the peptides can act.
Similar to the HR1-T20/T1249 interaction, the HR1-HR2 interaction can be affected by the drug resistance mutations. Indeed, as for the V38A T20-resistant mutant (5), a decrease in melting temperature of the six-helix bundle was seen for the V38E, V38R, and V38K variants. Consistent with these results, limited proteolytic experiments reveal not only a decrease in overall proteolysis resistance relative to that of the wild type but also a major change in the proteolytic pattern. This suggests that charged side chains at position 38 of gp41 perturb the six-helix bundle structure more dramatically than noncharged residues. Indeed, we measured a significantly destabilized six-helix bundle, reduced infectivity, and delayed replication for the resistant variants.
We analyzed the gp41 sequences after only one month of culturing under T1249 pressure. Upon prolonged culturing, we expect that further evolution will take place. It is likely that additional and/or compensatory mutations in gp41 or gp120 may provide further resistance to T1249 and/or improve viral fitness. This possibility is currently under investigation.
We initiated our in vitro T1249 escape studies with wild-type and T20-resistant virus variants. The input type of amino acid 38 appears to determine the outcome of evolution. Specifically, the V38R variant was generated exclusively from the T20-resistant V38G (three mutants) and V38W (four mutants) variants, whereas 38E was derived exclusively from the V38 wild type (one mutant) and the T20-resitant V38A variant (two mutants). Inspection of the underlying codon changes provides a likely explanation (Table (Table1).1). Evolution of a 38E-encoding codon is relatively easy starting from V38-encoding and 38A-encoding codons (GTG→GAG and GCG→GAG, respectively), which require only a single transversion (T-to-A and C-to-A, respectively) (6, 27, 28). However, both codons require double-hit mutations to make a 38R-encoding codon (GTG→CGG or AGG for V38; GCG→CGG or AGG for 38A). Interestingly, the situation is reversed for the G38- and W38-encoding codons, which prefer to evolve toward 38R. The G38E change requires only a single transition (GGG→GAG), but there are two simple routes toward R (GGG→CGG or AGG). The GGG→AGG change was in fact seen exclusively (three mutants), and it is linked to the most frequent G-to-A mutation that is needed (6, 7, 27, 28). Starting with a 38W-encoding codon also provides a route to a 38E-encoding codon (TGG→GAG, a double mutation) that is more difficult than that to 38R-encoding codon (TGG→CGG or AGG), two single mutation routes, of which the transition type (T to C; three mutants) is preferred over the transversion type (T to A; one mutant). Thus, the mutational bias of HIV-1 determines the precise evolution path toward drug resistance (28).
The findings reported here are of potential clinical relevance as T20 therapy may trigger the selection of resistant viruses that influence resistance development under subsequent T1249 therapy. Although the further clinical development of T1249 has been halted (23, 37), the same selection phenomenon may occur with new entry inhibitors that use a similar mechanism of action. However, our observation that T2635 is not affected by T20 and T1249 resistance mutations at position 38 may disprove this argument. The results of this study also underscore the possibility that HIV-1 will lose fitness in the process of becoming resistant to potent fusion inhibitors, which may impact disease progression. Newer fusion inhibitors, including T2635, may reduce resistance development by a combination of improved potency and loss of Env function upon the acquisition of resistance. As such, the further development of this class of antivirals is warranted.
We thank Trimeris and Roche for providing us with the T20 and T1249 peptides. We are grateful to Ilja Bontjer and Stef Heynen for technical assistance.
This research was supported in part by grant number 2005021 from the AIDS Fund (Amsterdam, The Netherlands) to B.B. and the National Institutes of Health grant AI42382 to M.L. R.W.S. is a recipient of an Anton Meelmeijer fellowship, a VENI fellowship from The Netherlands Organization for Scientific Research (NWO)—Chemical Sciences, and an amfAR Mathilde Krim research fellowship.
Published ahead of print on 23 April 2008.