Cells, virus and compounds
The MT-2 T-cell line [obtained through the NIH AIDS Research and Reference Reagent Program (ARP) from Dr. D. Richman] and the Sup-T1 T-cell line (obtained from Dr. James Hoxie)  were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. TZM-bl cells (a HeLa cell line derivative that expresses CD4, CXCR4 and CCR5 and expresses luciferase and ß-galactosidase under control of the HIV-1 promoter, obtained through the NIH ARP from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc) [55, 56], HEK 293T cells (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% FBS, penicillin and streptomycin. PBMCs were isolated and cultured as previously described . The HIV-1 molecular clones HIV-1IIIB and pNL4-3 were obtained through the NIH ARP from Dr. R. Gallo and M. Martin, respectively. Plasmid pNL4-3.Luc.R-.E- (Dr. N. Landau) [58, 59] and pSG3Δenv DNA (Drs. J.C. Kappes and X. Wu) [56, 60], were obtained through the NIH ARP. Env expression vector pHXB2-env (X4) was obtained through the NIH ARP from Dr. K. Page and Dr. D. Littman  and Env expression vector pSVIIIenv-ADA was kindly provided by Dr. JG Sodroski . Clones representing the standard HIV panels A, A/D, A2/D and D were obtained through the NIH ARP from Dr. Julie Overbaugh [63, 64]. The panel of subtype A/G Env clones were also obtained through the NIH ARP from Drs. Ellenberger, D., Li, B., Callahan, M., and Butera, S. . The HIV-1 Env panel of standard reference subtype B Env clones were obtained through the NIH ARP from Drs. David Montefiori, Feng Gao and Ming Li (PVO, clone 4 (SVPB11), TRO, Clone 11 (SVPB12), SC422661, clone B (SVPB8)); from Drs. B.H. Hahn and J.F. Salazar-Gonzalez (pREJO4541 clone 67 (SVPB16), pRHPA4259 clone 7 (SVPB14)); from Drs. B.H. Hahn and D.L. Kothe (pTHRO4156 clone 18 (SVPB15), pCAAN5342 clone A2 (SVPB19)) [39, 56, 60]. The subtype C HIV-1 reference panel of Env clones were also obtained through the NIH ARP from Drs. D. Montefiori, F. Gao, S. Abdool Karim and G. Ramjee (Du172.17); from Drs. D. Montefiori, F. Gao, C. Williamson and S. Abdool Karim (Du422.1), from Drs. B. H. Hahn, Y. Li and J.F. Salazar-Gonzalez (ZM214M.PL15); from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12 and ZM109F.PB4); from Drs. L. Morris, K. Mlisana and D. Montefiori, (CAP210.2.00.E8) [39, 66, 67]. The subtype C Indian Env clones (HIV-25925-2, clone 22 and HIV-26191-2, clone 48) were obtained from Drs. R. Paranjape, S. Kulkarni and D. Montefiori .
Full-length V3 peptide (Cat# 1840), cyclic V3 peptide (Cat# 1837) and 15-mer overlapping V3 peptides (Cat# 1832, 6282, 6283, 6284, 6285, 6287, 6288, 6289, 6290 and 6291, for amino acid sequence see HIV-1 Subtype B (MN) Env Peptides - Complete Set Cat# 6451) were obtained through the NIH ARP.
Molecular cloning, protein expression and purification
pET14b or pET28a plasmids encode Gag-derived proteins from the HIV-1 NL4-3 strain. The full-length gag expression vector was subcloned from HIV-1 NL4-3 strain. The CA coding region was obtained by PCR amplification and was inserted into the pET28a vector. The C-CA (CTD) DNA fragment was provided by Drs. Ming Luo and Peter E Prevelige Jr . The C-CA (CTD) DNA fragment was subcloned into the pET14b vector. The CTD mutant (W184A/M185A) was generated from pET14b-C-CA using Stratagene's QuikChange® Site-Directed Mutagenesis Kit following the manufacture’s protocol. The corresponding proteins (including 15N or 15N/13C labeled mutant C-CA and C-CA) were expressed and purified as described previously . Protein concentrations were determined with the A280 molar extinction coefficients of 2,980 M-1cm-1(CTD, mutant), 8,480 M-1cm-1(CTD), 33,460 M-1cm-1(CA) and 64,400 M-1cm-1 (Gag), respectively.
Synthesis of the i,i + 7 stapled peptides
The peptides were synthesized manually by Fmoc solid phase synthesis using Rink amide MBHA resin (0.3-0.4 mmol/g). For the normal amino acids, the couplings were performed with four-fold excess of amino acids. Fmoc-amino acids were activated using the ratio of Fmoc-amino acid:HBTU:HOBt:DIEA, 1:1:1:2. For (S)-N-Fmoc-2-(4′-pentenyl)alanine (Fmoc-S
5-OH, Okeanos Tech Co. Ltd) and (R)-N-Fmoc-2-(7′-octenyl)-alanine (Fmoc-R
-OH, Okeanos Tech Co. Ltd), the double coupling was performed with two-fold excess of amino acid which was activated with DIC:HOAt (1:1) or HCTU. For peptide olefin metathesis, the peptide resin with N-terminal protected by Fmoc group was treated with degassed 1, 2 dichloroethane containing Bis(tricyclohexylphosphine)-benzylidine ruthenium (IV) dichloride (10 mM) at room temperature for two hours and the reaction was repeated once for completion. After de-Fmoc, the resin bound peptide was cleaved using standard protocols (95% TFA, 2.5% water, 2.5% TIS). The cleaved peptide was purified by RP-HPLC using 0.1% (v/v) TFA/water and 0.1% (v/v) TFA/acetonitrile and its identity was confirmed using electrospray mass spectroscopy.
Pseudoviruses capable of single-cycle infection were obtained by transfecting HEK 293T cells as previously described . Briefly, 5x106 HEK 293T cells were seeded in a T75 flask and 24 h later, transfected in 15ml medium with a mixture of 10 μg of an env-deleted proviral backbone plasmid, pNL4-3.Luc.R-.E- DNA or pSG3Δenv DNA and 10 μg of an Env expression vector using FuGENE 6 (Roche) following manufacturer’s instructions. Pseudovirus-containing supernatants were collected 2 days after transfection, filtered and stored in aliquots at -80°C.
Virus assembly and release assays
293T cells, plated at 3 × 105 cells/well in 12 well plates, were transfected with the HIV-1 molecular clone pNL4-3 . Six h after transfection, cells were treated with indicated concentrations of NYAD-36, -66, and -67 for 16–20 h. One day posttransfection, cells were metabolically labeled with [35S]Met/Cys for 2 h. The labeled virions were pelleted in an ultracentrifuge, and the virus and cell lysates were immunoprecipitated with pooled sera from HIV-1-infected patients (HIV-Ig, obtained from the ARP) and subjected to SDS-PAGE. Protein band intensities were quantified by phosphorimager analysis, and virus release was calculated as the amount of virion-associated Gag as a fraction of total (cell- plus virion-associated) Gag synthesized during the metabolic labeling period. Accumulation of Pr55Gag in cells was measured by calculating the ratio of Pr55Gag to p24 (CA).
Single-cycle infection assay
The inhibitory activity of i,i + 7 stapled peptides NYAD-36, NYAD-66 and NYAD-67 was measured on HIV-1 pseudotyped viruses expressing Env from the panel of standard reference subtype A, A/D, A2/D, AG, B, C and D. Pseudoviruses were obtained by transfecting HEK 293T cells with a mixture of an Env-deleted backbone proviral plasmid pSG3Δenv and an Env expression vector DNA. Briefly, for the neutralization assay 100 μl of TZM-bl cells at 1 × 105 cells/ml were added to the wells of a 96-well tissue culture plate and cultured at 37°C overnight. 50 μl of a staple peptide at graded concentrations was mixed with 50 μl of the HIV-1 pseudovirus at about 100 TCID50 (50% tissue culture infectious dose). After incubation at 37°C for 30 min, the mixture was added to the cells and incubated at 37°C for 3 days. Cells were washed 2 times with PBS and lysed with 50 μl of cell culture lysis reagent. 20 μl of lysates were transferred to a white 96-well plate and mixed with 100 ul of luciferase assay reagent. The luciferase activity was immediately measured as above to calculate IC50 (Luciferase Assay System, Promega). The luciferase activity was immediately measured with a Tecan Infinite M100 reader (Tecan, USA) and the IC50 values were calculated by the GraphPad Prism software (GraphPad Software, Inc., USA).
TZM-bl cells were seeded at 1 × 104 cells/well in 96 well plates; one day later cells were infected with reverse-transcriptase (RT)-normalized virus stocks for 2 h. Stapled peptides (NYAD-1, -36, -66, -67) were added during the 2 h infection period at indicated concentrations. Two days postinfection, cells were washed with PBS, lysed in luciferase lysis buffer (Promega) and luciferase activity was measured with luciferase assay substrate (Promega). In experiments where virus-producing cells were treated with the peptides, six-hour posttransfection 293T cells were treated with indicated concentrations of NYAD-36, -66, or -67, and virus supernatant was collected after 2 days. Env pseudotyped viruses were generated by cotransfecting 293T cells with an Env-defective pNL4-3 derivative (pNL4-3/KFS)  and HIV-1 Env expression vector pIIINL4env [71, 72], pIIINL4env-V120Q/A327P, or VSV-G expression vector pHCMV-G .
Multi-cycle infection assay
The inhibitory activity of i,i + 7 stapled peptides on infection by laboratory-adapted HIV-1IIIB strain was determined as previously described . In brief, 104 MT-2 cells were infected with HIV-1IIIB at 100 TCID50 (0.01MOI) in the presence or absence of test compounds at graded concentrations and incubated overnight. The culture supernatants were then replaced with fresh media. On the fourth day post-infection, 100 μl of culture supernatants were collected from each well, mixed with equal volume of 5% Triton X-100 and tested for p24 antigen by sandwich ELISA. The percentage of inhibition of p24 production and IC50 values were calculated with the GraphPad Prism software (GraphPad Software Inc.).
Determination of cytotoxicity
In MT-2 cells
Cytotoxicity of stapled peptides in MT-2 cells was measured with the XTT (Sodium 3’-[1-phenylamino-carbonyl]-3,4-tetrazolium]-bis[4-methoxy-6-nitro]benzenesulfonic acid hydrate) method as previously described . Briefly, 100 μl of a stapled peptide at graded concentrations was added to an equal volume of cells (105cells/ml) in 96-well plates followed by incubation at 37°C for 4 days. Following addition of XTT (PolySciences, Inc.), the soluble intracellular formazan was quantitated colorimetrically at 450 nm 4 h later. The percent of cytotoxicity and the CC50 values were calculated with the GraphPad Prism software (GraphPad Software Inc.).
In TZM-bl cells
The cytotoxicity of stapled peptides in TZM-bl cells was also measured by the colorimetric method using XTT. Briefly, 100 μl of a compound at graded concentrations was added to equal volume of cells (105/ml) in wells of 96-well plates followed by incubation at 37°C for 3 days. Following addition of XTT the soluble intracellular formazan was quantitated as described above.
Selection and characterization of NYAD-resistant viruses and virus replication assays in the Jurkat T-cell line and PBMCs
NYAD-36-resistant viral isolates were selected by prolonged serial passage of wild-type (WT) NL4-3 in Jurkat cells in the presence of 25, 37.5 and 50 μM NYAD-36. Virus replication during the selection process was monitored by RT activity as previously described . Virus supernatants and cell pellets were collected on the days of peak RT activity. RT-normalized viruses were used to infect fresh Jurkat cells, and virus replication was examined as above to confirm acquisition of NYAD-36 resistance. To identify the mutations conferring resistance to NYAD-36, genomic DNA was extracted from cells on the day of peak RT activity using a whole blood DNA purification kit (Qiagen). The entire Gag and Env coding region was amplified by PCR and sequenced. Putative resistance-conferring mutations were introduced into the Env expression vector pIIINL4env by site-directed mutagenesis (Stratagene) using mutagenic oligonucleotides. Following sequence confirmation, the EcoRI-NheI fragment was cloned back into the WT pNL4-3 to generate a molecular clone containing the Env mutations, which were confirmed by sequencing. PBMCs isolated from healthy donors were activated by phytohemagglutinin (PHA) and were infected with RT-normalized WT- and V120Q/A327P mutant viruses and replication was carried out in the presence of 30 M NYAD-36, -66, or -67. Virus supernatant was collected every 2 or 3 days, and RT activity was monitored as above.
A pET11a plasmid containing DNA for a monomeric HIV-1 CA, p24-W184A/M185A, with a C-terminal His tag was transformed into Rosetta 2 (DE3) cells (EMD) for protein expression. The His-tag was not removed prior to NMR and turbidity studies. 15N isotopically labeled protein was expressed in a minimal media solution supplemented with 15NH4Cl. Protein overexpression was induced with 1mM IPTG at an OD600 of 0.6 overnight at 37°C. After expression, the cells were harvested by centrifugation and lysed using a microfluidizer (Microfluedics) using lysis buffer containing 50 mM NaPO4 [pH 7.4] 5 mM BME and 10 mM imidazole. Lysed cells were then centrifuged and the soluble fraction was applied to a cobalt resin (Thermo Scientific) for purification. The resin was washed with lysis buffer and CA was eluted with lysis buffer containing 250 mM imidazole. Pure protein was dialyzed in NMR buffer containing 50 mM NaOAc [pH 5.5] 5 mM DTT with 10%D2O.
The NMR-based titrations were collected at 35°C on a Bruker AVANCE III 600 MHz equipped with a cryogenic probe. Staple peptides were dissolved in 100% DMSO and titrated into the monomeric HIV-1 CA, p24-W184A/M185A, in a buffer containing 50 mM NaOAc [pH 5.5] 5 mM DTT with 10%D2O. Increasing molar ratios of staple peptides were added into the protein and chemical shift perturbations were monitored.
Circular dichroism spectroscopy
Circular dichroism (CD) spectra were obtained on an Aviv model 62DS CD spectrometer (Aviv, Lakewood, NJ) at 25°C using the standard measurement parameters in 1× PBS in the presence of 1–20% (v/v) acetonitrile at a final concentration of 100 μM. In all samples, the final concentrations of peptides and salt were always the same, and the spectra were corrected by subtracting the CD spectrum of the appropriate reference solvent.
For the cell penetration study, 293T cells were seeded in 4-well chamber plates and incubated at 37°C with 5 μΜ of FAM-conjugated peptides for 20 h in medium containing serum. After three washes with 1X PBS, live cells were examined and imaged under a Zeiss LSM510 laser scanning confocal microscope (Zeiss).
FACS Analysis of FAM-conjugated Peptide Treated Cells
297-T cells were grown in RPMI 1640 (Gibco), 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 50 mM Hepes pH 7, and 50 μM ß-mercaptoethanol. 293-T cells (6x105/well) in 2 ml serum-free media were treated with FAM-conjugated NYAD-1, NYAD-66, NYAD-67, NYAD-36 and NYAD-41. The final concentration of the corresponding FAM-conjugated peptides is 2 μM, 4 μM or 8 μM. After 4 hours (data not shown) or 20 hours incubation with the FAM-conjugated peptides at 37°C, the treated cells were washed twice with 1x PBS. After a treatment with 0.25% Trypsin-EDTA (GIBCO) for 30 min at 37°C and two washes with 1× PBS, the cells were subjected to FACS analysis. The data were analyzed by using Flowjo Software.
Electron microscopy to study inhibition of in vitro assembly by peptides
In vitro assembly experiments were set up as described [9, 75–77] with minor modification. We used 50 mM Na2HPO4, pH 8.0 as the dialysis buffer. The buffer used for assembly studies also contained 0.1 ~ 2 M of NaCl. 500-Da-MWCO dialysis tubes (Spectra/Por) were used for the peptide dialysis. Briefly, stock proteins were adjusted to the appropriate concentration (25 μΜ for Gag protein or 50 μΜ for CA protein) with the Na2HPO4 buffer at pH 8.0. After incubation with varied doses of NYAD-36, NYAD-66 and NYAD-67 for 30 min at 4°C, the samples were dialyzed overnight in Na2HPO4 buffer at pH 8.0 containing 100 mM NaCl at 4°C. Negative staining was used to check the assembly. Carbon-coated copper grids (200 mesh size; EM Sciences) were treated with 20 μl of poly-L-lysine (1 mg/ml; Sigma) for 2 min. 20 μl of reaction solution was placed onto the grid for 2 min. Spotted grids were then stained with 30 μl of uranyl acetate solution for 2 min. Excess stain was removed, and grids were air-dried. Specimens were examined with a TECNAI G2 electron microscope (FEI, OR, USA).
In vitro CA assembly
Wild-type HIV-1 CA was overexpressed and purified as previously described . Pure protein was dialyzed in NMR buffer containing 50 mM NaPO4 [pH 8.0]. Turbidity assays were carried out in triplicate using a Beckman spectrophotometer at a wavelength of 350 nm to monitor HIV-1 in vitro assembly conducted at ambient temperatures . Protein samples were prepared in an assembly buffer [50 mM NaPO4 pH 8.0]. In vitro assembly reactions were prepared with 250 μl of assembly buffer containing 60 μM protein. To catalyze CA assembly 250 μl of an assembly buffer containing 5M NaCl was added. The solution was mixed by agitation and transferred a cuvette for a total delay of 20 seconds. Assembly was monitored every 10 sec for the first min and every min thereafter for a total 10 min. Assembly reactions with stapled peptides were incubated in the assembly buffer for 10 min and then centrifuged for 1 min to separate any insoluble fractions before catalyzing assembly.
Isothermal titration calorimetry (ITC)
The binding affinity between CTD (W184A/M185A) and NYAD-36, NYAD-66 or NYAD-67 was measured by ITC at 30°C using a Microcal titration calorimeter (MicroCal VP-ITC). Both CTD (W184A/M185A) protein and stapled peptides were exhaustively dialyzed against 25 mM sodium phosphate buffer (pH 7.3) prior to experimental measurements. In a typical experiment, the ITC injection syringe was loaded with 625 μM CTD (W184A/M185A) protein, dissolved in the dialysis buffer. The calorimetric cell (ca. 1.4 ml active volume) initially contained only 25 μM stapled peptide in the identical dialysis buffer. Typically titrations consisted of 27 injections of 10 μl into the calorimetric cell, with 240-s equilibration between injections. Both forward and reverse titrations were used to confirm the interactions between V3 loop peptide and stapled peptides.
For forward titration, 90 μM full-length V3 peptide, full-length cyclic V3 peptide or 90 μM 15-mer V3 peptides were titrated into 4.5 μM of stapled or linear peptides. Reference measurements were performed by titrating 90 μM full-length V3 peptide, full-length cyclic V3 peptide or 15-mer V3 peptide into buffer without peptide. For reverse titration, 90 μM stapled peptide was titrated into 4.5 μM full-length V3 peptide, full-length cyclic V3 peptide or 90 μM 15-mer V3 peptide. Reference measurements were performed by titrating 90 μM stapled peptide into buffer without peptide.
Surface plasmon resonance (SPR)
The binding kinetics and affinity of stapled peptides to HIV-1 Yu2gp120 (kindly provided by Dr. Peter Kwong, Vaccine Research Center, NIH) were analyzed by SPR (BIAcore 3000, Piscataway, NJ). The Yu2gp120 or Yu2gp120 protein with V3 loop deletion (Yu2gp120ΔV3) was covalently immobilized to a CM5 sensor chip via amine groups using the amine coupling kit (BIAcore) in 10 mM sodium acetate buffer at pH 5.0. The immobilization level of gp120 reached about 1,500 response units (RU).
For the binding affinity assay, the stapled peptides were prepared as a 10 mM stock solution in 100% DMSO. Immediately prior to analysis, the compounds were diluted with Dulbecco's PBS to a final DMSO concentration of 3%. Experiments were run at 30 μl/min in running buffer (Dulbecco's PBS containing 3% DMSO). The stapled peptides at different concentrations were injected over the target protein (gp120) and reference surfaces for 2 min, followed by a 5 min dissociation period. The surface was regenerated with 6.25 mM NaOH and 50 μl/min for 30 s.
The binding kinetic parameters were evaluated with BIA-Evaluation software (BIAcore 3000, Piscataway, NJ) in which all data sets were fit to a simple 1:1 (Langmuir) binding model including a term for mass transport. The data selection of the binding and dissociation curves for fitting was based on the instruction manual of the BIAcore 3000 instrument, in which 3–5 s of data at association and dissociation start were eliminated.