CHO cells stably transfected with either the HIV-1HXB2 Env-expressing vector pEE14 (CHO-WT) or control pEE14 vector (CHO-EE) were cultured in Glutamine-deficient minimal essential medium (GMEM-S) containing 400 μM Methionine sulfoximine (Sigma, St. Louis, MO). Cf2Th/syn CCR5 cells stably expressing CCR5 receptors were cultured in DMEM complemented medium with 10% FBS, pen/strep, 500 μg/ml G418, 500 μg/ml zeocin and 3 μg/ml puromycin (Invitrogen, Carlsbad, CA). MT-2 and TZM-b1 cells, as well as laboratory-adapted HIV-1 strains IIIB and Bal, and primary HIV-1 isolates were obtained from the AIDS Research and Reference Reagent Program of NIH. The N-peptide N36 (aa 546–581), and C-peptides C34 (aa 628–661), T1144, and T20 (aa 638–673), used in this study were derived from the NHR and CHR, respectively, of the HIV-1HXB2 gp41 (Figure
1A). These peptides (> 95% purity) were synthesized by a standard solid-phase FMOC method using an Applied Biosystems model 433A peptide synthesizer.
Construction of vectors encoding D1D2 and 2DLT
To create the expression plasmid pD1D2-PDI and p2DLT-PDI, DNA fragments encoding D1D2 (aa 1–185 of CD4), the 35-mer linker (GGGGS)7, and T1144 were linked together by three-step overlapping PCR. We took p2DLT-PDI as an example. First, the D1D2 (FD1D2his: 5′-CGCGGATCCCATCACCATCACCATCATAAGAAAGTGGTGCTG-3′, RD1D2: 5′-CACTTCCTCCTCCTCTATGCTGGAGGCCTTCTGGAA-3′), L35 (FL35: 5′-GGAGGAGGA GGAAGTGGCGGCGGCGGCTCGGGTGGTGGTGGTTCTGGAGGTGGCGGTAGCGGAGGTGGAGGTAGTGGAGGC-3′, RL35: 5′-GCTACCTCCGCCTCCCGAACCTCCGCCTCCA CTACCTCCACCTCCGCTACCGCCACCTCCAGAACCACCACCACCCGAG-3′) and T1144 (FT1144: 5′-GAGGCGGAGGTAGCACGACCTGGGAAGCATGGGACAGAGCTATTGCTG AATACGCAGCTAGGATAGAAGCTTTACTCAGAGCTTTA-3′, RT1144: 5′-CGGAGAT CTCTATAATTCCCTTAAGGCTGCTTCATTCTTTTCTTGCTGTTCTTGTAAAGCTCTGAGTAAAGCTTCTATCC-3′) DNA fragments were genera-ted by overlapping PCR using the corresponding primer pairs. Second, the DNA fragments coding for L35 and T1144 were linked by overlapping PCR with the primers FL35 and RT1144. Third, the two DNA fragments encoding D1D2 and L35-T1144 were linked by overlapping PCR with the DNA fragment D1D2 and the primers FD1D2his and RT1144. Finally, the amplified DNA fragment coding for 2DLT was digested by BamHI and EcoR I and inserted into the expression vector pGEX-6p-1 to generate the p2DLT plasmid. In order to prevent the formation of the inclusion bodies in E. coli, we inserted a protein disulfide isomerase (PDI)
[21, 22] DNA sequence (aa 18–508) with PreScission protease cutting site (called ppase site) in the N terminus into the EcoR I and Xho I sites located at the C terminus of His-2DLT gene in the plasmid p2DLT to extend the GST-his-2DLT reading frame, resulting in the generation of chimeric GST-his-2DLT-ppase-PDI. This plasmid is called p2DLT-PDI. The sequences were confirmed by DNA sequencing.
Protein expression and purification
To express D1D2 and 2DLT fusion proteins, Escherichia coli strain Rosetta 2 (DE3) pLysS (Novagen) was transformed with pD1D2-PDI and p2DLT-PDI, respectively, cultured at 37°C to OD600 = 0.4, then induced at 16-22°C for 8–12 h with 0.4 mM IPTG. The cells were harvested and lysed by sonication in the presence of protease inhibitor mixture (Roche). After centrifugation, supernatants containing the fusion protein were collected. The protein was purified with Glutathione-Sepharose 4B affinity columns and cleaved with PreScissionTM Protease (GE Healthcare). These fusion proteins were purified by His·Bind® Purification Kit (Novagen) and fast protein liquid chromatography (FPLC), and then analyzed by SDS-PAGE.
SDS-PAGE and Western blot analysis
Purified fusion proteins were analyzed by SDS-PAGE as previously described
. Briefly, D1D2 or 2DLT was mixed with 4X SDS sample buffer (Novagen, Gibbstown, NJ) and boiled for 5 minutes or kept at room temperature (RT) before loading onto a 10-20% Tricine-Glycine gel (Invitrogen, Carlsbad, CA). The electrophoresis was conducted in SDS-PAGE running buffer with 125 V constant voltage at 4°C. In Western blot, the anti-human CD4 and anti-T1144 polyclonal antibodies were used.
Enzyme-linked immunosorbent assay (ELISA)
D1D2 and 2DLT fusion proteins were characterized by ELISA as previously described
. Briefly, they were coated onto a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) (10 μg/ml in 0.1 M Tris–HCl, pH 8.8), which was blocked with 2% non-fat milk in PBS. The polyclonal antibodies against CD4, conformation-dependent monoclonal antibody against CD4 (Sim.4) and anti-T1144 polyclonal antibody, respectively, were added to the plate. After incubation at 37°C for 60 min, horseradish peroxidase (HRP)-labeled antibodies (ZYMED Laboratories, S. San Francisco, CA) and the substrate TMB (Sigma) were added, sequentially. The binding of D1D2 and 2DLT to gp120 or gp41 NHR, and their inhibitory activity on gp41 6-HB formation were determined by ELISA as previously described
The binding of D1D2 and 2DLT to gp120/gp41 expressed on the cell surface was detected by flow cytometry as previously described
[49, 51]. Briefly, the cultured CHO-WT (with Env) and CHO-EE (with no Env) cells were detached from plate and washed with wash buffer (PBS containing 5% GBS) three times and incubated with the testing protein for 1 h at 4°C. After three washes, anti-CD4 or anti-T1144 polyclonal antibody was added for 1 h at 4°C. After three washes, FITC-conjugated anti-rabbit or mouse antibody was added and incubated for 1 h at 4°C. After three washes, the cells were examined by flow cytometry and the fluorescence intensity was recorded by FACSCalibur (Becton Dickinson).
Surface plasmon resonance (SPR) assay
The binding affinity of D1D2 and 2DLT to gp120 was measured by SPR using the BIAcore3000 system (Pharmacia, Piscataway, NJ), following the Manual of the Biomolecular Interaction Analysis (BIA) Technology as described previously
. Briefly, gp120 (100 μg/ml) was immobilized onto the CM3 sensor chip by amine coupling, and the unreacted sites were blocked with ethanolamine. The dissociation reaction was done by washing with running buffer (10 mM HEPES pH7.4 containing 0.15 M NaCl, 3.4 mM EDTA and 0.005% v/v surfactant) for at least 2 min.
Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE)
FN-PAGE for detecting 6-HB formation as described before
. Briefly, a testing peptide or protein (100 μM) was pre-incubated with N36 (100 μM) at 37°C for 30 min, followed by addition of C34-FAM (100 μM) at 37°C for 30 minutes. The mixtures were added into Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA). The samples (20 μl) were then loaded onto Tris-glycine gels (18%; Invitrogen, Carlsbad, CA), which were run under 120 V constant voltage at room temperature for 1 h. The gels were stained and visualized with the FluorChem 8800 Imaging System (Alpha Innotech Corp., San Leandro, CA) using a transillumination UV light source with excitation wavelength at 520 nm and then with Coomassie Blue.
Inhibition of HIV-1 infection
Inhibitory activities of D1D2 and 2DLT on HIV-1 infection were determined as previously described
[53, 54]. For inhibition of HIV-1 IIIB (subtype B, X4) infection, 100 TCID50 of the virus was added to 1 × 104/ml MT-2 cells in RPMI medium 1640 containing 10% FBS in the presence or absence of the test peptide or protein overnight. The culture supernatants were removed, and fresh media were added. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. For inhibition of infection by the HIV-1 strain Bal (subtype B, R5), TZM-bl cells (1 × 105/ml) were pre-cultured overnight and infected with Bal at 100 TCID50 in the presence or absence of the test peptide or protein overnight. The cells were harvested and lysed on the fourth day post-infection with lysing reagent. The luciferase activity was analyzed using a luciferase kit (Promega, Madison, WI) and a luminometer (Ultra 386, Tecan, Durham, NC) according to the manufacturer’s instructions. For testing the effect of pre-incubation times on the inhibitory activity of 2DLT, the TZM-b1 assay was performed after pre-incubation of the HIV-1 Bal virus with the inhibitors for 0, 15, 60 and 240 min. For inhibition of primary HIV-1 isolate infection, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors. The PHA-stimulated cells were infected with a primary HIV-1 isolate at a multiplicity of infection (MOI) of 0.01 in the absence or presence of peptide or protein at graded concentrations. The supernatants were collected on the 7th day post-infection and tested for p24 antigen by ELISA as previously described
[54, 55]. The percent inhibition of p24 production or luciferase activity was calculated.
Inactivation of HIV-1 virions
The virus inactivation by D1D2 and 2DLT was determined as previously described
[25, 56, 57]. Briefly, 100 μl of the protein or peptide at graded concentration were added to 100 μl of an HIV-1 strain (200 TCID50/ml), followed by incubation at 4°C for 1 h. Then, PEG-6000 was added to the treated virus at final concentration of 3%, 4°C for 1 h. The mixture was centrifuged on a microfuge at 15,000 rpm for 30 min. The supernatants were removed and the pellet was washed with 3% PEG in PBS containing 10 mg/ml BSA. The viral pellet was then resuspended in 100 μl of PBS, before addition of 100 μl MT-2 or TZM-bl cells (1 × 105/ml). After cultur at 37°C for 3 days, p24 production in MT-2 cell culture or luciferase activity in TZM-bl cell culture was tested as previously described
To measure the effects of D1D2 and 2DLT on induction of short-lived PFI of HIV-1 Env, we used a cell-based ELISA as previously described
. Briefly, CHO-WT cells steadily expressing HIV-1 Env were seeded in 96-well plates (5 × 104/well). Cells were then harvested and washed twice with blocking buffer (35 mg/ml BSA, 10 mg/ml non-fat dry milk, 1.8 mM CaCl2, 1 mM MgCl2, 25 mM Tris, pH 7.5 and 140 mM NaCl). For pulse activation experiments, the cells were incubated with D1D2 (2.5 μM) or 2DLT (2.5 μM) suspended in blocking buffer for three minutes, washed three times with blocking buffer and incubated with the C34-biotin (2 μM). To study the temperature dependence of NHR groove exposure, the D1D2- or 2DLT-pulsed cells were incubated at the requisite temperature for different lengths of time. The cells were subsequently returned to room temperature for incubation with C34-biotin. Cells were then washed four times with blocking buffer and four times with washing buffer (140 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2 and 20 mM Tris, pH 7.5). A horseradish peroxidase-conjugated streptavidin (ZYMED Laboratories, S. San Francisco, CA) was then incubated with the samples for 45 minutes at room temperature. Cells were washed 5 times with blocking buffer and 5 times with washing buffer. HRP enzyme activity was determined after the addition of 33 μl per well of a 1:1 mixture of Western Lightning oxidizing and luminal reagents (Perkin Elmer Life Sciences) supplemented with 150 mM NaCl. Light emission was measured.
HIV-1 infectivity in CD4-/CCR5+ cells treated by D1D2 or 2DLT
The effects of D1D2 and 2DLT on HIV-1 infection of CD4-/CCR5+ target cells were evaluated as previously described
. Briefly, HIV-1 Bal (100 TCID50/well was cultured with Cf2Th-CCR5 cells (1 × 106 cells per well) at room temperature for 1 h, then was resuspended, followed by addition of D1D2 or 2DLT at different concentrations. After a short centrifugation, the mixture was incubated for 8–12 h at room temperature. The viral infectivity was measured three days later.
Inhibition of HIV-1-mediated cell-cell fusion
HIV-1 mediated cell-cell fusion was measured with a dye transfer assay as previously described
[27, 29]. In brief, the HIV-1IIIB chronically infected H9 (H9/HIV-1IIIB) cells were labeled with Calcein-Am (Molecular Probes, Inc., Eugene, OR). After washes, the fluorescence-labeled H9/HIV-1IIIB cells were incubated with MT-2 cells at 37°C for 2 h in the absence or presence of an inhibitor at a graded concentration. The percentage of fused cells was counted under a fluorescence microscope (Zeiss, Germany), and the 50% inhibitory concentration of each drug was calculated with the Calcusyn software program