Cells and viruses
MT-4, Jurkat A72, CEM, HuT-78 and Raji/DC-SIGN cells were grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate and 20 μg gentamicin per ml. The HIV-1(IIIB) strain was provided by R.C. Gallo and M. Popovic (at that time at the NIH, Bethesda, MD, USA). HIV-2(ROD) was obtained from L. Montagnier (at that time at the Pasteur Institute, Paris, France) and SIV(Mac251) from C. Bruck. Raji/DC-SIGN were kindly provided by L. Burleigh (Paris, France).
In vitro antiviral assays
Evaluation of the antiviral activity of the compounds against HIV-1 strain IIIB in MT-4 cells was performed using the MTT assay as previously described
[18, 19]. Stock solutions (10 x final concentration) of test compounds were added in 25 μl volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell samples were included as controls. HIV-1(IIIB) stock (50 μl) at 100-300 CCID50 (50% cell culture infectious doses) or culture medium was added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 minutes at 220 g, and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/ml and 50 μl volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically using the MTT assay. The MTT assay is based on the reduction of yellow coloured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median absorbance value of three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50).
The antiviral activity of the compounds against HIV was evaluated in Jurkat cells stably transformed to express the LTR-GFP (A72 cells)
[20, 50]. In 96-well plates, 3 × 104 A72 cells were infected with HIV in the presence of various concentrations of test compound. Three days post infection, cells were harvested and fixed in 3% paraformaldehyde. GFP-expression was quantified on a single cell basis by flow cytometry
[51, 52]. Toxicity of the compounds was tested using an MTT-based method.
HIV-1 core antigen (p24 Ag) in the supernatant was analyzed by the p24 Ag enzyme-linked immunosorbent assay (Perkin Elmer).
Aliquots of a HIV stock (IIIB or ROD) were incubated with various concentrations of compound in a final volume of 100 μl RPMI-1640 culture medium with 10% FCS for 1 hour at 37°C. For the clinical isolates of different clades, stock was incubated with 125 μM WDO-217 for 1 hour at 37°C. Subsequently, the samples were diluted 4000 times with complete medium so that the residual concentration of compound present was far below its IC50. The drug-treated and diluted virus suspension was then used to infect susceptible MT-4 T-cells to quantify the viral infectivity by titration and CCID50 calculation
. The different clinical isolates were titrated on freshly isolated PBMCs from a healthy donor. Control experiments with AZT indicated that this procedure effectively diluted the compound to concentrations well below its effective antiviral concentration.
Inhibitory effect on virus production from HuT-78/IIIB persistently infected cells, virion analysis and western blot
Chronically-infected HIV-1 IIIB HuT-78 cells (HuT-78/IIIB) were washed four times with PBS to remove all free virions before treatment and 2 × 105 cells were resuspended in 1 ml compound-containing medium for 43 hours at 37°C. Then, virions were prepared from clarified supernatants (10 min at 300 g) by centrifugation at 36670 g for 2 hours at 4°C. Protein from lysed virions were separated by SDS-PAGE on a NuPage® Novex 4-12% Bis-Tris gel (Invitrogen) and transferred on a hydrophobic polyvinylidene difluoride (PVDF) membrane (Amersham Hybond™-P). The blot was blocked overnight at 4°C by 5% dry milk powder in Western blot wash solution (WBWS; PBS + 0.5% Tween 20), washed three times for 5 minutes with WBWS and incubated with a mouse anti-HIV-1 p24 antibody (1:5000) from Abcam. The blot was then washed three times for 5 minutes with WBWS and incubated for 1 h with a goat anti-mouse IgG-HRP secondary antibody (1:2500) from Santa Cruz Biotechnology. The blot was washed three times for 5 minutes with WBWS and after 5 minutes of incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) it was developed.
Effect of WDO-217 on the exposure of HIV-1 to Raji/DC-SIGN cells
In a first set of experiments (procedure A), HIV-1 (NL4.3) was exposed to WDO-217 at 105 μM (in 0.5 ml culture medium) for 60 minutes at 37°C. Then, 0.5 ml exponentially growing Raji/DC-SIGN cells (106 cells) was added, and the suspension further incubated at 37°C for 60 minutes. Subsequently, 39 ml medium was added and the cell suspension was centrifuged at 300 g for 10 minutes. The obtained pellet was washed again with 40 ml medium and after centrifugation, the cell pellet (containing DC-SIGN-bound virus) was analyzed for p24 antigen content by ELISA. In a second set of experiments (procedure B), Raji/DC-SIGN cells treated as in procedure A were co-cultured in the presence of an equal amount of C8166 cells (106 cells) (total volume of 1 ml). Replication in C8166 was measured after ~ 20 hr of incubation. In a third set of experiments (procedure C), Raji/DC-SIGN cells were given the opportunity to capture HIV-1/NL4.3 by mixing Raji/DC-SIGN cells with virus (106 cells/ml). After one hour of incubation at 37°C, C8166 cells (106 cells/ml) were added in the presence of WDO-217 at different concentrations and giant cell formation in the cultures was examined microscopically after approximately 24 hr. In the above-described experiments, the mannose-binding entry inhibitor HHA was included as a reference compound.
Total mRNA from virus stock was extracted using the QIamp viral RNA kit (Qiagen) followed by DNA digestion using RNase-free DNase I (Invitrogen). DNase I treated mRNA was used to generate cDNA along with Thermoscript reverse transcriptase (Invitrogen) and oligo(dT)20. qRT-PCR for genomic unspliced HIV mRNA was performed according to a protocol described earlier
, using 0.2 mM primers TCAGCCCAGAAGTAATACCCATGT and TGCTATGTCAGTTCCCCTTGGTTCTCT, and 0.2 mM FAM-BHQ1 fluorescent probe ATTAACAGAAGGAGCCACCCCACAAGA. Control reactions omitted reverse transcriptase, and the number of cDNA copies was determined using a HIV-1NL4.3 molecular clone DNA standard.
Zinc ejection and inhibition of NC(11-55)-induced destabilization of cTAR monitored by fluorescence techniques
The NC(11-55) peptide was synthesized by solid phase peptide synthesis on a 433A synthesizer (ABI, Foster City, CA), as previously described
. The lyophilized peptide was dissolved in water, and its concentration was determined using an extinction coefficient of 5,700 M–1 x cm–1 at 280 nm. Next, 2.5 molar equivalents of ZnSO4 were added to the peptide and pH was raised to its final value, by adding buffer. The increase of pH was done only after zinc addition to avoid oxidization of the zinc-free peptide. Zinc ejection was monitored through the changes in the intrinsic fluorescence of the Trp37 residue of NC(11-55)
[23, 24], after addition of a 10-fold excess of WDO-217 (10 μM) to 1 μM NC(11-55).
To monitor the inhibition by WDO-217 of the NC(11-55)-induced destabilization of cTAR, we used doubly labelled cTAR, synthesized at a 0.2 μmol scale by IBA GmbH Nucleic Acids Product Supply (Göttingen, Germany). The 5′ terminus of cTAR was labeled with 6-carboxyrhodamine (Rh6G) via an amino-linker with a six carbon spacer arm. The 3′ terminus of cTAR was labeled with 4-(4′-dimethylaminophenylazo)benzoic acid (Dabcyl) using a special solid support with the dye already attached. The doubly labeled cTAR was purified by reverse-phase HPLC and polyacrylamide gel electrophoresis. An extinction coefficient at 260 nm of 521,910 M–1 x cm–1 was used for cTAR. All experiments were performed at 20°C in 25 mM Tris–HCl, pH 7.5, 30 mM NaCl, and 0.2 mM MgCl2. The effect of WDO-217 on the NC(11-55)-induced destabilization of cTAR was observed after addition of 10 μM WDO-217 to 0.1 μM Rh6G-cTAR-Dabcyl preincubated with 1 μM NC(11-55).
Absorption spectra were recorded on a Cary 400 spectrophotometer. Fluorescence spectra were recorded at 20°C on a Fluorolog spectrofluorometer (Horiba, Jobin-Yvon), equipped with a thermostated cell compartment. Excitation wavelength was 295 nm and 520 nm, for NC(11-55) and Rh6G-5′
-Dabcyl, respectively. The spectra were corrected for dilution and buffer fluorescence. The protein spectra were additionally corrected for screening effects due to the zinc ejecting agent, using:
where Im is the measured fluorescence of the protein, Ip is the fluorescence intensity of the protein in the absence of inner filter, dp is the absorbance of the protein, ds is the absorbance of WDO-217 at the excitation wavelength, and dr is the absorbance of WDO-217 at the emission wavelengths.
Zinc ejection monitored by supramolecular mass spectrometry
Before mass spectrometry (MS) analysis, NC(11-55) was dissolved and buffer exchanged with 50 mM ammonium acetate pH 7.0 using 4 cycles of microcentrifuge size exclusion filtering (Vivaspin 500 5kD, Sartorius Stedim biotech, Aubagne, France) and peptide concentration was measured by a Bradford assay.
ESI-MS measurements were performed in the positive ion mode on an electrospray time-of-flight mass spectrometer (LCT, Waters, Manchester, UK) equipped with an automated chip-based nanoESI source (Triversa Nanomate, Advion Biosciences, Ithaca, NY). Calibration of the instrument was performed using multiply charged ions of a 2 μM horse heart myoglobine solution. For analysis in denaturing conditions, samples were diluted to 2 μM in a 1/1 water/acetonitrile mixture (v/v) acidified with 1% formic acid and standard interface parameters were used to obtain best mass accuracy. In these conditions, noncovalent interactions are disrupted, allowing the measurement of the molecular weight of the monomer with a good accuracy (better than 0.01%).
Analyses under non-denaturing conditions were carried out after careful optimization of instrumental settings to avoid dissociation of noncovalent bonds and obtain sensible detection of protein/zinc complexation states. The accelerating voltage (Vc) was fixed to 20 V, and the pressure in the first pumping stage of the instrument (Pi) to 6 mbar. Zinc ejection measurements were performed after 30 minutes incubation at room temperature of a 20 μM solution of NC(11-55) with either 40 μM or 100 μM WDO-217. Data analysis was performed with the MassLynx 3.5 software (Waters, Manchester, UK). Peak intensities were used to estimate the ratios of the different ions detected.