A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions
- Thomas Vercruysse†1,
- Beata Basta†2,
- Wim Dehaen3,
- Nicolas Humbert2,
- Jan Balzarini1,
- François Debaene4, 5,
- Sarah Sanglier-Cianférani4, 5,
- Christophe Pannecouque1,
- Yves Mély2Email author and
- Dirk Daelemans1Email author
© Vercruysse et al.; licensee BioMed Central Ltd. 2012
Received: 31 August 2012
Accepted: 24 October 2012
Published: 12 November 2012
Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides.
Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed.
This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.
Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS (Acquired Immune Deficiency Syndrome), still represents a serious global public health problem. Although established anti-HIV treatments are relatively effective, they are sometimes poorly tolerated, highlighting the need for further refinement of the existing antiviral drugs and the development of novel anti-HIV strategies. In this respect, the use of topical virucides would be an interesting therapeutic strategy to prevent HIV transmission. Several topical agents for preventing HIV transmission have been described, including i) agents that inactivate HIV such as detergents (e.g. Nonoxynol-9 or SAVVY®) or pH modifiers (e.g. BufferGel; ReProtect ), ii) agents that target viral replication (e.g. the reverse transcriptase inhibitors UC-781, TMC-120, tenofovir) and iii) agents that target viral entry (e.g. PRO 2000, cellulose sulfate). With the exception of tenofovir , most microbicide candidate drugs that have been subject of large clinical trials, have proved to be non-effective or even toxic upon long-term exposure of the vaginal environment to these products.
The HIV-1 nucleocapsid NCp7 is essential for and plays multiple roles in virus replication . NCp7 contains two CCHC zinc-finger motifs and binds the viral genomic RNA in the interior of the virion. The binding of NCp7 to nucleic acids results in their condensation and protection from nuclease degradation [4–6]. Therefore, this nucleoprotein complex protects the genomic RNA ensuring viral stability. A number of classes of compounds targeting the retroviral NCp7 have been described, including, 3-nitrosobenzamide (NOBA) , 2,2′-dithiobisbenzamides (DIBA) , cyclic 2,2′-dithiobisbenzamides (e.g. SRR-SB3) , 1,2-dithiane-4,5-diol-1,1-dioxide , azadicarbonamide (ADA) [11, 12], pyridinioalkanoyl thiolesters (PATEs) , bis-thiadiazolbenzene-1,2-diamines  and S-acyl-2-mercaptobenzamide thioesters (SAMTs) . The latter class of compounds was recently considered for testing as topical microbicide for the prevention of HIV transmission . These SAMT compounds were able to efficiently prevent vaginal transmission of SHIV upon exposure of nonhuman primates . Here, we have identified a low-molecular-weight molecule, 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine, WDO-217, that inhibits HIV replication. Mechanism-of-action studies reveal WDO-217 as a potent NCp7 zinc ejector that directly inactivates HIV-1 and HIV-2 virions and inhibits the transmission of DC-SIGN captured virus to CD4+ lymphocytes. WDO-217 qualifies as a potential microbicide lead compound for further (pre)clinical studies.
Results and discussion
Inhibition of HIV and SIV in cell culture
Antiretroviral activity and cytotoxicity of WDO-217 in the MT-4/MTT-assay
7.9 ± 3.3
2.3 ± 0.3
5.3 ± 1.5
72 ± 11
0.007 ± 0.001
0.005 ± 0.0008
0.016 ± 0.007
Anti-HIV-1 and -HIV-2 activity and cytostatic properties of WDO-217 in human T-lymphocyte (CEM) cells
8.3 ± 1.8
15 ± 6.7
94 ± 15
0.058 ± 0.030
0.055 ± 0.020
Antiretroviral activity and cytotoxicity of WDO-217 in the anti-HIV
1.04 ± 0.2
0.9 ± 0.05
1.2 ± 0.1
1.7 ± 0.9
1.5 ± 0.6
1.3 ± 0.4
1.04 ± 0.3
75 ± 11
Inactivation of HIV-1, including clinical isolates, and HIV-2 virions by 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine
Inactivation of isolated HIV particles by WDO-217
1 × 106
5.4 × 106
AZT (3.7 μM)
1 × 106
5.4 × 106
Triton X-100 (0.5%)
WDO-217 (0.2 μM)
5.4 × 106
1.6 × 106
WDO-217 (1 μM)
1.6 × 106
1 × 106
WDO-217 (5 μM)
1 × 106
2.1 × 106
WDO-217 (25 μM)
WDO-217 (125 μM)
WDO-217 (625 μM)
Inactivation of clinical isolates from different HIV-1 subtypes by WDO-217
WDO-217 (125 μM)
Effect of 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine on the capture of HIV-1 by Raji/DC-SIGN cells and on subsequent virus transmission to CD4+T cells
Inhibition of HIV-1 NL4.3 capture and transmission* by Raji/DC-SIGN cells, quantified by p24-ELISA
HIV-capture procedure A
Transmission procedure B
Transmission procedure C
2.2 ± 1
8.3 ± 0.4
0.014 ± 0.01
0.002 ± 0.001
Next, we evaluated whether WDO-217 could prevent the transmission of captured HIV-1 from DC-SIGN-expressing cells to CD4+ T cells. Therefore, the DC-SIGN+ cells that efficiently captured drug-treated virus were co-cultured with uninfected C8166 cells. In these co-cultures, WDO-217 dose-dependently inhibited syncytium formation at an IC50 of 2.2 μM, whereas abundant syncytium formation occurred within 24 to 48 h post co-cultivation when the captured virus had not been pre-exposed to the drug (Table 6, procedure B). When virus was first given the opportunity to be captured by Raji/DC-SIGN cells in the absence of compound and then the Raji/DC-SIGN cells were co-cultured with C8166 cells in the presence of various concentrations of compound, WDO-217 still prevented the transmission of DC-SIGN-captured virus with an IC50 value of 8.3 μM. This suggests that WDO-217 can inactivate virus particles even when they are bound to DC-SIGN (Table 6, procedure C). All together, these results demonstrate that 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine does not inhibit the viral capture by DC-SIGN but is able to inactivate captured virus and efficiently prevents the transmission of HIV-1 from DC-SIGN-expressing cells to CD4+ T-lymphocytes, underlining its potential use as a microbicide agent.
Treatment of HIV-1 with WDO-217 decreases the virion-associated viral RNA content
Zinc ejection from the retroviral zinc fingers
Inhibition of NCp7(11-55)-induced destabilization of cTAR
Effect of WDO-217 on the interaction of NCp7(11-55) with SL3 or PBS
Next, we monitored the changes in the emission spectrum of 3HC-NCp7(11-55) that was preincubated with WDO-217 for 30 minutes, before addition to SL3 (Figure 7C). The incubation of 3HC-NCp7(11-55) with WDO-217 for 30 minutes leads to a decrease in the overall intensity of the 3HC probe as well as its N*/T* ratio (from 1.0 to 0.86) in respect with the free 3HC-NCp7(11-55). This is likely the result of a stronger interaction of the 3HC probe with the peptide backbone when it is in the zinc-free form. Addition of SL3 to the zinc-depleted 3HC-NC(11-55) induced a further decrease of the fluorescence emission resulting in a spectrum similar to that obtained when WDO-217 was added to the preformed 3HC-NC(11-55)/SL3 complex (Figure 7B). This result confirms that WDO-217 is able to eject zinc from the NC/ODN complex.
WDO-217 does not cause accumulation of unprocessed Gag polyprotein
The retroviral zinc fingers of the HIV-1 NCp7 are strictly conserved and functionally obligatory in the viral replication and virion stability. In contrast to the zinc fingers, the N-terminal domain is less conserved, but the positions of the basic residues are mainly conserved; also the basic linker between the two zinc fingers is highly conserved [36, 37]. Early studies have demonstrated that disruption of the zinc fingers by removal of the zinc led to a loss of viral replication . Different classes of zinc ejectors have been reported to efficiently eject zinc from the NCp7 zinc fingers. More specifically, the N-substituted S-acyl-2-mercaptobenzamides (SAMTs) are suggested as candidate pluripotent, HIV-specific, virucidal microbicides . Such nucleocapsid inhibitors were directly virucidal by preventing the initiation of reverse transcription and modifying the CCHC amino acid domain conformation within Gag [11, 39]. Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a very potent ejector of zinc ions from the HIV-1 NCp7 zinc fingers, inactivating HIV-1 and HIV-2 virions and relieving the protection of the viral RNA by the retroviral nucleocapsid protein. The exact detailed mechanism by which the RNA is degraded is currently under study. Interestingly, WDO-217 is able to dissociate zinc ions from NCp7 even when it is bound to nucleic acids. In agreement with the comparable affinities of the native and zinc-depleted NCp7 forms for oligonucleotides [29, 40, 41], ejection of zinc from NCp7 by WDO-217 should not dissociate the protein from the oligonucleotide. However, a change in the binding mode is expected since native NCp7 was shown to strongly interact with the bases of its nucleic acid targets through van der Waals, H-bonding and stacking interactions [32, 33, 42–44], while the zinc-depleted forms of NCp7 are thought to interact mainly through electrostatic interactions with the phosphate groups of the DNA or RNA backbone [45, 46]. The differences in the binding modes of the native and zinc-depleted forms have been clearly evidenced by the fact that only the former can modify the dynamics of the bases and structurally rearrange the oligonucleotides [32, 45, 46]. In contrast to the DIBA compounds  WDO-217 is found not to form a covalent complex with NCp7. This could be of interest because it would in principle allow using a lower concentration of compound as one molecule can react with more than one NCp7 as compared to compounds that react covalently. A disadvantage of a non-covalent binder could be that zinc ejection may be reversible if it is not followed by oxidation of the cysteines in the zinc fingers. As a consequence, the concentration of WDO-217 should be high enough to maintain an efficient depletion of zinc, so that oxidation of cysteines will occur. Although its antiviral selectivity is somewhat moderate, WDO-217 represents the first lead compound among zinc-ejecting compounds with a unique mode of action. Due to its moderate therapeutic index this lead compound would be more appropriate as microbicide then as a systemic therapeutic agent. Future structure activity relationship studies and molecular modeling studies, however, will likely enable improvement on activity, selectivity and pharmacological properties of the compound. Although WDO-217, as such, is a simple molecule there is room for further modifications and subsequent SAR studies. Modifications are possible introducing substituents at different positions on the phenyl ring. It is certainly worthwhile to further investigate the importance of the relative positions and nature of the substituents on the 1,2,4-thiadiazol ring. Furthermore it would be interesting switching to other heterocyclic rings, the most obvious being a series of 4,5-disubstituted -2-aminothiazole derivatives.
Interestingly, WDO-217 treatment does not inhibit the direct gp120-mediated capture of virus on DC-SIGN suggesting that it is able to inactivate HIV but with a preservation of the conformational and functional integrity of the surface envelope proteins. This preservation of surface proteins has earlier been demonstrated with the prototypical NCp7 zinc ejector AT-2 . Moreover, WDO-217 inactivates virus particles even when they are bound to DC-SIGN and prevents the transmission of DC-SIGN-captured HIV to CD4+ cells. This makes WDO-217 suitable, not only as a valuable component in topical microbicides, but also for the inactivation of virions to be used as vaccine antigens . Indeed, HIV vaccine strategies using DCs are currently being investigated. DCs detect viruses in peripheral tissues and, following activation and virus capture/uptake, migrate to lymph nodes to trigger adaptive immune responses . However, HIV is able to modulate these DCs to facilitate infection and transmission to T-cells causing their disregulation . This justifies the need to develop strategies that prevent this modulation and disregulation. In this respect, WDO-217 appears of high interest, since it does not prevent the capture by DC SIGN but prevents subsequent transmission and infection.
This new compound is useful as a lead for the design of a future new generation NCp7 inhibitors that could be applied as an agent in microbicide formulations and vaccine strategies.
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 vitroantiviral 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/IIIBpersistently 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).
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.
We thank L. Bral, C. Heens, K. Erven K. Uyttersprot and L. Ingels for excellent technical assistance; a number of reagents were obtained through the NIH AIDS Reagent Program.
The work was supported by grants from the KU Leuven (GOA 10/14 and PF 10/18), the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO)” number 126.96.36.199 and the Agence Nationale de la Recherche sur le Sida (ANRS). BB is supported by an ANRS fellowship.
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