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The high rate of HIV-1 mutation and the frequent sexual transmission highlight the need for novel therapeutic modalities with broad activity against both CXCR4 (X4) and CCR5 (R5)-tropic viruses. We investigated a large number of natural products, and from Sargassum fusiforme we isolated and identified palmitic acid (PA) as a natural small bioactive molecule with activity against HIV-1 infection. Treatment with 100μM PA inhibited both X4 and R5 independent infection in the T cell line up to 70%. Treatment with 22μM PA inhibited X4 infection in primary peripheral blood lymphocytes (PBL) up to 95% and 100μM PA inhibited R5 infection in primary macrophages by over 90%. Inhibition of infection was concentration dependent, and cell viability for all treatments tested remained above 80%, similar to treatment with 10−6M nucleoside analogue 2′, 3′-dideoxycytidine (ddC). Micromolar PA concentrations also inhibited cell-to-cell fusion and specific virus-to-cell fusion up to 62%. PA treatment did not result in internalization of the cell surface CD4 receptor or lipid raft disruption, and it did not inhibit intracellular virus replication. PA directly inhibited gp120-CD4 complex formation in a dose-dependent manner. We used fluorescence spectroscopy to determine that PA binds to the CD4 receptor with Kd ~1.5±0.2μM, and we used one-dimensional saturation transfer difference NMR (STD-NMR) to determined that the PA binding epitope for CD4 consists of the hydrophobic methyl and methelene groups located away from the PA carboxyl terminal, which blocks efficient gp120-CD4 attachment. These findings introduce a novel class of antiviral compound that binds directly to the CD4 receptor, blocking HIV-1 entry and infection. Understanding the structure–affinity relationship (SAR) between PA and CD4 should lead to the development of PA analogs with greater potency against HIV-1 entry.
In most patients, highly active antiretroviral therapy (HAART) substantially improves clinical outcomes in treated populations.1,2 However, the high rate of HIV-1 mutation increases the likelihood and frequency of generating drug-resistant HIV-1 strains.3 Consequently, some reports show that as many as 20% of all new HIV-1 infections are with viruses resistant to the currently available drugs,4,5 which highlight the need for continued discoveries of novel inhibitors of HIV-1 infection.6 New classes of drugs, including fusion inhibitors, provide new therapeutic options, especially in the antiretroviral (ARV) treatment-experienced population. Inhibitors of virus entry are desirable therapeutic modalities, since in addition to limiting viral spread in the already infected host, they also block the virus from entering its target cell and thereby prevent de novo infection.6,7 Worldwide, heterosexual intercourse remains the principal route of new HIV infections, with women bearing a disproportionate burden of new infections,7 and viral entry inhibitors can be used in microbicide formulations aimed at the prevention of sexual transmission.8
Products derived from natural sources represent a potential source of novel and therapeutic agents that have been shown to inhibit HIV-1 infection at different stages of the virus life cycle.9,10 We investigated a large number of different natural products (NP), including plants and marine products, and identified an aqueous extract from Sargassum fusiforme (S. fusiforme) as a potent inhibitor of HIV-1 infection.11 We undertook a large-scale, bioactivity-guided fractionation on a complex S. fusiforme mixture that generated 600 fractions, with one bioactive fraction (SP4-2) exhibiting activity against global HIV-1 infection by ~87%.12 The SP4-2 fraction blocked viral entry by 53%, and it also inhibited postentry replication up to 71% that was specific against reverse transcriptase (RT). Inhibition of entry was reversed by the addition of the sCD4 receptor, suggesting an interaction of the SP4-2 bioactive molecule with the CD4 receptor.12 From the SP4-2 fraction we isolated and identified two unsaturated fatty acids, oleic and linoleic acid, and two saturated fatty acids, myristic and palmitic acid (PA).13 Our initial results showed that the two unsaturated fatty acids, oleic and linoleic acid, inhibit HIV-1 reverse transcriptase.13 However, to date, only myristic acid has been report to have activity against HIV-1 infection, which inhibits virus budding.14,15
In the present study we undertook a detailed investigation of HIV-1 inhibition of infection by PA, a saturated 16-carbon (16:0) fatty acid. Here we report that treatment of cells with PA results in specific PA-to-CD4 receptor binding and subsequent inhibition of HIV-1 entry.
Purification of S. fusiforme and the SP4-2 fraction to its individual components and identification of a straight-chain saturated PA (hexadecanoic acid, CH3(CH2)14COOH, MW=256.42) by nuclear magnetic resonance (NMR) was previously described.12,13 PA (Sigma) was solublized at 100mM in ethanol (EtOH)16 and stored at −20°C. Working aliquots were kept at 4°C for up to 4 weeks.
SupT1, GHOST (3) X4/R5, CEM, and 1G5 cells were obtained from the HIV AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and were cultured and maintained as specified by the reagent protocol. Monocyte-derived human macrophages (Ms) and peripheral blood leukocytes (PBL) were recovered from peripheral blood mononuclear cells (PBMCs) by countercurrent centrifugal elutriation as previously described.17 Monocytes were cultured as adherent monolayers (1×106 cells/well in 24-well plates), differentiated for 7 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% human serum, and recombinant human macrophage colony-stimulating factor (rhM-CSF, Wyeth, Cambridge, MA). PBL were cultured in RPMI and 10% fetal bovine serum (FBS), stimulated for 24h with 4μg/ml phytohemagglutinin (PHA), and cultured in the presence of 10U interleukin (IL)-2.
Cell cultures were treated overnight (18h) with different PA concentrations, or with different treatment as indicated in the figure legends for each experiment, washed three times, and HIV-1 infected in the absence of PA, except for macrophage cultures that were infected in the presence of PA. Cells were infected with HIV-1 at the indicated multiplicity of infection (moi), washed three times, and returned to culture with same concentration of each treatment for the duration of the experiment, and then analyzed as indicated.
Inhibition of gp120-CD4 complex formation was investigated by gp120 capture ELISA, in accordance with the manufacturer's instructions (ImmunoDiagnostics, Inc., MA). Envelope gp120 (IIIB) protein was captured on 96-well plates, washed, and incubated in the presence of CD4-biotin alone or in the presence of increasing concentrations of PA, as indicated. Strepavidin-HRP was added, and then developed by addition of a chemiluminescent substrate, o-phenylenediamine dihydrochloride (OPD). The reaction was stopped by adding 1N HCl and read at 490nm. Percent of gp120-CD4 binding was calculated from gp120-CD4 complex formation in the absence of any inhibitor.
Lipid rafts of 1G5 T cells were labeled with 1μg/ml cholera toxin-B subunit (CTB)-HRP and isolated as previously described.18 Briefly, labeled cells were then incubated for 1h in media containing 20μM PA, 20μM myristic acid (MA), or no fatty acid (No Treatment). Cells were then lysed in cold 0.1% Triton X-100 and lipid rafts isolated by sucrose density gradient centrifugation. Five gradient fractions were collected (top to bottom) and assayed for HRP activity. The (CTB-HRP) distribution was reported as a percent of total activity.
HIV-1 X4-tropic molecular clone NL4-3, which expresses all known HIV-1 proteins,19 and the R5-tropic molecular clone 81A,20 which has Ba-L Env sequences on the backbone of NL4-3, were obtained from the HIV AIDS Research and Reference Reagent Program. Macrophage HIV-1 R5-tropic primary isolate ADA was prepared as previously described.11,17 Envelope expression-deficient and luciferase-positive pNL4-3.HSA.R+.E− (HIV-Env−Luc+) was obtained from Dr. Nathaniel Landau21,22 and was pseudotyped with the VSV-G envelope. The pL-VSV-G vector was obtained from Dr. M. Emerman; it contains a VSV-G insert in the pcDNA expression vector modified by replacing the cytomegalovirus promoter with the HIV-1 long terminal repeat.23 We generated native and pseudotyped virus by calcium phosphate transfection of 293T cells, as previously described.24 Cell-free viral stock was quantified for HIV-1 p24 core antigen content by enzyme-linked immunosorbent assay (ELISA, AIDS Vaccine Program, NCI-Frederick), and was also quantified for titers of infectious virus by multinuclear activation of a β-galactosidase indicator (MAGI) assay.25
Fusion-sensitive BlaM-Vpr chimera DNA plasmid was a kind gift from Dr. W. Greene,26 and HIV-1 virions containing the BlaM-Vpr chimera were produced by cotransfection of 293T cells with pNL4-3 proviral DNA (60μg), pCMV-BlaM-Vpr (20μg), and pAdVAntage vectors (10μg) (Invitrogen), as previously described.26 After 48h at 37°C, the virus-containing supernatant was centrifuged at low speed to remove cellular debris and then concentrated at 72,000×g for 90min at 4°C, and viral stocks were normalized by p24 content measured by ELISA, and stored at −80°C before use.
Standard HIV-1 fusion assay was done as previously described.26 Briefly, Sup T1 cells were first infected for 2h with BlaM-Vpr-X4 (NL4-3) chimera at 0.5moi, washed in CO2-independent media, loaded for 1h at room temperature (rt) with the CCF2/AM dye as specified by the manufacturer (Gibco), and washed in developing buffer; the reaction was allowed to developed overnight. After development, cells were washed in phosphate-buffered saline (PBS) and fixed in 1.2% paraformaldehyde solution. The BlaM reaction was detected by the change in the emission fluorescence of CCF2 after cleavage by the BlaM-Vpr chimera, which was monitored by FACS with a three-laser Vantage SE (Becton Dickinson, San Jose, CA). A coherent krypton laser operating at 200mW and generating light at 406.7nm was used to excite the CCF2 dye. Blue emission was detected with an HQ455/50 filter and green emission was detected with an HQ545/90 BP filter; for light splitting, a 505 SP filter was used. Data were collected with CellQuest and analyzed with FlowJo software (Treestar, San Carlos, CA).
GHOST X4/R5-expressing cells were plated in 24-well plates at a concentration of 5×104 cells/well in DMEM, 10% FBS, 500mg/ml G418, 100mg/ml hygromycin, 1mg/ml puromycin, and 1% penicillin/streptomycin. The next day, cells were treated with dilutions of PA for 1.5h, and cells were infected at 0.3moi with either X4-tropic (NL4-3) or with R5-tropic (81A) HIV-1 clones; cell were then washed and returned to media containing each respective treatment. Cells were collected 40–48h after infection, washed in PBS, and incubated in 200μl 1.2% paraformaldehyde in PBS for 2–3h at 4°C prior to FACS analysis. Cell counting was performed on a BD FACSCanto FACS system and analyzed with BD FACSDiva software.
SupT1 cells were pretreated overnight with different concentrations of PA or treated for 5h with 10ng/ml phorbol 12-myristate 13-acetate (PMA), which served as a positive control for CD4 internalization.27 All systems were surface labeled with phycoerythrin (PE)-conjugated anti-CD4 antibody (Santa Cruz, CA) and analyzed on a BD LSR II flow cytometer. Internalization of CD4 was measured by the shift in the mean fluorescent intensity of labeled cells.
To perform the STD-NMR experiment, we used commercially available sCD4 (200μg, 4.4nmol, Progenics Pharmaceuticals, Inc.), which was exchanged into the NMR buffer (160μl of D2O and 40μl d6-DMSO, containing 10mM KPO4 buffer, pH 7.0). A 1mM solution of PA was dissolved in the NMR buffer and used for the titration of the NMR sample containing 18μM sCD4; 20% of d6-DMSO in the NMR buffer was used to prevent formation of PA micelles. All NMR experiments were performed at room temperature. The data were acquired on a Bruker Avance 700-MHz spectrometer equipped with a z-gradient cryoprobe. The on-resonance irradiation of the protein during the 1D STD NMR experiment was performed at a chemical shift of 0ppm. Off-resonance irradiation was applied at 30ppm. A sequence of Gauss-shaped pulses with a strength of 86Hz and a length of 50ms separated by a 1ms delay was applied for 2.04s during selective presaturation of the sCD4. The total number of scans was 1024. The NMR data were processed and analyzed using Topspin 2.0 (Bruker, Inc).
For fluorescence titration experiments, 2μM sCD4 (Progenics Pharmaceuticals, Inc.) dissolved in the NMR buffer was used, and a 1mM solution of PA dissolved in the NMR buffer was added in 1μM steps. Titrations in the absence of sCD4 were performed as a reference. Tryptophan fluorescence was measured using an excitation wavelength of 290nm. Measurements were performed on a Fluorolog-3 fluorescence spectrophotometer (HoribaJobin Yvon) at 25°C in a 1-ml stirred cuvette. The fluorescence emission signal was subtracted from the signal of the reference titrations, and the differences adjusted by the dilution factor were plotted against the final concentration of added PA. Curve fitting (OriginLab) was performed to find the best values for Kd using a single-site binding isotherm approximation.28
Previous studies with whole S. fusiforme extract and with the bioactive SP4-2 fraction demonstrated inhibition of HIV-1 infection in several primary and transformed cell lines.11,12 To test the ability of PA, which was isolated from the SP4-2 bioactive fraction,13 to specifically block productive X4 and R5-tropic HIV-1 infection, we first tested for virus inhibition in GHOST X4/R5-expressing human osteosarcoma cells by flow cytometry (Fig. 1A). Cells were treated with increasing micromolar PA concentrations and infected with HIV-1, NL4-3 (X4 infection, upper panels), or 81A (R5 infection, lower panels). In the X4 virus-infected cells, PA treatment gradually reduced the total number of infected cells from 20% to 5.9% (X4 infection, upper panels). This reduction in the total number of infected cells translated to 13, 24, 40, 52, and 70% inhibition of X4 infection due to the corresponding PA treatment of 10, 20, 40, 60, and 100μM, respectively. Similarly, infection with R5 virus gradually reduced the total number of infected cells from 52.5% to 14.45%, which translated to 15, 31, 43, 56, and 73% inhibition of infection due to the corresponding PA treatment of 10, 20, 40, 60, and 100μM, respectively (R5 infection, lower panels). This result demonstrated that treatment with free PA blocks both X4 and R5 tropic HIV-1 infection to similar levels, which is in agreement with our previous results for the SP4-2 fraction inhibition of both X4 and R5 infection in the same cell line.12 To ascertain the specificity of PA for inhibition of HIV-1, we also tested MA, which did not inhibit HIV-1 infection as compared to PA (data not shown).
Human peripheral blood mononuclear cells (PBL) and macrophages (Ms) are the primary target for HIV-1 infection in vivo; we examined PA inhibition of HIV-1 infection in these primary cells (Fig. 1B and C). In PBL, measurements of HIV-1 p24 antigen levels in cell culture supernatants revealed a peak of p24 production at 8897pg of p24/ml for NL4-3-infected and untreated cultures (0μg PA), which was followed by a gradual decline to approximately 435pg p24/ml (Fig. 1B, left panel). Treatment with 1.5, 3, 6, 12, and 22μM PA resulted in 70, 75, 77, 84, and 95% inhibition of NL4-3 infection (Fig. 1B, middle panel). Treatment with 10−6M ddC inhibited infection by 84%, somewhat lower than 95% maximal PA inhibition. In parallel, we measured cell viability, which remained above 96% for all PA treatments tested and measured slightly above 89% viability for 10−6M ddC treatment (Fig. 1B, right panel).
In human Ms, peak p24 production was quantified at 30,717pg p24/ml for HIV-1 primary R5 isolate ADA-infected and untreated (0μg PA) cells, which was approximately 3.4-fold higher as compared to NL4-3 infection in PBL. This p24 production was followed by a gradual decline to 373pg p24/ml (Fig. 1C, left panel). Treatment with 25, 50, 100, 150, and 200μM PA resulted in 35, 67, 91, 95, and 99% inhibition of ADA infection, and treatment with 10−6M ddC resulted in 99% inhibition (Fig. 1C, middle panel). Cell viability increased with the increase in inhibition of infection and was similar to the viability of ddC-treated cells (Fig. 1C, right panel).
In both cell types, a dose-dependent inhibitory effect of PA was observed throughout productive HIV-1 infection, most notably at the peak of virus replication. However, it is not clear why PA inhibitory concentrations differed between these two primary cell types. Possible differences in the level of infection between laboratory-adapted NL4-3 and primary isolate ADA, and expression of receptor or coreceptor between these primary cells, may account for these differences.
HIV-1 infection is spread either by free viral particles or 100 times more efficiently by direct cell-to-cell fusion,29 and we previously demonstrated that whole S. fusiforme extract was capable of inhibiting cell-to-cell fusion in GHOST cells.11 We next determined the ability of PA to block cell-to-cell fusion by treating uninfected GHOST cells with 20 or 40μM PA and cocultivating these cells together with NL4-3-infected CEM cells for 24h (Fig. 2A). Tat-driven GFP expression measured in uninfected GHOST cell cultures can arise only from fusion of these cells with HIV-1-infected CEM cells, and we monitored this mixed cell culture system quantitatively for GFP expression by flow cytometry. Untreated (0μM PA) GHOST mixed cell cultures resulted in 6% GFP fusion-positive cells. Treatment with 20 and 40μM PA resulted in GFP reduction to 2.94% and 2.19%, which translated to 56% and 70% inhibition of cell-to-cell fusion (indicated at the top of each panel).
The SP4-2 fraction was also shown to block HIV-1 binding and fusion, which was reversed by the addition of sCD4, suggesting interaction between the SP4-2 bioactive molecule and CD4 receptor.12 To begin to assess the mechanism by which PA inhibits HIV-1 replication we investigated virus-to-cell fusion in a standard and specific HIV-1 fusion assay30,31 as the first step in the infection process (Fig. 2B). In contrast to untreated (0μM PA) T cells that allowed HIV-1 particles to fuse and enter into 37.6% of the cells, treatment with 2, 4, and 8μM PA restricted virus fusion to 24.6, 19.7, and 14.4% cells, which translated to 3.5, 48.2, and 62.5% inhibition of HIV-1 fusion, respectively (indicated inside each panel). Inhibition of fusion in this system was confirmed by the addition of X4 coreceptor inhibitor AMD3100 that limited virus fusion to 2.8% of cells, which translated to 93% inhibition.
Because CD4 is the main receptor responsible for HIV-1 attachment, fusion, and entry into the cell, and PA-mediated inhibition of HIV-1 fusion could result from internalization of the cell surface CD4 receptor, we checked this possibility by treating SupT1 cells overnight with increasing PA concentrations, or treating cells for 5h with positive control for receptor internalization, 10ng/ml PMA27 (Fig. 3A). Cells were surface labeled with PE-conjugated anti-CD4 antibody (Santa Cruz, CA) and analyzed on a BD LSR II flow cytometer. Percent internalization (Int) or percent surface (Sur) CD4 was measured by the shift in the mean fluorescent intensity of labeled cells, and is indicated inside each box. Treatment with 0, 25, 50, and 100μM PA internalized the CD4 receptor by 1.3, 1.4, 4.7, and 7.4%, respectively. Cell surface CD4 receptor expression changed from 98, 98, 94, and 91%, which corresponded to the same micromolar PA treatment. In contrast, PMA treatment internalized the cell surface CD4 receptor by 94%, and cell surface receptor expression was 1.8%. Based on these results we conclude that treatment with up to 100μM PA does not appear to significantly alter CD4 receptor cell surface expression.
To evaluate whether the effect of PA on HIV infectivity might be due to the impact on lipid rafts, which are necessary for HIV infection, we investigated whether PA treatment has the ability to isolate cholera toxin-B (CTB)-labeled rafts from 1G5 T cells (Fig. 3B). As shown by the results presented in Fig. 3B, treatment of T cells with 20μM PA, which inhibited HIV infection by 65% (Fig. 4B) or treatment with 20μM MA, which failed to inhibit HIV infection (not shown), failed to inhibit the ability to isolate CTB-HRP-labeled lipid rafts by sucrose density gradient centrifugation.18 Treatment with 100μM PA also failed to inhibit isolation of lipid rafts (not shown). These results are consistent with the hypothesis that PA inhibits HIV infection by binding to the CD4 receptor and blocks HIV uptake, and also suggest that the inhibitor effect of PA is not due to disruption of the lipid raft structure or perturbation of the cellular membrane.
We next investigated the ability of PA to specifically inhibit envelope gp120-CD4 receptor complex formation by gp120 capture ELISA (Fig. 4A). Then 96-well plates were coated with gp120 (IIIB) and incubated with biotin-sCD4 in absence or presence of increasing PA concentrations. Percent inhibition was calculated in the absence of PA, which was taken as 100% gp120-CD4 complex formation. Treatment with 25, 50, and 100μM PA inhibited gp120-CD4 binding by 23, 45, and 71%, respectively. This result clearly indicated that PA inhibits HIV-1 infection by blocking gp120-CD4 interaction. In reverse experiments, we coated plates with sCD4, incubated gp120 with increasing concentrations of PA and tested for inhibition of gp120–CD4 complex formation, which was not inhibited, indicating that PA does not bind to soluble gp120 (not shown).
To confirm that PA specifically inhibits CD4-dependent entry, we utilized infectious envelope-deficient and luciferase-positive HIV-1 (HIV-Env−Luc+), which we pseudotyped with a CD4-independent vesicular stomatitis virus (VSV-G) protein envelope,24 or with a CD4-dependent HXB2 envelope (Fig. 4B). To examine single-cycle viral replication, CEM cells were treated with 10 or 20μM PA and infected with each pseudotyped virus; luciferase expression was quantified 72h after infection. Inhibition of infection with CD4-dependent HXB2-enveloped virus was quantified at 32% and 65%. However, inhibition of productive infection with CD4-independent VSV-G-enveloped virus was limited to 2% and 12%, also suggesting that PA treatment posed no intracellular restriction to CD4-independent virus replication once the virus was allowed to bypass CD4 receptor restriction and enter the host cell. Collectively, these results indicate that PA blocks HIV-1 infection by associating with the CD4 receptor and thus limiting virus entry into cells.
To conclusively determine the association of PA and CD4, we used one-dimensional STD-NMR32 to characterize binding of PA to sCD4, in vitro. STD-NMR experiments are typically used to probe low-affinity interactions (Kd~10−8 to 10−3M) between small molecules (ligand) and proteins, and are routinely used in drug discovery screening tests.32 Saturation transfer from protein to ligand protons identifies ligand binding to a protein, and takes place only when the ligand specifically binds to the protein, with a rate that depends on the protein mobility, ligand–protein complex lifetime, and binding geometry. Protons of the ligand having the strongest contact with the protein show the most intense STD-NMR signals, enabling the mapping of the ligand's binding epitope. During the STD-NMR experiment, increasing concentrations of PA were titrated into the sCD4 solution, and we observed PA binding to sCD4 as shown by an increase in the intensity of the ligand STD-NMR signal (Fig. 5A and B). The amount of STD was quantified by calculating the difference between the intensity of one signal in the off resonance, or reference NMR spectrum (I0), and the intensity of a signal in the on-resonance NMR spectrum (Isat), for various concentrations of the ligand (Fig. 5C). As the concentration of PA increased, we observed a steady increase of the STD signal from the CH2 and CH3 groups of PA (Fig. 5B). Methylene groups located close to the carboxyl end of PA did not exhibit an STD signal during titration. Based on these results, we concluded that PA binds to sCD4 by utilizing a hydrocarbon chain located at a distance from the negatively charged end of the fatty acid. No STD-NMR signal was observed during titration of MA into the sCD4 solution, which was consistent with our results showing no HIV-1 inhibition by MA (data not shown). This negative result indicates that PA binds to sCD4 specifically. We used tryptophan fluorescence of sCD4 to estimate the binding affinity of PA. Saturating concentrations of PA quenched 30% of the sCD4 tryptophan fluorescence and resulted in a red shift of the emission peak of 2nm. Based on the fluorescence titration experiments (Fig. 5D and 5D insert), we estimated the dissociation constant (Kd) to be ~1.5±0.2μM.
We demonstrated the ability of PA to block X4 and R5-tropic HIV-1 infection and virus replication in T cells, in primary PBL and Ms (Fig. 1). The reason for the observed differences in the inhibitory concentrations of PA between PBL and Møs is unclear; it is possibly due to intrinsic differences in receptor and coreceptor frequencies in these primary cells. Differences between laboratory-adapted NL4-3 infection of PBL as opposed to primary isolate ADA infection of Møs, and the level of infection that was approximately 3.4-fold higher in Møs as compared to PBL, may also explain differences in inhibition rates between these primary cells. PA also blocked cell-to-cell fusion (Fig. 2A), which has physiological relevance considering that this is one of the mechanisms of T cell depletion in vivo.29 Cell-to-cell fusion is normally mediated between infected cells expressing a viral envelope gp120 protein that fuses with the cell surface CD4 receptor of the host cell and thus form syncytia; PA treatment limited this specific gp120-CD4 interaction up to 70%. We further established PA inhibition specificity by demonstrating the ability of PA to explicitly block virus-to-cell fusion (Fig. 2B). We also determined that HIV-1 inhibition by PA was not due to internalization of the cell surface CD4 receptor or to lipid raft disruption (Fig. 3A and B, respectively).
PA treatment inhibited specific gp120-CD4 complex formation in a dose-dependent manner (Fig. 4A), suggesting that PA blocks HIV-1 infection by interfering with virus gp120 envelope-efficient attachment to the CD4 receptor. Inhibition was mediated against the CD4-dependent HXB2 envelope but not against the CD4-independent VSV-G envelope (Fig. 4B). Productive infection by the CD4-independent HIV-1 envelope also demonstrated that PA does not pose an intracellular restriction to virus replication, once receptor restriction is bypassed. This is in agreement with data showing partial inhibition of HIV-1 reverse transcriptase by oleic and linoleic acid contained in the SP4-2 fraction but not by PA.12,13
The definitive specificity of the PA and CD4 interaction was demonstrated by STD NMR experiments, which showed that the binding epitope of PA for sCD4 consists of methyl and methelene groups located away from the carboxyl terminal (Fig. 4), suggesting that these groups are responsible for CD4 binding affinity and that, in addition, the PA carboxyl group may block viral attachment to the receptor. The binding affinity between PA and sCD4 was estimated to be ~1.5±0.2μM. MA, which has the same saturated structure and is only two carbon atoms shorter than PA, did not block HIV infection or bind to CD4 (not shown), which demonstrated a high level of specificity of PA to CD4 binding and consequent inhibition of virus entry. Collectively, these results demonstrate that PA binds to sCD4 by utilizing a hydrocarbon chain located away from the negatively charged carboxyl group, which blocks efficient gp120-CD4 interaction.
PA is a natural small molecule that specifically inhibits HIV-1 fusion to the CD4 receptor and probably functions independently of viral coreceptors. However, ARV drugs that interact with host cell receptors could have profound immunomodulatory effects, such as T lymphocyte proliferation, T cell activation, cytokine production, apoptosis, and B cell production of immunoglobulins. In contrast to systemically administered ARV therapeutics, vaginal microbicides that are normally applied topically in a gel formulation may have different pharmacodynamics,33 as was recently demonstrated with glycerol monolaurate (GML), which prevented mucosal SIV transmission.34 It is of interest to note that GML has physical and chemical properties similar to PA, indicating that PA is a lead molecule for microbicide development. Although PA efficacy of inhibition is limited, understanding the structure–affinity relationship between PA and the CD4 receptor should allow for the rational design and development of PA analogs with enhanced potency against HIV-1 entry; these possibilities with respect to PA applications are currently under investigation.
This work was supported by NIH Grant AT003371 to M.C. and American Diabetes Association Career Development Award 1-06-CD-23 to A.S. The authors wish to thank Drs. Paul R. Skolnik and Simon Hirschl for valuable discussions and help with manuscript editing. D.Y.W. Lee and X. Lin contributed equally to this study.
No competing financial interests exist.