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Originally published In Press as doi:10.1074/jbc.M603609200 on August 1, 2006

J. Biol. Chem., Vol. 281, Issue 39, 28699-28711, September 29, 2006
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Inhibition of HIV-1 Replication by Amphotericin B Methyl Ester

SELECTION FOR RESISTANT VARIANTS*

Abdul A. Waheed{ddagger}, Sherimay D. Ablan{ddagger}, Marie K. Mankowski§, James E. Cummins§, Roger G. Ptak§, Carl P. Schaffner, and Eric O. Freed{ddagger}1

From the {ddagger}Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, the §Infectious Disease Research Department, Southern Research Institute, Frederick, Maryland 21701, and the Department of Microbiology and Biochemistry, Waksman Institute, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08903

Received for publication, April 14, 2006 , and in revised form, June 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human immunodeficiency virus type 1 (HIV-1)2 replication cycle both begins and ends with events that take place at the host cell plasma membrane. Viral entry is initiated upon binding of the surface Env (envelope) glycoprotein gp120 to the CD4 receptor molecule on the target cell membrane. Following CD4 binding and interaction of gp120 with the coreceptor (generally CCR5 or CXCR4), conformational changes in gp120 and in the transmembrane Env glycoprotein gp41 lead to fusion of the virion lipid bilayer with the target cell plasma membrane (for review, see Ref. 1). This fusion event allows the viral nucleoprotein complex to enter the host cell and permits the establishment of a productive infection. Late in the replication cycle, viral assembly and release are directed by the Gag precursor protein Pr55Gag. During or immediately after particle release, Pr55Gag is cleaved by the viral protease to generate the mature infectious virion (2).

Studies from our laboratory and others have suggested that specific cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts (3) are involved in both early and late phases of the HIV-1 replication cycle (for review, see Refs. 4 and 5). Pr55Gag and the HIV-1 Env glycoproteins have been reported to associate with detergent-resistant, cholesterol/sphingolipid-rich membrane in biochemical assays (6–13) and to colocalize with raft markers by confocal microscopy (9, 11, 13). Disruption of lipid rafts interferes with both HIV-1 fusion and entry (14) and viral particle production (10, 11). Treatment of virus-producing cells with statins also inhibits viral assembly and release and reduces the infectivity of virions produced from treated cells (10). Exposing target cells to compounds that inhibit the synthesis of glycosphingolipids, another essential lipid raft component, impairs HIV-1 infection (15). The HIV-1 lipid bilayer is enriched in cholesterol and raft-specific sphingolipids relative to the host cell plasma membrane (16, 17), and depleting cholesterol from viral particles also results in impaired HIV-1 infectivity (18–22). Other viruses have also been reported to utilize lipid rafts to facilitate their replication; these include retroviruses such as murine leukemia virus (MLV) and human T-cell leukemia virus type 1 and other enveloped viruses such as the orthomyxoviruses, paramyxoviruses, and filoviruses (for review, see Ref. 5).

The involvement of lipid rafts in multiple stages of the HIV-1 replication cycle raises the possibility that disruption of these membrane microdomains could serve as an antiviral therapy. Indeed, Khanna et al. (23) showed that topical application of 2-hydroxypropyl-beta-cyclodextrin blocks transmission of cell-associated HIV-1 in a severe combined immunodeficient mouse model of vaginal HIV-1 transmission. del Real et al. (24) reported that statin treatment reduces viral loads in HIV-1-infected patients; this effect, however, was not observed in other studies (25–27).


Figure 1
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  		FIGURE 1.
Replication kinetics of HIV-1 in the absence or presence of AME. Viral stocks obtained by transfection of 293T cells with HIV-1 molecular clones were normalized for reverse transcriptase (RT) activity and used to infect T-cell lines (CEMx174, CEM(12D-7), and MT-2), primary human PBLs, and MDMs. T-cell line and PBL infections were performed with the T-cell line-tropic molecular clone pNL4-3; MDM infections were performed with the macrophage-tropic pNL4-3 derivative (pNL(AD8)). Jurkat cells were transfected with the pNL4-3 molecular clone. T-cell lines were split every 2 or 3 days, and RT activity was monitored at each time point. The results are representative of at least two independent experiments. Cell viability was not significantly affected by treatment with 10 µM AME as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium toxicity assays. {blacksquare}, untreated; x, 1 µM AME; {blacktriangleup}, 5 µM AME; •, 10 µM AME; {diamondsuit}, mock-infected.

 
Inconsistent effects of statin therapy on viral loads in vivo, together with the cytotoxicity associated with cyclodextrins, prompted us to examine the antiviral activity of other compounds that target cholesterol. To this end, we investigated the effect of amphotericin B methyl ester (AME) treatment on HIV-1 replication. This compound is a relatively non-cytotoxic derivative of the cholesterol-binding polyene antifungal antibiotic amphotericin B (28). AME has been shown in the past to be active against a variety of lipid-enveloped viruses, including HIV-1 (29–33); however, the mechanism of action of this compound, the breadth of its antiviral activity against diverse clades of HIV-1, the viral determinants of drug sensitivity, and the step(s) in the replication cycle affected have not been defined. In this study, we demonstrate the potent antiviral activity of AME against a wide range of HIV-1 isolates. We show that this compound inhibited both viral egress and entry. To understand further the mechanism of action of AME, we selected for, isolated, and characterized HIV-1 isolates that acquired AME resistance. Changes that conferred resistance to AME mapped to the cytoplasmic domain of the gp41 transmembrane Env glycoprotein. We further demonstrate that the infectivity of HIV-1 virions bearing either the amphotropic MLV Env glycoprotein or the vesicular stomatitis virus glycoprotein (VSV-G) were insensitive to AME. This study sheds light on the target and mechanism of action of AME and provides support for the development of cholesterol-binding compounds as antiretroviral drugs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The infectious full-length HIV-1 molecular clone pNL4-3 (34) and the macrophage-tropic (pNL(AD8)) (35), Envdefective (pNL4-3/KFS) (36), and protease-defective (pNL4-3/PR–) (37) derivatives have been described previously. pNL4-3 derivatives containing the gp41 endocytosis motif mutations Y712A, Y712S, and Y712del (38) were kindly provided by M. Thali (University of Vermont). For nomenclature consistency, these mutants were renamed Y201A, Y201S, and Y201del, respectively. The full-length rhesus macaque simian immunodeficiency virus (SIVmac) molecular clone SIVmac239 (39) and the pB239Q734 derivative encoding a truncated gp41 cytoplasmic tail were kindly provided by B. Crise and Y. Li (AIDS Vaccine Program, NCI-Frederick, National Institutes of Health). The plasmid pSV-A-MLV-env (40), encoding amphotropic MLV Env, was obtained from D. Littman and N. Landau through the AIDS Research and Reference Reagent Program of the National Institutes of Health. HIV-1 Env expression vectors pIIINL4env, pIIINL4envCTdel144-2, and pIIINL4envCTdel104 have been described previously (36, 41, 42). The VSV-G expression vector pHCMV-G (43) was generously provided by J. Burns (University of California, San Diego, La Jolla, CA). pCMV-BlaM-Vpr (44) was a gift from W. Greene (University of California, San Francisco).

Cells, Transfections, and Infections—Jurkat, CEMx174, MT-2, CEM(12D-7), and HeLa cell lines were maintained as described (45). Primary human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs) were isolated and cultured as detailed previously (35). Viral stocks were prepared by transfection and normalized for reverse transcriptase activity, and infections of T-cell lines, PBLs, and MDMs were performed as described (35, 45). In the cultures treated with AME (generously provided by Karykion Corp., Princeton, NJ), the compound was present continuously. For luciferase-based, single-cycle infectivity assays, reverse transcriptase-normalized viral stocks were used to infect the CD4+/CXCR4+/CCR5+ HeLa cell derivative TZM-bl (obtained from J. Kappes through the AIDS Research and Reference Reagent Program). This indicator cell line contains integrated copies of the beta-galactosidase and luciferase genes under the control of the HIV-1 long terminal repeat (46). Infection efficiency was determined by measuring luciferase activity 2 days post-infection as described (45).

Mutagenesis and DNA Cloning—Molecular clones encoding gp41 amino acid substitution mutants P203L and S205L were constructed by QuikChange mutagenesis (Stratagene) using mutagenic oligonucleotides.3 The Env expression vectors pIIINL4env/P203L and pIIINL4env/S205L were constructed by cloning the 2.5-kb KpnI-XhoI fragment (pNL4-3 nucleotides 6343–8887) from pNL4-3/P203L or pNL4-3/S205L, respectively, into pIIINL4env. The reverse transcriptase- and Env-defective pNL4-3 derivative (pNL4-3/RT–/KFS) was constructed by cloning the SphI-SalI fragment (pNL4-3 nucleotides 1443–5785) from pNL4-3/RTD186N and the SalI-BamHI fragment (pNL4-3 nucleotide 5785–8465) from pNL4-3/KFS into the SphI-BamHI sites of pNL4-3.


Figure 2
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  		FIGURE 2.
AME inhibits HIV-1 infectivity by blocking viral entry. A, effect of AME on HIV-1 infectivity. TZM-bl cells were infected with HIV-1 for 2 h in the absence or presence of AME. Cells were washed and cultured for 2 days and lysed, and luciferase activity was measured. Error bars indicate S.D. B, effect on HIV-1 infectivity of AME treatment of virions, target cells, or both. TZM-bl cells or virions alone were treated with 10 µM AME for 2 h prior to infection or were infected in the absence or presence of AME as described for A, and luciferase activity was measured. Error bars indicate S.E. C, effect of AME on viral entry. Jurkat cells were infected with virions containing BlaM-Vpr in the absence or presence of AME and loaded with CCF2-AM dye. The BlaM reaction was allowed to develop, and fluorescence ratios were determined as described under "Materials and Methods." Error bars indicate S.E.

 
Viral Assembly and Release Assays—For metabolic radiolabeling assays, Jurkat cells were infected with high titer viral stocks pseudotyped with VSV-G, and HeLa cells were transfected with proviral DNA. Two days post-transfection or post-infection, cells were metabolically labeled for 2 h with [35S]Met/Cys. Preparation of cell lysates, pelleting of labeled virions in an ultracentrifuge, and immunoprecipitation of cell- and virion-associated proteins with HIV Ig (obtained through the AIDS Research and Reference Reagent Program) have been described (47). Quantification was performed by PhosphorImager analysis, and viral release efficiency was calculated as the amount of virion-associated Gag as a fraction of total (cell- and virion-associated) Gag.

Viral Entry and Env-induced Fusion Assays—To measure the efficiency of viral entry, HIV-1 virions containing the BlaM-Vpr chimeric protein were produced, and the virion-based fusion assay was conducted as described previously (44). Briefly, Jurkat cells were incubated with virions containing BlaM-Vpr (50–500 ng of p24); the BlaM substrate CCF2-AM was loaded, and the BlaM reaction was allowed to develop for 12 h as described (44). The cells were washed and fixed in 1.2% paraformaldehyde, and fluorescence was quantified at 447 nm (blue) and 520 nm (green) using excitation at 409 nm in a Fluoromax-3 fluorometer (Horiba). Fluorescence ratios were determined after subtracting the blue fluorescence of cells that were not infected with virus and the green fluorescence of cells that were neither infected nor loaded with CCF2-AM.


Figure 3
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  		FIGURE 3.
HIV-1 particle production is inhibited by AME. A, Jurkat cells were infected with VSV-G-pseudotyped viral stocks and, 2 days post-infection, metabolically labeled for 90 min with [35S]Met/Cys. Labeled viral proteins in cell and virion lysates were immunoprecipitated with HIV Ig and analyzed by SDS-PAGE, followed by fluorography. B, viral release efficiency was calculated as the amount of virion-associated p24 relative to total (cell plus virion) Gag. Data from two independent experiments were quantified by PhosphorImager analysis and are shown as the means ± S.D.

 
Syncytium formation was monitored by transfecting TZM-bl cells with HIV-1 Env expression vector pIIINL4env, pIIINL4env/P203L, or pIIINL4env/S205L. After transfection, cells were treated or not treated with AME for 2 days, and syncytia were counted after staining the cells with a solution of methylene blue and pararosaniline (48).

Selection and Characterization of AME-resistant Viruses—AME-resistant isolates were selected by prolonged serial passage of wild-type (WT) pNL4-3 in Jurkat cells in the continual presence of an inhibitory concentration (10 µM) of AME. Viral DNA was prepared from cultures infected with putative AME-resistant virus using the QIAamp blood kit (Qiagen Inc.). The entire Gag and Env coding regions were amplified by PCR and sequenced. Putative resistance-conferring mutations were introduced into pNL4-3 by site-directed mutagenesis as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AME Inhibits HIV-1 Replication—To examine the potential anti-HIV-1 activity of AME, we monitored its effect on replication of the HIV-1 pNL4-3 clone in a range of cell types at varying concentrations of the compound. We observed that, in a number of human T-cell lines, 5 and 10 µM AME significantly inhibited HIV-1 replication (Fig. 1A–D). Cell viability was not significantly affected by treatment with 10 µM AME (data not shown). To extend this analysis to primary cell types that constitute natural targets for HIV-1 infection, we tested the replication of HIV-1 in primary human PBLs and MDMs. The replication of HIV-1 was blocked at AME concentrations of 5 and 10 µM in both PBLs and MDMs (Fig. 1, E and F).

AME Inhibits an Early Stage of the HIV-1 Replication Cycle—To define the step in the HIV-1 replication cycle inhibited by AME, we carried out single-cycle infectivity assays in TZM-bl cells, a CD4+/CXCR4+/CCR5+ HeLa cell derivative that harbors a stably transfected luciferase reporter gene under the control of the HIV-1 long terminal repeat (46). TZM-bl cells were infected with HIV-1 in the presence or absence of AME, and luciferase activity was measured 2 days post-infection. As shown in Fig. 2A, we observed a dose-dependent inhibition of viral infectivity, with concentrations above 4 µM reducing HIV-1 infectivity by >100-fold.

To explore further the mechanism by which AME inhibits viral infectivity, we performed TZM-bl infectivity assays following AME treatment of the target cells, the virions, or both (Fig. 2B). We observed that pretreatment of the target cells had a minimal effect, whereas pretreatment of the virions severely inhibited infectivity. Treatment of both target cells and virions resulted in a level of inhibition that was similar to that observed upon treatment of virions alone. These results suggest that the primary target of AME inhibition is the virion, not the target cell membrane. Consistent with this hypothesis, we observed a minimal effect of AME on cell-cell fusion (syncytium formation) in TZM-bl cells (data not shown).


Figure 4
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  		FIGURE 4.
Selection for AME-resistant virus. A, Jurkat cells were transfected with pNL4-3 and cultured in the presence or absence of AME. B, the virus that replicated and peaked at day 31 in the presence of 10 µM AME in A was repassaged in parallel with the WT virus and cultured in the presence or absence of AME. The results are representative of at least three independent experiments. RT, reverse transcriptase.

 
The inhibitory effect of AME on viral infectivity could result from either an entry or a post-entry defect. To define the step at which AME inhibition is imposed, we carried out viral entry assays (44) in the presence or absence of AME. In this assay, beta-lactamase-Vpr (BlaM-Vpr) is packaged into virions. Upon fusion between the viral and target cell membranes, BlaM is delivered into the target cell cytoplasm, where it cleaves preloaded CCF2, a fluorogenic BlaM substrate. Cleavage of CCF2 can be detected by fluorometry. As indicated in Fig. 2C, AME inhibited viral entry in a dose-dependent manner. These results suggest that viral entry (the fusion between the viral and target cell membranes) is the major step at which AME inhibition is imposed.

AME Disrupts HIV-1 Particle Production—The data presented in Fig. 2 indicate that AME potently inhibits early events in the HIV-1 replication cycle. Because AME is a cholesterol-binding compound and because we reported previously that cholesterol depletion significantly inhibits HIV-1 particle production (10), we determined whether AME might also disrupt a late stage of HIV-1 replication. Viral release assays were performed in the Jurkat T-cell line (Fig. 3, A and B) and in HeLa cells (data not shown). In both cell lines, viral release efficiency was inhibited in a concentration-dependent manner. The effect of AME on viral production was independent of viral protease activity, as the release of virus-like particles produced by a protease-defective pNL4-3 derivative (pNL4-3/PR–) (37) was also significantly reduced by AME (data not shown).

Selection of AME-resistant Virus—The data presented thus far indicate that AME disrupts both early and late stages of the HIV-1 replication cycle, with effects on the early stages being particularly profound. To elucidate further the mechanism(s) by which AME interferes with HIV-1 replication, we sought to select for AME-resistant HIV-1 variants. We observed in Jurkat cells that AME markedly delayed viral replication (Fig. 4A). To determine whether the delayed viral replication was due to the emergence of AME-resistant viral variants, we repassaged virus obtained from the AME-treated Jurkat cultures in parallel with virus obtained from untreated cultures. We observed that the virus obtained from AME-treated cells replicated with comparable kinetics in the presence or absence of 10 µM AME. In contrast, the WT virus again showed a significant delay in replication in the presence of AME (Fig. 4B). The observation that the replication of virus obtained from AME-treated cultures was not inhibited by AME upon repassage indicated that this virus had acquired AME resistance.

Characterization of AME-resistant Mutants—To identify the change(s) responsible for AME resistance, we amplified the Gag and Env coding regions by PCR using as a template HIV-1 genomic DNA from infected cells obtained from two independent AME selections. Upon DNA sequence analysis, no changes were observed in the gag gene. In env, two single amino acid changes were repeatedly observed in or adjacent to the YSPL motif in the membrane-proximal region of the gp41 cytoplasmic tail (P203L or S205L) (Fig. 5A). This motif has been implicated in endocytosis of Env from the plasma membrane through its binding to the clathrin-associated adaptor protein complex AP-2, basolateral targeting of Env in polarized epithelial cells, and polarized viral budding from a localized region of the lymphocyte plasma membrane (38, 49–51).


Figure 5
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  		FIGURE 5.
Mutations in the gp41 cytoplasmic domain confer resistance to AME. A, identification of mutations that confer AME resistance. The entire Gag and Env coding regions were amplified by PCR from the genomic DNA of cells producing AME-resistant virus and sequenced. Changes that were observed in three independent experiments mapped to the gp41 cytoplasmic tail. TM, transmembrane domain. B, multiple-round replication of AME-resistant mutants. The P203L and S205L mutations were introduced into pNL4-3, and viral stocks were prepared and used to infect PBLs. Viral replication was monitored as described in the legend to Fig. 1. The results are representative of at least two independent experiments. RT, reverse transcriptase. C, single-cycle infectivity of the P203L and S205L mutants. TZM-bl cells were infected with WT and AME-resistant mutant viruses in the absence or presence of the indicated concentrations of AME as described in the legend to Fig. 2A. Cells were lysed, and luciferase activity was measured. Data shown are means ± S.E. from five independent experiments.

 
To determine whether the identified changes in gp41 conferred AME resistance, we constructed pNL4-3 derivatives containing the P203L and S205L substitutions. The replication kinetics of the mutants in PBLs were compared with those of the WT parental virus in both the presence and absence of AME. Interestingly, the replication kinetics of the P203L and S205L mutants were unaffected by AME (Fig. 5B), demonstrating that these mutations conferred AME resistance. To confirm that the P203L and S205L mutants were AME-resistant, we performed single-cycle infectivity assays in TZM-bl cells. The infectivity of the P203L and S205L mutants was reduced by only 20% in the presence of 4 µM AME during infection, whereas the infectivity of the WT virus was reduced by 50-fold (Fig. 5C). Treatment with 10 µM AME reduced the infectivity of P203L and S205L by only ~2-fold, whereas the infectivity of WT pNL4-3 was reduced by >100-fold. These results establish that the P203L and S205L mutations confer resistance to AME. We also tested the P203L and S205L mutants in the BlaM viral entry assay. Consistent with their resistance to AME, entry of P203L and S205L mutant virions was not inhibited even at 10 µM AME, whereas entry of virions bearing WT Env was significantly reduced (data not shown). Particle production of the WT virus and the P203L and S205L mutants was reduced to a similar extent by treating virus-producing cells with 10 µM AME (data not shown).

As mentioned above, the YSPL motif has been implicated in endocytosis of Env from the plasma membrane. Mutations in this motif, particularly substitution of Tyr at gp41 amino acid 201 (gp160 amino acid 712), have been shown to block AP-2 binding, to increase the levels of Env on the cell surface, and to increase Env-mediated syncytium formation (38, 49). To test whether the P203L and S205L changes affected syncytium formation, we transfected TZM-bl cells with WT, P203L, and S205L Env expression vectors and scored the number of syncytia 2 days post-transfection. No significant differences were observed between the WT virus and mutants in this assay (data not shown). We also observed that the single-cycle infectivity in TZM-bl cells of virions bearing P203L or S205L mutant Env was comparable with that of virions bearing WT Env (data not shown).

To monitor the possible effects of the AME resistance-conferring mutations on Env expression, processing, and incorporation into virions, we measured the cell-associated gp160/gp120 ratio and the levels of virion gp120 by radioimmunoprecipitation analysis. No significant differences were observed in the cell-associated gp160/gp120 ratio, indicating that the mutations did not affect Env processing (data not shown). The levels of virion gp120 also did not differ significantly between WT Env and P203L or S205L mutant Env, and AME treatment did not affect the levels of virion gp120 (Fig. 6).


Figure 6
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  		FIGURE 6.
AME-resistant mutants display WT levels of Env incorporation into virions. HeLa cells were transfected with WT or AME-resistant molecular clones and treated or not with 10 µM AME. One day post-transfection, cells were metabolically labeled with [35S]Cys for 5 h. Virion-associated material was obtained by pelleting the cell supernatant in an ultracentrifuge, lysed, and immunoprecipitated with HIV Ig. The ratio of gp120 to p24 in virions was quantified by PhosphorImager analysis. The data from three independent experiments are shown as the means ± S.D.

 
Because depletion of cholesterol from HIV-1 virions with cyclodextrin has been shown to significantly reduce viral infectivity (18–22), we sought to determine whether the inhibitory effect of AME on particle infectivity was due to cholesterol depletion. Supernatants collected from virus-producing cells labeled with [35S]Met/Cys and [3H]cholesterol were divided into two equal parts and treated or not treated with 10 µM AME. The virion-associated material was collected by ultracentrifugation, and the ratio of [3H]cholesterol to [35S]Met/Cys in virions was calculated. We detected no effect of AME on the levels of cholesterol in virions following AME treatment (Fig. 7). In contrast, cholesterol depletion from virions with the cholesterol-binding compound 2-hydroxypropyl-beta-cyclodextrin disrupted the integrity of the virion lipid bilayer (data not shown). These results indicate that the mechanisms by which cyclodextrin and AME interfere with particle infectivity are distinct. We also tested the possibility that the gp41 mutations might influence the levels of cholesterol in viral particles, thereby altering particle sensitivity to this cholesterol-binding compound. We observed that the levels of cholesterol in the P203L and S205L virions were slightly reduced relative to those in the WT virions, but this reduction was not statistically significant (Fig. 7). These results suggest that resistance does not arise through modulation of virion cholesterol levels. Finally, we tested whether the AME resistance-conferring mutations also reduced sensitivity to 2-hydroxypropyl-beta-cyclodextrin. We found that the infectivity of the P203L and S205L mutants was as sensitive as that of the WT virus to 2-hydroxypropyl-beta-cyclodextrin treatment (data not shown). These results again indicate that the mechanisms by which AME and cyclodextrins inhibit HIV-1 infectivity are distinct.


Figure 7
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  		FIGURE 7.
AME treatment does not deplete cholesterol from WT or AME-resistant virions. HeLa cells were transfected with WT or AME-resistant molecular clones. One day post-transfection, cells were metabolically labeled overnight with [35S]Met/Cys and [3H]cholesterol, and the cell supernatant was collected and divided into two halves, one not treated and the other treated with 10 µM AME at 37 °C for 2 h. Virion-associated material was obtained by pelleting the supernatant in an ultracentrifuge, and the ratio of [3H]cholesterol to [35S]Met/Cys in virions was quantified by liquid scintillation counting. Data from four independent experiments are shown as the means ± S.D.

 
Because AME resistance maps to residues in or adjacent to the YSPL endocytosis motif, we sought to determine whether disruption of this motif would confer AME resistance. To this end, we tested three well characterized mutants of the YSPL motif (Y201A, Y201S, and Y201del), all of which remove the critical Tyr at gp41 amino acid 201 (gp160 amino acid 712) (38). We observed that all of these Tyr mutants were fully sensitive to inhibition by AME in single-cycle TZM-bl infectivity assays (Fig. 8). Together, these data indicate that disruption of the gp41 membrane-proximal YSPL endocytosis motif does not confer resistance to AME. Furthermore, although the AME resistance-conferring mutations are located within and adjacent to the YSPL motif, these changes are distinct from those previously reported to disrupt the function of this motif (38).

Truncation of the Cytoplasmic Tails of Both HIV-1 and SIVmac gp41 Confers AME Resistance—Because the changes responsible for AME resistance are located within the long cytoplasmic tail of gp41, we investigated whether truncation of the cytoplasmic tail would confer resistance to inhibition of viral infectivity by AME. Interestingly, although the infectivity of virions bearing WT Env was markedly inhibited by AME, the infectivity of virions bearing the Env truncation mutants was only minimally affected (Fig. 9A).

Like HIV-1, SIV encodes a gp41 with a long cytoplasmic tail (~150 amino acids). We therefore examined whether SIV is sensitive to AME and, if so, whether its gp41 cytoplasmic tail influences AME sensitivity. The results showed that SIVmac239 infectivity was severely inhibited by AME (Fig. 10A). Interestingly, as we observed for HIV-1, truncation of the SIVmac239 gp41 cytoplasmic tail relieved sensitivity to AME (Fig. 10A). To corroborate these results in the context of a spreading infection, CEMx174 cells were infected with SIVmac239 stocks, and the effect of AME on viral replication was determined (Fig. 10, B and C). This analysis indicated that the replication of WT SIVmac239 was potently inhibited by AME (Fig. 10B). In contrast and consistent with the single-cycle infectivity data presented above, the replication of an SIVmac239 clone encoding a truncated gp41 cytoplasmic tail was not delayed in the presence of AME (Fig. 10C).


Figure 8
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  		FIGURE 8.
Mutation of the Tyr residue in the YSPL motif does not confer resistance to AME. Viral stocks were prepared using the WT virus, YSPL Tyr mutants (Y201A, Y201S, and Y201del), and AME-resistant mutants (P203L and S205L). TZM-bl cells were infected with these viruses in the absence or presence of the indicated concentrations of AME as described in the legend to Fig. 2A. Cells were lysed, and luciferase activity was measured. Data are shown as the means ± S.E. from four to six independent experiments.

 
HIV-1 Virions Bearing MLV Env or VSV-G Are AME-resistant—The data presented above indicate that both HIV-1 and SIVmac are sensitive to AME and that, in both cases, deletions in the gp41 cytoplasmic tail relieve inhibition by AME. To evaluate the effect of AME on the infectivity of virions containing highly divergent Env glycoproteins, we pseudotyped virions with either amphotropic MLV Env or VSV-G and tested their infectivity in the TZM-bl assay. Interestingly, we observed that, following AME treatment, the infectivity of VSV-G- or amphotropic MLV Env-pseudotyped virions was 70 and 115%, respectively, of that measured in the absence of AME (Fig. 9B). In contrast, the infectivity of virions bearing WT HIV-1 Env was potently inhibited by AME. These results demonstrate that the infectivity of HIV-1 virions pseudotyped with either amphotropic MLV Env or VSV-G is not significantly inhibited by AME. These findings suggest that the anti-HIV activity of AME is highly selective for virions bearing native HIV-1 or SIV Env glycoproteins.

AME Inhibits the Replication of a Broad Panel of HIV-1 Isolates—To extend the analysis of AME antiviral activity to other HIV-1 isolates, we examined the effect of the compound on the replication in peripheral blood mononuclear cells of a diverse panel of 25 HIV-1 strains (Table 1). These viruses include representatives from groups M (clades A–G) and N (obtained through the AIDS Research and Reference Reagent Program) as well as three multidrug-resistant viruses (52). The tropism of these isolates was X4, R5, or dual-tropic (X4/R5) and included both syncytium-inducing and non-syncytium-inducing viruses. In nearly all cases, marked inhibition was observed. These results indicate that AME inhibits the replication of diverse HIV-1 isolates, irrespective of their clade, target cell tropism, or drug resistance status.


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  		TABLE 1
AME inhibits the replication of divergent HIV-1 isolates

Phytohemagglutinin-stimulated peripheral blood mononuclear cells from at least two normal donors were pooled and diluted in fresh medium to a final concentration of 1 x 106 cells/ml. After infection with the viral stocks, peripheral blood mononuclear cell cultures were maintained for 7 days in the presence of 10 different concentrations of AME. Cell-free supernatant samples were collected for analysis of reverse transcriptase activity and/or HIV p24 content. Following removal of supernatant samples, compound cytotoxicity was measured by the addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. The 50% (IC50) and 90% (IC90) inhibitory concentrations are shown. The therapeutic index (TI) was calculated by dividing the TC50 by the IC50. Infections were performed in triplicate; data represent the means ± S.D. from the three dose-response curves. SI, syncytium-inducing; NSI, non-syncytium-inducing.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data obtained in this study demonstrate that AME blocks the replication of diverse HIV-1 isolates, irrespective of their clade, target cell tropism, or resistance to reverse transcriptase or protease inhibitors. We observed that AME inhibits both viral particle production and viral entry, with the defects in entry being particularly severe. Time-of-addition experiments suggested that the effect of AME on viral infectivity is imposed primarily on the virion, not on the target cell. A similar disruption of viral infectivity was also observed with SIVmac. These results demonstrate that AME inhibits multiple steps in the lentiviral replication cycle.

To further understand the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Interestingly, amino acid substitutions that conferred resistance to AME mapped within or adjacent to the YSPL endocytosis motif (38, 49, 53–56) in the membrane-proximal region of the gp41 cytoplasmic domain (Pro203 or Ser205). The YSPL motif is a highly conserved region of gp41 that is important for lentiviral replication in vivo (57). The P203L and S205L mutants were able to overcome the defects in viral infectivity imposed by AME and replicated efficiently in the presence of the compound. Although the P203L and S205L mutations are in or adjacent to the YSPL endocytosis motif, we observed that Tyr201 mutants were sensitive to AME, indicating that disruption of the YSPL endocytosis motif per se does not confer AME resistance. Intriguingly, truncation of the gp41 cytoplasmic tail also relieved the effect of AME on viral infectivity in the context of both HIV-1 and SIVmac. This suggests that the effects of P203L and S205L mutations on Env function with respect to AME resistance are similar to those induced by gp41 truncation. We also observed that HIV-1 virions pseudotyped with amphotropic MLV Env or VSV-G displayed AME-resistant infectivity.

Several studies have revealed an "inside-out" regulation of lentiviral Env function by the gp41 cytoplasmic domain. Cytoplasmic domain mutations have also been shown to contribute to CD4 independence and to affect the sensitivity of the Env complex to neutralizing antibody (54, 58–63), presumably via effects of the cytoplasmic tail mutations on gp120 conformation. In addition, we (64) and others (65) have demonstrated an effect of virion maturation on fusion activity, suggesting that interactions between the gp41 cytoplasmic tail and unprocessed Gag can restrict fusion. In light of these previous studies, several models could be proposed to explain the resistance to AME induced by the P203L and S205L mutations and by gp41 cytoplasmic domain truncations. AME binding to the virion lipid bilayer could prevent the Env glycoprotein complex from undergoing conformational changes necessary for fusion. According to this model, the P203L and S205L mutations or gp41 cytoplasmic tail truncations would render virions resistant to AME either because these mutations allow the requisite conformational changes to occur even in the presence of AME or because the Env mutants are less dependent on the conformational changes that AME disrupts to achieve a fusogenic state. Alternatively, although AME is a cholesterol-binding compound known to interact with lipid bilayers, AME-mediated inhibition could be due to direct binding of the compound to gp120 or to the ectodomain of gp41. In this case, conformational changes induced by gp41 cytoplasmic domain mutations could prevent AME binding via the inside-out conformational regulation discussed above. Our initial studies indicated that the sensitivity of the P203L and S205L mutants to neutralizing antibodies, soluble CD4, and the fusion inhibitor T20 (66–68) was indistinguishable from that of WT Env.4 We are continuing to probe the conformation of WT and AME-resistant Env with the goal of determining whether AME affects gp120 or gp41 conformation and whether the resistant mutants are differentially affected with respect to such AME-induced conformational changes.


Figure 9
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  		FIGURE 9.
Virions bearing truncated HIV-1 Env, VSV-G, or amphotropic MLV Env are AME-resistant. A, truncation of the gp41 cytoplasmic domain relieves inhibition by AME. TZM-bl cells were infected with virions bearing WT Env or gp41 truncation mutants (CTdel-144 and CTdel-104) in the absence or presence of 10 µM AME as described in the legend to Fig. 2A. Cells were lysed, and luciferase activity was measured and normalized to untreated controls. HIV-1 CTdel-144 and CTdel-104 lack 144 and 104 residues, respectively, from the C terminus of gp41. Data are presented as the means ± S.D. from four independent experiments. B, virions bearing VSV-G or amphotropic MLV Env are AME-resistant. HIV-1 virions were pseudotyped with amphotropic (Ampho) MLV Env or VSV-G by cotransfecting the Env-defective pNL4-3 derivative (pNL4-3/KFS) with amphotropic MLV Env or VSV-G expression vectors. Viral infectivity was measured in TZM-bl cells in the presence or absence of 10 µM AME as described in the legend to Fig. 2A. Data are presented as the means ± S.D. from three to four independent experiments.

 
Antiviral activity has been observed with another amphotericin B derivative, MS8209 (an N-methylglucamine salt of 1-deoxy-1-amino-4,6-O-benzylidene-D-fructosyl amphotericin B) (69, 70). Although it has not been established that AME and MS8209 possess similar mechanisms of action, naturally occurring resistance to this compound maps to the V3 loop of gp120, a domain of the glycoprotein known to participate in post-CD4-binding interactions with coreceptor (for review, see Ref. 71). If AME and MS8209 have similar mechanisms of action, it is possible that the ability of mutations in the gp41 cytoplasmic tail to confer resistance to AME could be due to effects of the gp41 changes on the activity of the gp120 V3 loop. It is significant that AME did not disrupt cell-cell fusion (syncytium formation) between Env-expressing and receptor/coreceptor-bearing cells, again arguing that the virion is the major target for the antiviral activity of AME. Although our data indicate that AME potently inhibits viral entry (i.e. fusion between the lipid bilayer of the virion and the target cell plasma membrane) (Fig. 2C), we cannot exclude the possibility that a post-entry block could also contribute to the 50–100-fold infectivity defect observed at high concentrations of AME.

Although the anti-HIV-1 activity of amphotericin B derivatives has been described previously, this study has provided significant and novel data that offer new insights into the antiviral properties of this class of cholesterol-binding compounds. 1) We have demonstrated that AME inhibits the replication of a broad panel of HIV-1 isolates, a finding that is highly relevant to any contemplated use of AME in a therapeutic setting. 2) Using a recently described viral entry assay (44), we defined viral entry as the step in the infection pathway that is blocked by AME. 3) We have shown that inhibition by AME extends to both HIV-1 and SIVmac, but that HIV-1 virions pseudotyped with VSV-G or MLV Env are resistant. 4) Unlike a previous study that mapped resistance to an amphotericin B derivative to gp120 (70), in this study, we have shown that mutations in the cytoplasmic domain of gp41 confer resistance. Furthermore, we found that truncation of the gp41 cytoplasmic domain also relieves the AME-imposed infectivity block and that the ability of gp41 truncation to confer AME resistance extends to SIVmac. 5) In addition to a block in viral entry, we observed that treatment of virus-producing cells with AME significantly inhibits viral particle production. 6) We have demonstrated that AME does not block viral entry by reducing the levels of gp120 on virions or by depleting virion cholesterol and that resistance to AME does not arise through altered gp120 or cholesterol content of viral particles. Finally, 7) we found that the infectivity of AME-resistant mutants remains sensitive to cyclodextrin treatment, indicating that the mechanisms by which AME and cyclodextrins inhibit viral infectivity are distinct.


Figure 10
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  		FIGURE 10.
Truncation of the SIVmac239 cytoplasmic tail confers resistance to AME. A, single-cycle infectivity assay of SIVmac239 containing full-length gp41 (full Env) or truncated gp41 lacking 145 C-terminal amino acids (truncated Env). TZM-bl cells were infected in the absence or presence of 10 µM AME as described in the legend to Fig. 2A, and luciferase activity was measured and normalized to untreated controls. Data from four independent experiments are shown as the means ± S.D. B, AME blocks the replication of WT SIVmac239. CEMx174 cells were infected with viral stocks derived from the SIVmac239 molecular clone. Cells were cultured in the presence or absence of AME, and replication was monitored as described in the legend to Fig. 1. RT, reverse transcriptase. C, replication of an SIVmac239 clone encoding a truncated gp41 cytoplasmic tail (pB239Q734*) is resistant to AME. Data are representative of at least two independent experiments. The symbol key in C also applies to B.

 
It remains to be determined whether AME or related compounds could be used effectively as short-term antiretroviral drugs in a clinical setting. The parent compound (amphotericin B) has been used for decades to treat fungal infections in vivo (72). Its antifungal activity has been attributed to its ability to bind sterols in cell membranes and to induce the formation of sterol/amphotericin B aggregates (28). The elevated levels of cholesterol in the HIV-1 lipid bilayer compared with those in the cell plasma membrane (16, 17) could make the virion particularly sensitive to binding by amphotericin B and its derivatives. Indeed, previous findings have demonstrated a connection between sterol levels in membranes and sensitivity to amphotericin B (73, 74). In addition to possible short-term systemic use, we believe that AME could be effective as a topical microbicide to prevent HIV-1 transmission. Although resistance to AME can be selected for in culture, the high degree of sequence conservation observed at gp41 Pro203 and Ser205 (/www.hiv.lanl.gov/content/index) and the ability of AME to block the replication of highly diverse isolates of HIV-1 (Table 1) suggest that there may be a fitness cost in vivo associated with mutations that confer AME resistance. We feel that the results presented in this study should encourage efforts to consider AME and other cholesterol-binding compounds for use in antiretroviral therapy.


   FOOTNOTES
 
* This work was supported by the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, NIH, Bldg. 535, Rm. 108, Frederick, MD 21702-1201. Tel.: 301-846-6223; Fax: 301-846-6777; E-mail: efreed{at}mail.nih.gov.

2 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; MLV, murine leukemia virus; AME, amphotericin B methyl ester; VSV-G, vesicular stomatitis virus glycoprotein; SIVmac, rhesus macaque simian immunodeficiency virus; PBLs, peripheral blood lymphocytes; MDMs, monocyte-derived macrophages; WT, wild-type. Back

3 Sequences are available upon request. Back

4 A. A. Waheed and E. O. Freed, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank V. Pathak, V. KewalRamani, A. Ono, and members of the Freed laboratory for helpful discussions and critical review of the manuscript; M. Thali, B. Crise, Y. Li, D. Littman, J. Burns, and W. Greene for providing plasmids; and T. Merigan and S. Palmer for providing HIV-1 isolates MDR 807, MDR 1385, and MDR 3761.



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