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Originally published In Press as doi:10.1074/jbc.M403394200 on September 14, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49055-49063, November 19, 2004
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Polyarginine Inhibits gp160 Processing by Furin and Suppresses Productive Human Immunodeficiency Virus Type 1 Infection*

Karen V. Kibler{ddagger}§, Akiko Miyazato{ddagger}§, Venkat S. R. K. Yedavalli{ddagger}, Andrew I. Dayton¶, Bertram L. Jacobs||, George Dapolito{ddagger}, Seong-jin Kim**, and Kuan-Teh Jeang{ddagger}{ddagger}{ddagger}

From the {ddagger}Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460, the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892-0460, the ||Biodesign Institute, Center for Infectious Disease and Vaccinology, Arizona State University, Tempe, Arizona 85287, and the **Laboratory of Cell Regulation and Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892-0460

Received for publication, March 26, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency virus-1 envelope protein, gp160, into gp120 and gp41 has been attributed to the activity of the cellular subtilisin-like proprotein convertase furin. The prototypic furin recognition cleavage site is Arg-X-Arg/Lys-Arg. Arg-Arg-Arg-Arg-Arg-Arg or longer iterations of polyarginine have been shown to be competitive inhibitors of substrate cleavage by furin. Here, we tested polyarginine for inhibition of productive human immunodeficiency virus-1-infection in T-cell lines, primary peripheral blood mononuclear cells, and macrophages. We found that polyarginine inhibited significantly human immunodeficiency virus-1 replication at concentrations that were benign to cell cultures ex vivo and mice in vivo. Using a fluorogenic assay, we demonstrated that polyarginine potently inhibited substrate-specific proteolytic cleavage by furin. Moreover, we verified that authentic processing of human immunodeficiency virus-1 gp160 synthesized in human cells from an infectious human immunodeficiency virus-1 (HIV-1) molecular clone was effectively blocked by polyarginine. Taken together, our data support that inhibitors of proteolytic processing of gp160 may be useful for combating human immunodeficiency virus-1 and that polyarginine represents a lead example of such inhibitors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus (HIV)1/AIDS is a health problem of immense magnitude. Over 40 million individuals globally are infected with HIV. In 2002 alone, more than 3 million people died from AIDS. Arguably, the "best" current treatment regimen is highly active antiretroviral therapy (HAART), which uses combinations of inhibitors targeting HIV-1 protease and reverse transcriptase. Although HAART has achieved widespread acceptance, its efficacy is limited by severe drug side effects, poor patient compliance, and the emergence of drug-resistant virus. New chemotherapeutics aimed at additional HIV-1 targets may ameliorate some of the problems associated with HAART. Such new approaches need to be urgently considered.

The envelope (Env) glycoprotein of HIV-1 is essential for receptor binding and membrane fusion during infection. Env is synthesized as a precursor polypeptide (gp160) that oligomerizes to form a trimer (1, 2), which is transported through the trans-Golgi network. In the trans-Golgi network, Env is cleaved by the cellular protease furin into surface (gp120) and transmembrane (gp41) subunits. Cleavage of gp160 occurs at a conserved Arg-Glu-Lys-Arg sequence (3, 4). Mutagenesis of the Arg-Glu-Lys-Arg sequence produces noninfectious HIV-1 particles containing unprocessed gp160 (4). This finding establishes the importance of furin-mediated processing for virus infectivity. Accordingly, there is emerging interest in identifying and developing anti-HIV-1 compounds that inhibit gp160 processing (5, 6).

Furin is a member of the mammalian subtilisin-related proprotein convertases (PCs). This family of proteases is involved in cellular metabolism and in the processing of bacterial toxins as well as viral coat proteins. Two previously reported furin inhibitors are the family of chloromethylketone- and decanolyderivatized proteins (6, 7) and {alpha}1-PDX (anti-trypsin Portland), an engineered variant of the cellular serine protease inhibitor, {alpha}1-antitrypsin (8). These inhibitors can block gp160 processing. However, complications with toxicity of chloromethylketones have been reported (9–11), and derivatized proteins can be complex to synthesize and difficult to produce in large quantities. Additionally, {alpha}1-PDX has limited effectiveness in inhibiting PC7, the other major proprotein convertase expressed in lymphocytes (12, 13), and is unable to durably inhibit HIV replication (14). Besides those inhibitors, peptides that mimic the Arg-Glu-Lys-Arg sequence have been reported to block furin activity in vitro and in vivo (8, 9, 15, 16). Specifically, poly-L-arginine (polyR) has been published as a furin inhibitor (9).

Based on the requirement for furin cleavage of gp160 in HIV infectivity, we reasoned that polyR might be a useful anti-HIV-1 compound. Here, we report that productive HIV-1 replication in T-cell lines (MT4 and Jurkat), peripheral blood mononuclear cells (PBMCs), and macrophages is abrogated effectively by polyR treatment. We show that the mechanism of polyR inhibition is through its blocking of gp160 cleavage by furin. Further, concentrations of polyR effective against HIV-1 replication are non-toxic to cultured cells ex vivo or to mice in vivo, suggesting the utility of this compound as a novel candidate therapeutic for AIDS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—polyR (molecular weight 8,500–13,000) and poly-L-lysine (polyK; molecular weight 4,000–15,000) were purchased from Sigma. Azidothymidine (AZT) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health.

Plasmids—HIV-1 molecular clones pNL4-3 (17) and pAD8 (18) were used. The furin expression vector was kindly provided by Dr. Juan Bonifacino (National Institutes of Health) and expresses furin from the CMV immediate-early promoter. Porcine TGF-{beta} expression vector, pTGFb, was from Dr. Seong-jin Kim (NCI, National Institutes of Health).

Cell Culture and Infection—MT-4, Jurkat, and human PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 IU/ml)/streptomycin (100 µg/ml), and glutamine (4 mM). 5 x 105/ml MT-4 cells in a 5-ml volume of complete medium were preincubated for 15 min with polyR and AZT at the indicated concentrations and were infected with 102 or 103 RT counts/ml HIV-1 NL4-3 virus. Jurkat cells were infected using 1 x 106 RT counts/ml virus. 40% of the cell suspension was removed every 2–3 days and replaced with an equivalent volume of complete medium containing the same amount of polyR as at the start. Supernatants were stored at–20 °C until tested for RT activity. PBMCs were stimulated with phytohemagglutinin (2 µg/ml) and interleukin-2 (20 units/ml) in RPMI 1640 for 2 days and then maintained with interleukin-2 only. PBMCs (1 x 106 cells) were preincubated with polyR for 15 min and then infected with 1 x 105 RT counts/ml HIV-1 NL4-3. Samples were collected every 2 or 3 days for subsequent RT assays, and wells were replenished with an equivalent volume of complete medium with or without polyR and AZT. Human monocyte-derived macrophages (MDMs) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, gentamicin (1 mg/ml), and glutamine (4 mM). MDM (differentiated for 10 days in medium containing 1000 units/ml macrophage-colony stimulating factor) were infected with 2 x 104 RT counts/ml HIV-1 AD8 and incubated with or without polyR. Half of the medium was replenished every 3 days with complete medium with or without polyR.

Enzymatic Hydrolysis of Internally Quenched Fluorogenic Substrate—Purified human furin was purchased from New England BioLabs Inc. (Beverly, MA). PC7 cDNA was cloned into pET30c vector. Isopropyl-1-thio-{beta}-D-galactopyranoside induction and purification of His-tagged PC7 protein from Escherichia coli were performed using nickel beads according to standard protocols. Fluorometric assays were performed in 2 ml of assay buffer, containing 100 mM Hepes (pH 7.5), 0.5% Triton X-100, 1 mM CaCl2, 1 mM 2-mercaptoethanol, and 0.5 µM internally quenched fluorescent substrate Avz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH (Bachem, King of Prussia, PA) at room temperature. Furin (0.25 µM) was added (final enzyme concentration 0.5 nM) to the reaction, and the fluorescence of the cleaved substrate was measured with a fluorospectrometer (Photon Technology International, Lawrence, NJ) (320-nm excitation, 425-nm emission). Following furin addition, inhibitors were added at the indicated intervals. Relative inhibition was based on the change in the slope of fluorescence generated by cleavage of the fluorogenic substrate by furin. Purified PC7 was added to a 200-µl volume containing protease assay buffer (100 mM Hepes, pH 7.5, 0.5% Triton X-100, 1 mM CaCl2, and 1 mM 2-mercaptoethanol) and 50 µM Boc-RVRR-AMC (Bachem). Inhibition of proteolysis was measured by adding different concentrations of polyR. Reactions were stopped by adding EDTA to 10 mM. Liberated AMC was detected with Wallac 1420 Victor multilabel plate reader (380-excitation/460-emission) (PerkinElmer Life Sciences).

Western Blotting—Proteins (20 µg/lane) were transferred to polyvinylidene difluoride membranes using a semidry procedure (19), and bands were visualized using chemiluminescence (Tropix, Bedford, MA). Signals were detected using x-ray film and analyzed using the NIH Image 1.62 program. The antibodies used were as follows. Human HIV immunoglobulin (HIV-IgTM) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, rabbit anti-gp160 (R160) was kindly provided by R. L. Willey (20), and chicken anti-TGF-{beta} was from R&D Systems.

Toxicity Assays—5 x 105/ml MT-4 cells were incubated with and without polyR at concentrations of 0.5 or 1.0 µM or AZT at a concentration of 1.0 µM. 40% of the cell suspension medium was removed every 2 or 3 days and replaced with an equivalent volume of complete medium with the same amount of polyR or AZT as at the start. On each harvest, 100 µl of each cell suspension was measured for viability using the Counting Kit-8 (Dojindo, Gaithersburg, MD) according to manufacturer's instructions as described previously (21). Absorbance at 450 nm was measured in a microplate reader (Bio-Tek). The percentage of viability of cells incubated with compounds was determined by the formula (OD of treated cells)/(OD of untreated cells) x 100. MDMs were incubated with or without polyR, and cell viability was assayed 2 weeks later. For in vivo assays, 8–10-week-old BALB/c mice (Taconic; Germantown, NY) were divided into seven groups, and each group was administered intraperitoneally 10 µl/g of body weight of either polyR or polyK at concentrations of 35, 350, or 700 µM (to achieve final tissue concentrations of 0.5, 5, or 10 µM respectively) or normal saline. Mice were injected once/day, 5 days/week for 5 weeks and sacrificed for necropsy.

Fluorescent Labeling and Cellular Uptake—Rhodamine labeling of polyR and bovine serum albumin was performed by FluoReporter tetramethylrhodamine-5-maleimide protein labeling kit (Geneprobe) as described previously (22). Cells were incubated with 1 µM rhodamine-labeled polyR, 1 µM rhodamine-labeled bovine serum albumin, or 1 µM rhodamine alone. To verify that the uptake of polyR is saturable, non-labeled 1 µM polyR was added to the medium followed by the addition of the fluorescent-labeled polyR (1 µM). The intracellular distribution of rhodamine label was observed using a fluorescence microscope (Micro System).

Env Protein and in Vitro Enzymatic Digestion—Purified gp160 LAV (T-tropic) envelope protein was purchased from Protein Sciences Corp. (Meriden, CT). For furin digestions, 0.2 µg (~12 pmol) of gp160 was incubated at 30 °C for 16 h in assay buffer with 2 units of furin in the presence or absence of inhibitors at a final volume of 50 µl. The digests were then analyzed by Western blotting. Densitometry measurements were performed using Image Gauge V.3.45 (Fuji Photo Film Co. Ltd., Tokyo, Japan). Gp160 cleavage efficiency was calculated by the formula (density of gp120 band)/(combined gp120 + gp160 bands) after background density was subtracted. Expression of gp160 in transfected cells was performed using pCMVEnv + pCMVRev. Both are CMV immediate-early promoter driven plasmids containing the HIV-1 Env or Rev open reading frames, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
polyR Inhibits Productive HIV-1 Infection of T-cells and Macrophages—Based on the report that Arg-Arg-Arg-Arg-Arg-Arg (9) is a potent inhibitor of furin, we investigated whether polyR would inhibit HIV-1 replication. We treated the T-cell line, MT-4, with or without polyR for 15 min and then infected the cells with 103 or 102 RT counts/ml HIV-1 NL4-3, a T-cell tropic CXCR4-co-receptor utilizing virus (17). Cell culture supernatants were collected every 2 days, and RT activities were measured (20). polyR, at 0.5 µM, completely inhibited HIV-1 replication in MT-4 cells (Fig. 1A, left, and right, lanes 1–4), and this inhibitory profile was comparable with that achieved by AZT at 1.0 µM (Fig. 1A, left, and right, lanes 9–10). Next, we repeated the assay using Jurkat cells infected with 1 x 106 RT counts/ml HIV-1 NL4-3. Here, polyR (1.0 µM) also abrogated productive HIV-1 infection (Fig. 1B). Additionally, we asked whether polyR would be effective in primary T-cells. Thus, we treated human PBMCs with 1.0 µM polyR and then infected cells with 1 x 105 RT counts/ml HIV-1 NL4-3. When compared with mock-treated PBMCs, which replicated virus robustly, little to no HIV-1 production was seen in polyR-treated PBMCs (Fig. 1C). These results support a general property of polyR to inhibit HIV-1 replication in various T-cells.



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  		FIG. 1.
polyR inhibits productive HIV-1 infection of cells. A, 2.5 x 106 MT-4 cells were infected in duplicate with HIV-1 NL4-3 at 103 or 102 RT counts/ml and incubated with polyR at concentrations of either 0.5 or 1.0 µM (lanes 1–4 and 5–8, respectively, right panel). As controls, parallel infected cell cultures either were treated with 1.0 µM AZT (lanes 9–10, right panel) or were untreated (lanes 11–12, right panel). The left panel shows the graphic summary of the RT results from three independent experiments. The right panel shows a representative RT assay result. The darkness of the spots in the assay reflects the level of RT activity. In this assay, all infections were conducted in duplicates; thus, lane 2 is a duplicate infection of lane 1, etc. B, 2.5 x 106 Jurkat cells were infected with HIV-1 NL4-3 at 1 x 106 RT counts/ml and treated with polyR at either 0.5 (lane 1, right panel) or 1.0 µM (lane 2, right panel) or treated separately with 1.0 µM AZT (lane 3, right panel), or mock-treated (Mock) (lane 4, right panel). The left panel shows average values from three independent experiments. The right panel shows a representative assay. C,1 x 106 PBMCs were infected by 1 x 105 RT counts/ml HIV-1 NL4-3 and incubated with either 0.5 or 1.0 µM polyR or without polyR. The graph summarizes results from three independent experiments. D,1 x 106 macrophages were infected with 1 x 105 RT counts/ml HIV-1 AD8 and treated either with 2.0 µM polyR or without polyR. Data represent results from three independent experiments.

 
We wondered next whether polyR would also inhibit HIV-1 replication in macrophages. Macrophage-tropic HIV-1 uses the CCR5 co-receptor and is the predominant viral phenotype found in newly infected individuals (23–25). To query for effectiveness, we tested polyR against HIV-1 AD8 (a CCR5-using virus)-infected macrophages (MDMs). When compared with mock-treated macrophages, HIV-1 replication in polyR-treated macrophages was also significantly suppressed (Fig. 1D).

Lack of polyR Toxicity in Cell Culture and in Mice—The virus-inhibitory effects of polyR are meaningful only if they are not trivial manifestations of cytotoxicity. To address this issue, we employed a colorimetric assay (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)) to monitor the cellular viability of polyR-treated cells. We incubated MT-4 cells with polyR at either 0.5 or 1.0 µM or with 1.0 µM AZT (Fig. 2A) as control. When compared with AZT-treated cells, 0.5 or 1.0 µM polyR-treated cells were comparable in viability over the 12-day assay period. We also checked for toxicity in macrophages. At a polyR concentration of 2.0 µM, which effectively suppressed HIV-1 replication, no toxicity was observed in macrophages over a 2-week incubation period (Fig. 2B).



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  		FIG. 2.
Ex vivo polyR toxicity assay. 5 x 105 MT-4 cells in a 1-ml volume were incubated in culture medium containing 0.5 or 1.0 µM polyR or 1.0 µM AZT. A, 100 µl of each cell suspension was incubated with 10 µl of WST-8 (a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, and colorimetric measurements were done. B, MDMs were incubated with 0.5, 1.0, or 5.0 µM polyR. Half of the medium was removed every 3 days and replaced with fresh medium containing the same amount of compounds as at the start. Colorimetric measurements were done on day 14. The percentage of viability of the cells incubated with compounds was determined by the formula (OD of treated cells)/(average OD of untreated cells) x 100. Each experiment was performed six times; the error bars represent the S.E. Mock, mock-treated.

 
We next asked whether polyR has in vivo toxicity in mice. We injected mice (in groups of five) intraperitoneally with daily drug boluses calculated to achieve final body polyR (or polyK) distributions of 0.5, 5, and 10 µM. The injected animals were monitored for 5 weeks. polyR-dosed mice were compared with counterparts administered with either normal saline or polyK. As shown in Fig. 3, groups of mice injected with the highest concentrations of polyR or polyK such as to achieve final body distributions of 10 µM succumbed quickly. Necropsies of these mice, however, provided no evidence for systemic organ or tissue toxicity (data not shown). Interestingly, these mice did reveal localized chemical peritonitis, suggesting that deaths occurred not from generalized toxicity but from local tissue shock induced by bolus administration of highly concentrated poly-basic solutions (i.e. polyR or polyK). On the other hand, virtually all mice injected with a lower concentration of polyR or polyK (i.e. calculated to achieve final body distributions <5.0 µM) thrived. At the conclusion of 5 weeks (or at the time of unexpected death), all mice were sacrificed, and blood and major organ systems were examined. No abnormalities were found in any of the blood cell counts, liver, and renal functions or in the histological examinations of tissue sections from all major organs (data not shown). Thus, excluding localized chemical peritonitis induced by highly concentrated bolus injections, polyR and polyK administered daily over 5 weeks are not significantly toxic in mice.



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  		FIG. 3.
In vivo toxicity assay of polyR in mice. BALB/c mice were used at 8–10 weeks of age. Mice were divided into seven groups, and each group (n = 5) was injected intraperitoneally with a 10-µl volume/g of body weight of polyR or polyK in saline at concentrations of 35, 350, or 700 µM (to achieve final tissue distributions of 0.5, 5, or 10 µM, respectively) or injected with normal saline only. Mice were injected once/day, 5 days/week and followed for 5 weeks.

 
polyR Inhibits Furin Activity—Having documented above that polyR inhibited HIV-1 replication in various cells, we next sought to understand its mechanism of action. Based on findings in the literature, one reasonable hypothesis is that polyR interferes with the furin-mediated processing of HIV-1 gp160. To test this hypothesis, we asked whether polyR can directly inhibit furin activity in vitro.

Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH is a substrate that is efficiently cleaved by furin with a Kcat/Km value more than 2000-fold higher than that of the commonly used Boc-Arg-Val-Arg-Arg-AMC substrate (10). We examined whether polyR could inhibit furin cleavage of fluorogenic Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH. When commercially purified furin (final enzyme concentration 0.25 µM) was added to reaction buffer containing fluorogenic substrate, an increase in fluorescence, proportional to the amount of substrate cleavage by enzyme, was observed (Fig. 4A). We also compared the control addition of buffer vehicle (Fig. 4B) or the addition of polyR into the reaction to final concentrations of 0.01 or 0.05 µM (Fig. 4, C and D). In the latter instances, polyR caused a dose-dependent halt in fluorescence, consistent with inhibition of furin-mediated cleavage of substrate.



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  		FIG. 4.
Inhibition by polyR on furin processing of fluorogenic substrate. A, furin was added into buffer containing 100 mM Hepes (pH 7.5), 0.5% Triton X-100, 1 mM CaCl2, 1 mM 2-mercaptoethanol, and 0.5 µM fluorogenic substrate (Abz-RVKRGLAY(NO2)D-OH) at room temperature. B, vehicle solvent was added to the reaction at the indicated time. C and D, inhibitory effect on furin cleavage was examined by adding polyR at concentrations of 0.01 (C) or 0.05 (D) µM as indicated. The graphs represent fluorescence emitted as a consequence of substrate cleavage by furin.

 
polyR Is Taken up Efficiently into Cells—HIV-1 gp160 is synthesized and cleaved intracellularly (3, 4). To check that the observed polyR-effect on HIV-1 might be manifested through suppression of gp160 processing by furin, we wished to verify that polyR is efficiently taken up by cells. From as early as 1965, cationic macromolecules have been reported to enter cells (22, 26–29). Although the mechanism of uptake has not been conclusively defined, recent reports suggest that internalization of domains containing a high content of arginine or lysine is accomplished through endocytosis and subsequent vesicular egress (30, 31). We anticipated that polyR might behave similarly. To verify this, we conjugated polyR with tetramethylrhodamine creating a fluorescent moiety (rhodamine-polyR). Using fluorescent microscopy, we then monitored the uptake over time of rhodamine-polyR into live HeLa cells. Indeed, fluorescent polyR was efficiently internalized into cells within 3 h (Fig. 5C). By comparison, rhodamine-labeled bovine serum albumin or rhodamine dye alone failed to enter cells (Fig. 5, A and B). When we competed rhodamine-labeled polyR (1 µM) with unlabeled-polyR (1 µM), intracellular fluorescence was markedly diminished (Fig. 5D), suggesting that the cellular uptake of polyR was saturable.



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  		FIG. 5.
Uptake of rhodamine-labeled polyR into HeLa cells. HeLa cells were incubated for 3 h at 37 °C with rhodamine-labeled bovine serum albumin (BSA) (A), tetramethyl-rhodamine alone (B), rhodamine-labeled 1 µM polyR (C), or 1 µM non-labeled polyR followed by the addition of the rhodamine-labeled polyR (1 µM) (D). After incubation, cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and then visualized by fluorescent microscopy.

 
polyR Inhibits HIV-1 gp160 Processing by Furin—Above, we showed that polyR suppressed HIV-1 replication in T-cells and macrophages (Fig. 1) and that polyR inhibited furin cleavage of a fluorescent peptide substrate (Fig. 4). The two observations suggest, but do not prove, that suppression of HIV-1 replication by polyR may be mediated through its interference of gp160 processing by furin. To link more directly the two findings, we wished to determine whether polyR directly interferes with gp160 processing. We therefore asked whether polyR can inhibit in vitro cleavage of purified gp160 by furin. When we incubated gp160 with furin at 30 °C for 16 h, ~40% of the starting material was cleaved to gp120 and gp41 (Fig. 6, lane 2). When the same furin-assay was repeated but with the addition of 0.5 µM polyR to the reaction (lane 3), we observed that all input gp160 remained uncleaved. As a control for specificity, the addition of 1 µM AZT to a furin reaction did not impair gp160 cleavage (Fig. 6, lane 4).



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  		FIG. 6.
polyR inhibited furin cleavage of gp160 in vitro. Purified gp160 was incubated with 2 units of furin for 16 h at 30 °C with (lane 3) or without polyR (lane 2) or with 1 µM AZT (lane 4) in a total reaction volume of 50 µl. Lane 1 contains unreacted gp160 electrophoresed as a marker. The status of gp160 processing was analyzed by Western blotting after SDS-PAGE. The blot was probed with pooled anti-HIV patient sera. The upper numbers in each lane indicate the intensity of the gp41 bands, and the lower numbers indicate the value relative to the intensity of the gp41 band cleaved by furin in lane 2. The right panel shows graphic quantification of cleavage calculated by the formula (density of gp120 band)/(combined densities of gp120 + gp160 bands) after background intensities were subtracted in each case.

 
The inhibition of gp160 cleavage by furin in vitro prompted us to investigate next whether polyR suppresses intracellular gp160 processing. We transfected HeLa cells with the HIV-1 molecular clone, pNL4-3, and analyzed gp160 processing in the presence or absence of polyR treatment (Fig. 7). In pNL4-3-transfected cells, gp160 monitored over 48 (lane 1) and 72 h (lane 4) was processed into gp120 and gp41. By contrast, gp160 processing was distinctly reduced in cells treated with either 0.5 (lanes 2 and 5) or 1 (lanes 3 and 6) µM polyR. By 72 h after transfection, the total gp160/gp120/gp41 protein had a slight diminution in the presence of inhibitor when compared with untreated cells, whereas p55 Gag levels remained stable. This is consistent with reports that a portion of uncleaved gp160 is degraded within the cell over time (6, 32–34). When internally normalized against the levels of p55 Gag, gp160 processing in cells incubated with polyR at 0.5 and 1.0 µM was approximately one-half and one-third, respectively, of that in untreated cells.



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  		FIG. 7.
Inhibitory effect of polyR on intracellular processing of gp160. HeLa cells were transfected with pNL4-3 and incubated in the absence (lanes 1 and 4) or presence of polyR at concentrations of 0.5 (lanes 2 and 5) or 1.0 (lanes 3 and 6) µM for 48 (lanes 1–3) or 72 (lanes 4–6) h. Cells were lysed and analyzed by Western blotting with a rabbit anti-Env gp160 antiserum or pooled anti-HIV patient sera. The top numbers in each lane in the upper panel indicate the efficiency of gp160 cleavage calculated by the formula (density of gp120 band)/(combined densities of gp120 + gp160 bands) after background intensities were subtracted in each case. The bottom numbers above the gp41 bands in the presence of polyR at 48 or 72 h indicate values relative to that in the absence of polyR, which was arbitrarily set at 1. The numbers in the Pr55Gag panel show values relative to Gag protein in the absence of polyR at 48 or 72 h respectively.

 
polyR Also Inhibits the Enzymatic Activity of Other Proprotein Convertases—Besides furin, PC7 is another major proprotein convertase present in activated lymphocytes. We wondered whether our polyR conditions used for furin would also be effective against PC7. Since purified PC7 is not commercially available, we expressed the cDNA for this convertase in E. coli and purified overexpressed protein to homogeneity as assessed by Coomassie-blue staining (Fig. 8A, lane 3). When we used purified PC7 in an in vitro assay, cleavage of fluorogenic substrate was indeed potently inhibited by polyR (Fig. 8B).



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  		FIG. 8.
Inhibition of non-furin proprotein convertases by polyR. A, a Coomassie Blue-stained SDS-gel profile of overexpressed PC7 purified from E. coli. (lane 3). B, polyR inhibited the in vitro PC7 cleavage of a fluorogenic peptide substrate. The graph shows the inhibition of cleavage of the fluorogenic substrate Boc-RVRR-AMC by PC7 in the presence of polyR. The inhibition of activity was measured by the decrease in released fluorogenic AMC using a fluorometer (see "Experimental Procedures"). C, the inhibition of gp160 processing in furin-proficient (CHOPar6 and CHOFD-Fur) and furin-deficient (CHO FD11) cells. Cells were transfected with pCMVEnv + pCMVRev and treated with or without 1 µg/ml polyR for 24 h. Cell lysates were immunoblotted using a polyclonal rabbit antiserum specific for HIV-1 Env.

 
To further verify the activity of polyR against non-furin proprotein convertases, we next checked the intracellular processing of HIV-1 gp160 in a Chinese hamster ovary cell line genetically selected to not express furin, CHO FD11 (Fig. 8C). Previous studies have shown that gp160 processing can occur in CHO FD11 cells via non-furin convertases (35). Using CHO FD11 and a furin-proficient CHOPar6 parental cell line, and another cell line selected from CHO FD11 to re-express furin (CHO FD-Fur), we compared the effect of polyR on the processing of gp160 expressed from exogenously transfected pCMVEnv plasmid. As shown in Fig. 8C, the processing of gp160 to gp120 and gp41 by furin (i.e. CHOPar6 and CHOFD-Fur; Fig. 8C, lanes 3 and 5) or non-furin convertases (i.e. CHO FD11; Fig. 8C, lane 1) were equally inhibited by polyR incubation (Fig. 8C, compare lane 2 with lanes 4 and 6). These results are consistent with the inhibition of PC7 in vitro (Fig. 8B) by polyR and indicate that polyR can be similarly effective against HIV-1 gp160 processing by non-furin convertases.

polyR Can Inhibit TGF-{beta}1 Processing by Proprotein Convertase—TGF-{beta}1 is synthesized as an ~55-kDa pro-TGF-{beta}1 protein that is rapidly processed by furin to an ~44 kDa profragment and an ~12-kDa secreted form (36). To ask whether polyR can affect the processing of pro-TGF-{beta} protein, we transfected an expression plasmid for porcine pro-TGF-{beta}1 into CHOPar6 cells and treated transfected cells in parallel with or without polyR (Fig. 9). Minimal endogenous pro-TGF-{beta} was detected in untransfected CHOPar6 cells (Fig. 9, lane 1), and a higher, albeit small, amount of pro-TGF-{beta} was seen in transfected cells not treated with polyR (Fig. 9, lane 3). By contrast, a significantly enhanced amount of pro-TGF-{beta} was observed when plasmid-transfected cells were incubated with polyR (Fig. 9, lane 2). These findings support that polyR treatment can also suppress the processing of cellular substrates by proprotein convertases.



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  		FIG. 9.
polyR inhibited processing of pro-TGF-{beta}1. CHO cells were transfected or mock-transfected (Mock) with a porcine pro-TGFb expression vector. Cell lysates were probed by immunoblotting with a polyclonal chicken antiserum raised to TGF-{beta}1. After immunoblotting, the filter was stained with Coomassie Blue (right panel) to verify the equivalence of protein loading in each of the lanes. The asterisk indicates the migration position of the ~44 kDa pro-fragment of cleaved TGF-{beta}1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Current therapies for AIDS have improved the quality and longevity of life for many HIV-1-infected individuals. However, extant regimens are problematic with respect to toxicity, patient non-compliance, and viral resistance. Increasingly, findings indicate that, for long term use, the "triple HAART mixture" approach will become ineffective or intolerable in many people (37). Realistically, developing new classes of HIV inhibitors is urgently warranted. Because HIV-1 requires several host proteins for its normal life cycle, in searching for new inhibitors, it is reasonable that one also explores agents which target cellular factors needed by the virus. One added benefit from an inhibitor that targets a host protein is that HIV-1 is less likely to mutate in a way which can compensate for a loss of cellular function.

A step of the HIV life cycle intimately dependent on cellular function is the processing of the envelope precursor gp160 to gp120 and gp41. Failure of the proper cleavage of gp160 to its two smaller subunits results in a non-infectious virus (20). Based on the premise that polyR mimics the furin cleavage sequence conserved in gp160, we investigated whether polyR would inhibit productive HIV-1 infection. Our evidence indicates that polyR does abrogate effectively HIV-1 infection in T-cells and macrophages (Fig. 1) and that the mechanism for this is likely through its inhibition of the furin-mediated processing of gp160 (Fig. 6, 7, 8).

We observed polyR to efficiently inhibit furin cleavage of synthetic substrates as well as authentic viral gp160 (Figs. 4 and 6,7,8). Our data show that polyR is efficiently taken up into cells, where it likely competes with gp160 for furin cleavage (Fig. 5). In fact, polyR has been shown to be an efficient and safe absorption enhancer for nasal protein delivery, serving to facilitate the transport of proteins from the nasal mucosa to reach therapeutic levels in plasma (38). Such report agrees with our data showing polyR entry into cells and further suggests that polyR may also serve as a cell-penetrating protein carrier.

Elsewhere, it has been suggested that other proprotein convertases can redundantly substitute for furin in gp160 processing (10, 35). Because all proprotein convertases share the consensus (R/K)-X-R cleavage sequence (39–41) with furin and because polyR works as a substrate-competitive inhibitor (7), it is likely that polyR inhibits gp160 processing by all proprotein convertases in the same way. Indeed, in furin-deficient LoVo cells (Ref. 42 and data not shown) and CHO FD11 cells (Fig. 8), we observed that polyR treatment inhibited gp160 processing, suggesting its general efficacy on proprotein convertases. This suppressive effect is not limited to the processing of viral proteins by these proteases since cleavage of TGF-{beta}1 was also sensitive to polyR treatment (Fig. 9).

Inhibition of cellular enzymes is a commonly evoked principle in many medical therapies. For example, angiotensin-converting enzyme physiologically converts angiotensin I to angiotensin II. Binding of angiotensin II to its receptor results in vasoconstriction, which (in excess) manifests as hypertension. Angiotensin-converting enzyme inhibitors, first developed in 1967 (43), are widely used to treat not only hypertension but also congestive heart failure, myocardial infarction, endothelial dysfunction, and renal disease (44). Other therapeutics that target cellular factors include aromatase inhibitors to treat hormone-dependent breast cancer (43) and inhibitors of proteases required for secretory pathways in Alzheimer's disease (45, 46). Thus, at the correct dosage, it is possible to use drugs that inhibit cellular factors in ways that maximize benefits while minimizing side effects. Specific furin inhibition in and of itself has been shown to lack significant toxicity in mice (47). Based on our ex vivo tissue culture (Fig. 2) and our in vivo mouse (Fig. 3) results, we believe that there is a therapeutic level for polyR that can inhibit proprotein convertase activity to suppress HIV-1 replication without provoking significant toxicity for the host. The potential of polyR as an inexpensive (e.g. $135/100 mg; Sigma), easily available, and non-toxic HIV-1 inhibitor warrants further study of this agent as a candidate therapeutic for human use.


   FOOTNOTES
 
* 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

§ Both authors contributed equally to this study. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Bldg. 4, Rm. 306, 9000 Rockville Pike, NIAID, National Institutes of Health, Bethesda, Maryland, 20892-0460. Tel.: 301-496-6680; Fax: 301-480-3686; E-mail: kj7e{at}nih.gov.

1 The abbreviations used are: HIV, human immunodeficiency virus; HAART, highly active antiretroviral therapy; RT, reverse transcriptase; Env, envelope; PC, proprotein convertase; polyR, poly-L-arginine; polyK, poly-L-lysine; PBMC, peripheral blood mononuclear cells; AZT, azidothymidine; CMV, cytomegalovirus; MDM, monocyte-derived macrophages; Abz, 2-aminobenzoic acid; Boc, t-butoxycarbonyl; AMC, 7-amino-4-methylcoumarin; CHO, Chinese hamster ovary; TGF, transforming growth factor; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt. Back


   ACKNOWLEDGMENTS
 
We thank Ben Berkhout and Rogier Sanders for useful discussions and for sending us HIV-1 virus stocks, Stephen Leppla for cells, members of the Jeang laboratory for critical readings of manuscript, and Lan Lin and Anthony Elmo for help with the preparation of the manuscript.



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