Polyarginine Inhibits gp160 Processing by Furin and Suppresses Productive Human Immunodeficiency Virus Type 1 Infection*

Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency vi-rus-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

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 ␣1-PDX (anti-trypsin Portland), an engineered variant of the cellular serine protease inhibitor, ␣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, ␣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-Larginine (polyR) has been published as a furin inhibitor (9).
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
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-␤ expression vector, pT-GFb, 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 g/ml), and glutamine (4 mM). 5 ϫ 10 5 /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 10 2 or 10 3 RT counts/ml HIV-1 NL4-3 virus. Jurkat cells were infected using 1 ϫ 10 6 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 Ϫ20°C until tested for RT activity. PBMCs were stimulated with phytohemagglutinin (2 g/ml) and interleukin-2 (20 units/ml) in RPMI 1640 for 2 days and then maintained with interleukin-2 only. PBMCs (1 ϫ 10 6 cells) were preincubated with polyR for 15 min and then infected with 1 ϫ 10 5 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 ϫ 10 4 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 Bio-Labs Inc. (Beverly, MA). PC7 cDNA was cloned into pET30c vector. Isopropyl-1-thio-␤-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 CaCl 2 , 1 mM 2-mercaptoethanol, and 0.5 M 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 M) 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-l volume containing protease assay buffer (100 mM Hepes, pH 7.5, 0.5% Triton X-100, 1 mM CaCl 2 , and 1 mM 2-mercaptoethanol) and 50 M 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/460emission) (PerkinElmer Life Sciences).
Western Blotting-Proteins (20 g/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-Ig™) 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-␤ was from R&D Systems.
Toxicity Assays-5 ϫ 10 5 /ml MT-4 cells were incubated with and without polyR at concentrations of 0.5 or 1.0 M or AZT at a concentration of 1.0 M. 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 l 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) ϫ 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 l/g of body weight of either polyR or polyK at concentrations of 35, 350, or 700 M (to achieve final tissue concentrations of 0.5, 5, or 10 M 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 M rhodaminelabeled polyR, 1 M rhodamine-labeled bovine serum albumin, or 1 M rhodamine alone. To verify that the uptake of polyR is saturable, non-labeled 1 M polyR was added to the medium followed by the addition of the fluorescent-labeled polyR (1 M). 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 g (ϳ12 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 l. 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 ϩ gp160 bands) after background density was subtracted. Expression of gp160 in transfected cells was performed using pCMVEnv ϩ pCMVRev. Both are CMV immediate-early promoter driven plasmids containing the HIV-1 Env or Rev open reading frames, respectively.

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 10 3 or 10 2 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 M, 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 M (Fig. 1A, left, and right, lanes 9 -10). Next, we repeated the assay using Jurkat cells infected with 1 ϫ 10 6 RT counts/ml HIV-1 NL4-3. Here, polyR (1.0 M) 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 M polyR and then infected cells with 1 ϫ 10 5 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.
When compared with AZT-treated cells, 0.5 or 1.0 M polyRtreated 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 M, which effectively suppressed HIV-1 replication, no toxicity was observed in macrophages over a 2-week incubation period (Fig. 2B).
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 M. 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 M 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 Ͻ5.0 M) 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.
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 K cat /K m 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 M) 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 M (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.
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 tetramethylrho- damine 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 M) with unlabeled-polyR (1 M), intracellular fluorescence was markedly diminished (Fig. 5D), suggesting that the cellular uptake of polyR was saturable.
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, ϳ40% 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 M polyR to the reaction (lane 3), we observed that all input gp160 remained uncleaved. As a control for specificity, the addition of 1 M AZT to a furin reaction did not impair gp160 cleavage (Fig. 6, lane 4).
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 pNL4-3transfected 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) M 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)(33)(34). When internally 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) M 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 ϩ 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. normalized against the levels of p55 Gag, gp160 processing in cells incubated with polyR at 0.5 and 1.0 M was approximately one-half and one-third, respectively, of that in untreated cells.
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).
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 pCM-VEnv 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-␤1 Processing by Proprotein Convertase-TGF-␤1 is synthesized as an ϳ55-kDa pro-TGF-␤1 protein that is rapidly processed by furin to an ϳ44 kDa profragment and an ϳ12-kDa secreted form (36). To ask whether polyR can affect the processing of pro-TGF-␤ protein, we trans- 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 ϩ pCMVRev and treated with or without 1 g/ml polyR for 24 h. Cell lysates were immunoblotted using a polyclonal rabbit antiserum specific for HIV-1 Env.
fected an expression plasmid for porcine pro-TGF-␤1 into CHO-Par6 cells and treated transfected cells in parallel with or without polyR (Fig. 9). Minimal endogenous pro-TGF-␤ was detected in untransfected CHOPar6 cells (Fig. 9, lane 1), and a higher, albeit small, amount of pro-TGF-␤ was seen in transfected cells not treated with polyR (Fig. 9, lane 3). By contrast, a significantly enhanced amount of pro-TGF-␤ 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.

DISCUSSION
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 -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-␤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.