HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 35, 30924-30934, September 2, 2005

This Article Abstract Full Text (PDF) All Versions of this Article: 280/35/30924    most recent M500195200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jinno-Oue, A. Articles by Hoshino, H. Search for Related Content PubMed PubMed Citation Articles by Jinno-Oue, A. Articles by Hoshino, H. Social Bookmarking                      What's this?

The Synthetic Peptide Derived from the NH₂-terminal Extracellular Region of an Orphan G Protein-coupled Receptor, GPR1, Preferentially Inhibits Infection of X4 HIV-1^*

Atsushi Jinno-Oue, Nobuaki Shimizu, Yasushi Soda, Atsushi Tanaka, Takahiro Ohtsuki, Dai Kurosaki, Yasuo Suzuki¶, and Hiroo Hoshino||

From the Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 371-8511 and the ^¶Department of Biochemistry, University of Shizuoka School of Pharmaceutical Science, 52-1, Yada, Shizuoka 422-8526, Japan

Received for publication, January 6, 2005 , and in revised form, May 19, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Several G protein-coupled receptors (GPCRs) serve as co-receptors for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. Here we report that a synthetic peptide derived from the NH₂-terminal extracellular region of an orphan GPCR, GPR1 (GPR1ntP-(1-27); MEDLEETLFEEFENYSYDLDYYSLESC), inhibited infection of not only an HIV-1 variant that uses GPR1 as a co-receptor, but also X4, R5, and R5X4 viruses. Among these HIV-1 strains tested, viruses that can utilize CXCR4 as their co-receptors were preferentially inhibited. Inhibition of early steps in X4 virus replication was also detected in the primary human peripheral blood lymphocytes. GPR1ntP-(1-27) directly interacted with recombinant X4 envelope glycoprotein (rgp120). This interaction was neither inhibited nor enhanced by the soluble CD4 (sCD4) but inhibited by the anti-third variable (V3) loop-specific monoclonal antibody and heparin known to bind to the V3 loop. Although the conformational changes in gp120, including the V3 loop, have been reported to be required for its interaction with a co-receptor after binding of gp120 to CD4, it has also been reported that the V3 loop is already exposed on the surface of virions before interaction with CD4. We found that GPR1ntP-(1-27) blocked binding of virus to the cells, and this peptide equally bound to rgp120 in the presence or absence of sCD4. Because we detected the binding of GPR1ntP-(1-27) to the highly purified virions even in the absence of sCD4, GPR1ntP-(1-27) probably recognized the V3 loop exposed on the virions, and this interaction was responsible for the anti-HIV-1 activity of GPR1ntP-(1-27).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
HIV-1¹ is the causative agent of AIDS (1, 2). CD4 glycoprotein as a receptor and GPCRs as co-receptors are required for the entry of HIV-1, and the cell tropism of HIV-1 is mainly determined by its ability to use co-receptors expressed on the target cells (3). The major co-receptor for R5 viruses is CCR5, of which ligands are CC chemokines RANTES (regulated on activation normal T-cell expressed and secreted), MIP1, and MIP1. On the other hand, the major co-receptor for X4 viruses is CXCR4, of which ligand is the CXC chemokine SDF-1. Dualtropic viruses (R5X4 viruses) can efficiently utilize both of these co-receptors (4-9).

In addition, primary human brain-derived fibroblast-like cells (BT-cells) are isolated from microvessel segments derived from autopsy of human brain tissue, are positive for CD4 and highly susceptible to HIV-1 variants, but are resistant to R5, X4, and R5X4 HIV-1 strains (10, 11). BT-cells are thought to be originated from smooth muscle cells or pericytes. We have previously shown that the substitution of Pro to Ser at the Gly-Pro-Gly-Arg sequence in the V3 loop of envelope glycoprotein (gp120) is responsible for the BT-cell tropism of the HIV-1 variants (10, 12). We have recently identified orphan GPCRs, GPR1 and RDC1, as co-receptors for the HIV-1 variants in vitro (13, 14). Not only HIV-1 variants, but also several strains of HIV-2 and simian immunodeficiency virus have been shown to utilize GPR1 and RDC1 as their co-receptors (13-15).

During the course of our previous experiments to generate anti-GPR1 antibody (-GPR1) by immunizing rabbits with a synthetic peptide derived from the NH₂-terminal extracellular region (NH₂-ECR) of GPR1 (GPR1ntP-(1-27)), we found unexpectedly that not only -GPR1 but also GPR1ntP-(1-27) was able to block GPR1-mediated infection of BT-cells with HIV-1 variants. Because it has been demonstrated that the NH₂-ECRs of the co-receptors play an important role in the interaction with gp120 and a subsequent infection steps (16-21), we speculated that GPR1ntP-(1-27) might inhibit interaction of gp120 (HIV-1 variants) with its co-receptor, GPR1, on target cell membrane.

In the present study, we tested anti-HIV-1 activity of GPR1ntP-(1-27) by several assays, including focal infectivity assay, HIV-1 p24 Gag detection assay, PCR assay, and syncytium formation assay. These assays all demonstrated an inhibitory activity of GPR1ntP-(1-27) against HIV-1 infection. Surprisingly, anti-HIV-1 activity of GPR1ntP-(1-27) was detected not only in HIV-1 variants that utilize GPR1 as a co-receptor, but also in genetically diverse isolates, including X4, R5, and R5X4 viruses. Our findings suggest that synthetic peptides derived from the NH₂-ECR of GPR1 are novel candidates for the development of GPCR-based and peptide-based agents to inhibit HIV-1 infection.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cells—NP-2 cells were derived from a human glioma and kindly provided by Dr. T. Kumanishi (Niigata University, Niigata, Japan). NP-2/CD4 and NP-2/CD4 cells stably expressing human GPCRs (NP-2/CD4/GPCRs, such as NP-2/CD4/CCR3, NP-2/CD4/CCR5, NP-2/CD4/CXCR4, and NP-2/CD4/GPR1) as indicator cells for HIV-1 infection were described previously (13-15, 22, 23). NP-2/CD4 and NP-2/CD4/GPCRs cells were maintained in Eagle's minimum essential medium containing 10% fetal calf serum (FCS). The human T-cell lines C8166 (24), MOLT-4 clone 8 (25), and MOLT4/IIIB (26) were maintained in RPMI 1640 medium supplemented with 10% FCS (RPMI/FCS). Peripheral blood lymphocytes (PBLs) were isolated from the blood of healthy subjects by Ficoll-Paque gradient centrifugation. PBLs were stimulated with phytohemagglutinin prior to HIV-1 infection and cultured in RPMI/FCS and 100 units/ml of recombinant interleukin-2 (Roche Applied Science). All cells were maintained at 37 °C in a humidified, 5% CO₂ atmosphere.

HIV-1 Strains—The GUN-1WT strain is a clinical isolate and can infect both T-cell lines and macrophages, but not BT-cells (10, 12, 27). In contrast, the variant of GUN-1WT strain can infect the BT-cells and T-cell lines (10, 12). Similar to the variant of GUN-1WT strain, GUN-1Ser strain, which had been prepared by the ligation and transfection of cloned HIV-1 (GUN-1WT) DNA fragments containing a mutation of proline to serine made by the site-directed mutagenesis at the GPGR sequence in the V3 loop, can infect BT-cells as well as T-cell lines (10, 12). The GUN-1WT, GUN-1Ser, and IIIB (28) strains of HIV-1 were produced by persistently infected MOLT-4 cells. The BaL (29) and SF162 (30) strains of HIV-1 were produced by infected PBLs. The primary HIV-1 strains, GUN-4 and GUN-14, were isolated from Japanese hemophilia patients as described previously (12). To prepare virus samples, culture supernatants containing HIV-1 were harvested, centrifuged at a low speed, and passed through 0.45-µm pore size cellulose acetate syringe filters (Gellman Science, Ann Arbor, MI). The virus samples were stored at -80 °C until use.

Synthetic Peptides—All synthetic peptides used in this study were purchased from Sawady Technology (Tokyo, Japan). The purity of the peptides was more than 95%. The amino acid sequences of the synthetic peptides used in this study are shown in Table I.

HIV-1 Infectivity Assay—To determine HIV-1 infectivity, a focal infectivity assay was performed as described previously (33-37). Namely, NP-2/CD4 cells expressing one of GPCRs were seeded at 2 x 10⁴ per well into 24-well tissue culture plates (Corning, Acton, MA), infected with 200 µl of the virus (1,000-2,500 focus-forming units per ml) in the presence or absence of one of the synthetic peptides. After 2 h at 37 °C, the cells were washed, fresh culture medium was added, and the cultures were incubated for 48 h at 37 °C. The cells were fixed with methanol for 10 min at room temperature, rinsed with PBS, and then incubated with anti-HIV-1 sera from AIDS patients diluted 1:300 with 3% bovine serum albumin (w/v) in PBS (blocking buffer A) for 2 h at room temperature. After being washed with PBS, cells were incubated with horseradish peroxidase-labeled antibody to human IgG (Dako, Glostrup, Denmark) diluted 1:200 with blocking buffer A for 1 h at room temperature, washed with PBS, and rinsed with Tris-buffered saline. To develop the color, cells were reacted in the dark with a solution of aminoethyl carbazol (Sigma) and H₂O₂ in acetate buffer (pH 5.0) for 20 min at room temperature, rinsed with water, air dried, and examined under a microscope. To determine the inhibitory activity of peptide to HIV-1 infection (inhibition (%)), the following formula was used: [(the number of foci with no peptide - the number of foci with peptide)/the number of foci with no peptide] x 100%.

To examine the effect of GPR1ntP-(1-27) on the infectivity of HIV-1 primary isolates, the level of p24^Gag protein in culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) as described elsewhere (38). Azido-3'-deoxythymidine was purchased from Sigma.

PCR Assay to Detect Reverse-transcribed HIV-1 DNA—The virus preparation (IIIB strain) was treated with RNase-free DNase I (10 units/ml, Roche Applied Science) for 30 min at 37 °C to remove contaminating viral DNA or cellular DNA. DNase I-treated IIIB virus was incubated with one of the synthetic peptides for 1 h at 37 °C, and inoculated onto MOLT-4 cells (5 x 10⁵) or PBLs (5 x 10⁵) for 2 h at 37 °C. The cells were washed, and fresh medium was added. After incubation for 20 h, the cells were washed and lysed with 10 mM Tris-HCl (pH 8.3) containing 1 mM EDTA, 0.45% Nonidet P-40 (Sigma), 0.45% Tween 20 (Sigma), and 0.2 mg/ml proteinase K (Sigma). The cell lysates were incubated for 2 h at 52 °C, heated for 10 min at 96 °C to inactivate proteinase K, and used as templates for the subsequent PCR analysis to detect the formation of reverse-transcribed HIV-1 DNA within the cells. PCR was performed with HIV-1 gag-specific primers, SK38 and SK39 (39) (nucleotide sequence: SK38, 5'-AAGGGGAAGTGACATAGCAG-3'; SK39, 3'-GGACCAACAAGGTTTCTGTC-5') in a PerkinElmer Life Sciences Cycler under the following conditions: 1 cycle at 95 °C for 9 min, 30 cycles at 94 °C for 1 min, 60 °C for 45 s, 72 °C for 1 min, and one cycle at 72 °C for 5 min. The human -globin gene primers, KM29 and KM38 (nucleotide sequence: KM29, 5'-GGTTGGCCAATCTACTCCCAGG-3'; KM38, 5'-TGGTCTCCTTAAACCTGTCTTG-3') (40) were used as an internal control. DNA amplification was performed in the following conditions: 1 cycle at 95 °C for 9 min, 30 cycles at 94 °C for 1 min, 60 °C for 45 s, 72 °C for 1 min, and one cycle at 72 °C for 5 min. The PCR products were visualized by electrophoresis through 2% agarose gels containing 0.5 µg/ml ethidium bromide.

HIV-1 Cell Binding Assay—HIV-1-cell binding assay was essentially done according to the protocol described by Valenzuela et al. (41). MOLT-4 cells (6 x 10⁶) were incubated with HIV-1 (IIIB strain) (200 ng of p24^Gag) for 1 h at 37 °C in RPMI/FCS (final volume, 0.5 ml). When indicated, viral preparation was incubated with GPR1ntP-(1-27), GPR1ntP-(Y/A), 0.5, or heparin, while the cells were incubated with anti-CD4 mAb, NU-TH/I, before binding. After incubation of the cells with HIV-1, the cells were washed once with PBS containing 5 mM EDTA and twice with RPMI/FCS. Then, the cells were lysed with 150 µl of lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl₂, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 0.5% Triton X-100, and 7 mM 2-mercaptoethanol), and the cell lysates were centrifuged at 1000 x g for 2 min at 4 °C. Then, the concentration of HIV-1 p24^Gag in the supernatants was determined by ELISA. The amount of p24^Gag in these cell extracts was expressed as the total amount of viral p24^Gag associated with the cells as described by Valenzuela et al. (41), because this value showed both viral p24^Gag bound to the surface of cells and viral p24^Gag that has entered the cells. To detect only HIV-1 that entered MOLT-4 cells, extracellular bound virus was removed. For this, cells were treated with 2.5 mg/ml of trypsin in PBS containing 5 mM EDTA for 40 s at room temperature after the cells were washed with PBS containing 5 mM EDTA. Then, the cells were washed twice with RPMI/FCS, lysed, and the concentration of intracellular p24^Gag was determined by ELISA. As compared with a T-cell line, CEM cells, which have been reported to be treated with trypsin for 5 min (41), MOLT-4 cells were treated with it for 40 s because they were highly sensitive to trypsin.

Syncytium Formation Assay—MOLT-4/IIIB cells and C8166 cells were suspended in RPMI medium at 1 x 10⁵ cells per ml and 5 x 10⁵ cells per ml, respectively. Before addition of 50 µl of C8166 cell suspension to each well of a U-bottom 96-well plate (Corning), MOLT-4/IIIB cells (50 µl per well) were incubated with 100 µl of RPMI medium containing one of the synthetic peptides or the medium alone for 1 h at 37 °C. After incubation at 37 °C for 17 h, the number of syncytia per well was counted by light microscope observation (42). The inhibitory activity of peptide to syncytium formation (inhibition (%)) was calculated using the following formula: [(the number of syncytia with no peptide - the number of syncytia with peptide)/the number of syncytia with no peptide] x 100%.

Association of GPR1ntP-(1-27) with HIV-1 Virions—To concentrate virus particles (IIIB strain), the culture supernatant (10 ml) of MOLT-4/IIIB cells was layered onto a 20% (w/v) sucrose cushion (2.5 ml) and centrifuged at 100,000 x g for 2 h at 4 °C using a Hitachi SRP28 rotor (Hitachi Koki Co., Tokyo, Japan). The viral pellet was resuspended in 100 µl of TNE buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA), layered onto the top of a continuous sucrose gradient (20-60% (w/v) sucrose in TNE) (43, 44), and centrifuged at 100,000 x g for 16 h at 4 °C. Fractions (700 µl) were collected from the top of the gradient, and the density of each fraction was determined. An aliquot from each fraction (50 µl) was mixed with 50 µl of TNE buffer containing 2% (v/v) Nonidet P-40 and 0.2% (w/v) bovine serum albumin, the mixture was incubated for 1 h at 56 °C, and the HIV-1 p24^Gag protein was detected by dot blotting as described below. The fractions that contained HIV-1 virions were pooled, concentrated again by centrifugation at 100,000 x g for 2 h at 4 °C using an RP55S rotor (Hitachi Koki Co.), suspended in TNE buffer, and used for detection of the association of synthetic peptides (GPR1ntP-(1-27) or X4ntP-(1-28)) with the HIV-1 virions. The viral suspension (100 µl) was incubated with one of peptides (5 µg) for 1 h at 37 °C, subjected to sucrose density gradient sedimentation, and then fractionated. The dot blotting was carried out to detect the presence of the HIV-1 p24^Gag protein and peptides in each fraction using anti-p24^Gag mAb and anti-peptide antibodies, respectively.

Dot Blotting—The HIV-1 lysates prepared after sucrose density gradient ultracentrifugation were blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using a Minifol-Microsample Filtration Manifold (Schleicher & Schuell). They were then blocked with 5% nonfat dry milk (w/v) in PBS containing 0.1% Tween 20 (v/v) (PBST) (blocking buffer B) for 2 h at 37 °C. The membranes were then probed with -p24^Gag mouse mAb diluted 1:1000 with blocking buffer B, or anti-peptide antibodies (-GPR1 or -CXCR4; 2 µg/ml in blocking buffer B) for 1 h at room temperature. After being washed three times with PBST, the membranes were incubated with a horseradish peroxidase-labeled antibody to mouse or rabbit IgG (DAKO) diluted 1:1000 with blocking buffer B for 1 h at room temperature, and then washed three times. Immunoreactive spots were detected by the enhanced chemiluminescence (ECL) system (Amersham Biosciences) according to the manufacturer's instruction.

Detection of Binding of rgp120 to GPR1 Peptides by ELISA—The recombinant envelope glycoprotein (rgp120) of HIV-1 (IIIB strain) produced in a baculovirus expression system, >90% pure, was obtained from the AIDS Reagent Project of the United Kingdom Medical Research Council (Potters Bar, United Kingdom). ELISA to detect peptide-rgp120 or sCD4-rgp120 interaction was based on the protocol as described (45), with a few modifications. Wells of a 96-well microtiter plate (Corning) were coated with GPR1ntP-(1-27) or its fragment or mutant peptides (0.015-5 µM in PBS) for overnight at 4 °C, washed twice with PBS, and blocked with blocking buffer A for 2 h at room temperature. After being washed with PBS, rgp120 (0.016 µM) in PBS containing 1% (v/v) Triton X-100 was added, and the mixture was incubated for 2 h at room temperature and washed with PBST. To detect bound rgp120, anti-HIV-1 sera from AIDS patients diluted 1:2000 with 3% bovine serum albumin (w/v) in PBST (blocking buffer C) was added, and the mixture was incubated for 2 h at room temperature and washed with PBST. Then horseradish peroxidase-labeled antibody to human IgG diluted 1:5000 with blocking buffer C was added, and the mixture was incubated for 1 h at room temperature, washed with PBST, and rinsed with PBS. To develop color, 3,3',5,5'-tetramethylbenzidine solution (Bio-Rad) as a substrate was added for 20 min at room temperature, and 1 N H₂SO₄ was added to stop the reaction. Then the optical densities (A) at 450 nm were determined.

For detection of interaction of sCD4 with GPR1ntP-(1-27), microtiter wells were coated with GPR1ntP-(1-27) as described above. Then, sCD4 (0.016 µM), which was obtained from the AIDS Reagent Project of the United Kingdom Medical Research Council, was added for 2 h at room temperature. Bound sCD4 was detected by ELISA as described above using antibody against sCD4 and horseradish peroxidase-labeled antibody to sheep IgG (Dako).

To examine the effect of -GPR1 antibody on binding of GPR1ntP-(1-27) to rgp120, microtiter wells were coated with GPR1ntP-(1-27) as described above. Then purified -GPR1 antibody or normal rabbit IgG (NRG) was added for 2 h at room temperature prior to addition of rgp120 (0.016 µM), and then bound rgp120 was detected as described above. For detection of binding of GPR1ntP-(1-27) to rgp120-sCD4, rgp120 (0.016 µM) was incubated with sCD4 (0.016 µM) for 1 h at 37 °C before incubation with GPR1ntP-(1-27) and then bound rgp120 was detected as described above. For detection of the binding of rgp120 to GPR1ntP-(1-27) or sCD4 in the presence of heparin (Wako), microtiter wells were coated with either GPR1ntP-(1-27) (1.0 µM) or sCD4 (0.016 µM) as described, and rgp120 (0.016 µM), which had been preincubated with or without heparin (37 °C for 1 h) was added. Bound rgp120 was detected as described above.

To examine the effect of GPR1ntP-(1-27) on binding of V3-specific neutralizing mouse mAb (0.5) to rgp120, a microtiter plate was coated with rgp120 (0.016 µM) at 4 °C for overnight, blocked, and treated with or without different concentrations of GPR1ntP-(1-27) or X4ntP-(1-28) (5-100 µM) at room temperature for 2 h. After being washed with PBS, mAb 0.5 (ascites, 1:500 dilution) was added, and the plate was incubated at room temperature for 2 h, and washed. Captured 0.5 was detected as described above using a horseradish peroxidase-labeled antibody to mouse IgG (Dako).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effects of Synthetic Peptides Derived from the NH₂-ECR of GPCRs on Cell-free HIV-1 Infection—We have previously established human glioma-derived, new HIV-1 infection indicator cell lines, which are transduced with both CD4 and co-receptors (NP-2/CD4/GPCRs), and showed that infectivity assay using these cell lines were highly reproducible and sensitive (13-15, 22, 23). As shown in Fig. 1, detection of HIV-1 antigen-positive cells by immunostaining after infection with several HIV-1 strains is dependent on both CD4 and the types of co-receptors expressed on NP-2 cells.

We, therefore, examined the effects of synthetic peptides derived from NH₂-ECR of GPCRs (GPR1, CXCR4, CCR3, and CCR5) on the infectivity of several HIV-1 strains by focal infectivity assays with NP-2/CD4/GPCRs as indicator cells (Fig. 2). Because our initial experiments indicated that the treatment of cells with the peptides at 37 °C for 1 h and their removal by washing before virus inoculation did not affect the infectivity by HIV-1, virus was incubated with one of the peptides (37 °C for 1 h), and this virus-peptide mixture was added to target cells. Among the peptides, only GPR1ntP-(1-27) markedly inhibited the infection of NP-2/CD4/GPR1 cells with GUN-1Ser strain, which utilizes GPR1 as a co-receptor, in a dose-dependent manner. Surprisingly, infections of the IIIB (X4), GUN-1WT (R5X4), and BaL (R5) strains were also inhibited by GPR1ntP-(1-27). As for IIIB, GUN-1Ser, and GUN-1WT strains, 50% inhibitory concentrations (IC₅₀) were 0.4-0.8 µM and 90% inhibitory concentrations (IC₉₀) were 3-12 µM (Table II), whereas GPR1ntP-(1-27) had weaker effects on infection of R5 virus (BaL strain) as compared with other HIV-1 strains; its IC₅₀ and IC₉₀ were 10 µM and 50 µM, respectively. No inhibitory activities of X4ntP-(1-28), R3ntP-(1-24), and R5ntP-(1-20) against any HIV-1 strains tested were detected. As for R5ntP-(1-20), our results are consistent with previous reports showing that no inhibitory effects on HIV-1 infection are detected in unmodified synthetic peptides derived from the NH₂-ECR of CCR5 (46, 47).

View larger version (115K): [in this window] [in a new window]   FIG. 1. Infection of NP-2/CD4/GPCRs cells with HIV-1. The CD4-transduced NP-2 cells (NP-2/CD4) and those stably expressing GPCRs (CXCR4, CCR5, and GPR1) were infected with HIV-1 (IIIB, GUN-1WT, and GUN-1Ser strains), fixed with methanol at 2 days later, and stained for HIV-1 antigen-positive cells. Stained cells were photographed by light microscope (x200).

Inhibition of HIV-1 Infectivity to a T-cell Line and Primary PBLs by GPR1ntP-(1-27) at Early Infection Step before Reverse Transcription—We next examined which step of virus infection was inhibited by GPR1ntP-(1-27). For this, a human T-cell line, MOLT-4, was infected with cell-free HIV-1 (IIIB strain), DNA was harvested 20 h after infection, and then the synthesis of HIV-1 DNA in target cells was detected by PCR using primers specific for the gag region. The relative intensities of amplified DNA bands were correlated with dilutions of the inoculated virus, indicating that PCR was performed within a linear range (Fig. 4, A and B). When HIV-1 was inoculated into the target cells in the presence of GPR1ntP-(1-27), the formation of HIV-1 DNA was inhibited in a dose-dependent manner (Fig. 4, C, lanes 1-5, and D). The inhibitory activity was not again detected when X4ntP-(1-28), R3ntP-(1-24), or R5ntP-(1-20) was used (Fig. 4, C, lanes 6-8 and D). When GPR1ntP-(1-27) was added to MOLT-4 cells only after inoculation of the virus, the formation of HIV-1 DNA was not inhibited (data not shown), suggesting that the reverse transcription of HIV-1 RNA in the host cells was not inhibited by GPR1ntP-(1-27).

We next examined whether GPR1ntP-(1-27) could inhibit HIV-1 infection to primary human PBLs, which are natural targets for HIV-1. The formation of reverse-transcribed HIV-1 DNA in PBLs, prepared from two independent donors, was also clearly inhibited by GPR1ntP-(1-27) in a dose-dependent manner (Fig. 4, E and F). Thus, these findings indicate that GPR1ntP-(1-27) blocks the early step of HIV-1 infection before reverse transcription, such as virus attachment or its entry into target cells.

Effect of GPR1ntP-(1-27) on Syncytium Formation—To examine the effect of GPR1ntP-(1-27) on the formation of multinucleated giant cells (syncytia), which are considered to represent cell-to-cell infection, we performed syncytia assays using HIV-1-positive MOLT-4/IIIB cells and HIV-1-negative C8166 cells in the presence of one of the synthetic peptides (Fig. 5). Syncytium formation was clearly inhibited by GPR1ntP-(1-27) but not by the other peptides. IC₅₀ of GPR1ntP-(1-27) determined by the syncytia assay was 1.1 µM. Because little effect of 50 µM GPR1ntP-(1-27) on the growth and viability of MOLT-4 cells was observed for up to 4 days (data not shown), the anti-HIV-1 activity of GPR1ntP-(1-27) appears not to be due to its cell toxicity.

Identification of a Functional Domain and Amino Acids in GPR1ntP-(1-27) Responsible for Its Anti-HIV-1 Activity—To determine a functional domain in the GPR1ntP-(1-27) peptide more precisely, fragment peptides, overlapped by six amino acids (GPR1ntP-(1-13), GPR1ntP-(8-20), and GPR1ntP-(15-27)) were synthesized (Table I). We examined the effect of these fragment peptides on the infection of NP-2/CD4/GPCR cells with HIV-1 (IIIB, GUN-1Ser, and BaL strains) (Table III). Among a series of the fragment peptides, GPR1ntP-(15-27) inhibited the infection of the IIIB and GUN-1Ser strains but not that of the BaL strain. Although relatively high concentrations of GPR1ntP-(15-27) were required for the inhibition of HIV-1 infection as compared with GPR1ntP-(1-27), the inhibitory effect of GPR1ntP-(15-27) against HIV-1 was repeatedly detected. This result indicates that the amino acid sequence from 15 to 27 (YSYDLDYYSLESC) is pivotal to inhibit HIV-1 infection by GPR1ntP-(1-27). Because it has been implicated that the tyrosine residues in the NH₂-ECR of CXCR4 and CCR5 play an important role in the co-receptor activity for HIV-1 infection (16-21), we examined a mutant peptide, in which all tyrosine residues had been substituted with alanine residues (GPR1ntP-(Y/A); Y15A, Y17A, Y22A, and Y23A), on HIV-1 infection. The inhibitory activity of GPR1ntP-(Y/A) peptide against HIV-1 infection was completely abrogated, indicating that tyrosine residues play a crucial role for GPR1ntP-(1-27) to inhibit HIV-1 infection.

Specific Interaction of GPR1ntP-(1-27) with rgp120 through Its V3 Loop—We next examined whether GPR1ntP-(1-27) binds directly to envelope glycoprotein gp120. Because GPR1ntP-(1-27) efficiently inhibited infection of X4 HIV-1 (IIIB strain), we performed a binding assay using a baculovirus-derived recombinant gp120 (rgp120) (IIIB strain) and GPR1ntP-(1-27). Microtiter plate wells were coated with different concentrations of GPR1ntP-(1-27) (0.015-5 µM) and incubated with rgp120 (0.016 µM), and bound rgp120 was detected by ELISA (Fig. 7A). The binding of rgp120 to GPR1ntP-(1-27) was detected in a concentration-dependent manner, indicating an association of rgp120 with GPR1ntP-(1-27). This binding appears to be specific, because pretreatment of GPR1ntP-(1-27) with -GPR1, which had been generated by immunization of rabbits with GPR1ntP-(1-27), but not with normal rabbit IgG inhibited binding of rgp120 in a manner dependent on IgG concentrations (Fig. 7B). In contrast to rgp120, no binding of sCD4 to GPR1ntP-(1-27) was detected (Fig. 7A).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Inhibition of cell-free HIV-1 infection by GPR1ntP-(1-27). NP-2/CD4 cells expressing one of the GPCRs were infected with HIV-1 in the presence or absence of synthetic peptides derived from the NH2-ECR of GPCRs, and HIV-1-antigen-positive cells were detected by focal infectivity assay as described under "Experimental Procedures." The target cell and inoculated HIV-1 strain are indicated at the top of graph and within the graph, respectively. The average number of foci in triplicate wells formed in the presence of peptides was counted, and the inhibition (%) was determined by the comparison with the average number of foci formed in the absence of peptides (500 foci/well). Four independent experiments gave similar results.

Because it has been reported that the soluble polyanion such as heparin binds to rgp120 at the V3 loop region (48-50), we next examined the effect of heparin on binding of rgp120 to GPR1ntP-(1-27) (Fig. 7D). The rgp120 was incubated with different concentrations of heparin (37 °C for 1 h) before addition to microtiter wells, which had been coated with either GPR1ntP-(1-27) or sCD4, and bound rgp120 was detected by ELISA. We found that heparin could inhibit association of rgp120 with GPR1ntP-(1-27) in a concentration-dependent manner. In contrast, heparin had little effect on the binding of sCD4 to rgp120, which is in consistent with a previous report (49).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Effect of GPR1ntP-(1-27) on the infectivity of HIV-1 primary isolates. NP-2/CD4/CCR5 (4 x 103) cells were infected with one of R5X4-tropic HIV-1 primary isolates (GUN-4 or GUN-14) or R5-tropic SF162 strain (each 10 ng of p24Gag) in the presence or absence of GPR1ntP-(1-27) or R5ntP-(1-20). Then, p24Gag in culture supernatants at 3 days after infection were measured by ELISA. As a control, 5 µM azido-3'-deoxythymidine was added. Data are means obtained from duplicate samples. Experiments were repeated three times to confirm results.

To determine which region of GPR1ntP-(1-27) was necessary for rgp120 binding, we next examined the binding activities of the fragment peptides to rgp120 (Fig. 7F). Among the fragment peptides, a small peptide, GPR1ntP-(15-27) significantly bound to rgp120, with a lower affinity compared with GPR1ntP-(1-27), which is consistent with our finding that GPR1ntP-(15-27) inhibited HIV-1 infection at higher concentrations (Table III). In contrast, any apparent interaction of neither GPR1ntP-(1-13) nor GPR1ntP-(8-20) with rgp120 was detected. The tyrosine mutant GPR1ntP-(Y/A) completely lost its ability to bind to rgp120, indicating that tyrosine residues in GPR1ntP-(1-27) play an important role in its binding to rgp120 as well as in its inhibition of HIV-1 infection (Table III).

Inhibition of HIV-1 Binding to MOLT-4 Cells by GPR1ntP-(1-27)—It has been reported that the V3 loop is exposed on the surface of intact virions (52, 53). In addition, it has also been reported that anti-V3 loop mAbs inhibit the binding of HIV-1 to cells (41), suggesting that the V3 loop exposed on the surface of virion plays an important role for its binding to the target cells. Since our sucrose density gradient sedimentation assay indicated that GPR1ntP-(1-27) bound to intact virions (Fig. 6), and the ELISA assay revealed that there was a specific interaction between the V3 loop and GPR1ntP-(1-27) (Fig. 7), we examined whether GPR1ntP-(1-27) affected the binding of HIV-1 (IIIB strain) to MOLT-4 cells. For this, the cells were incubated with HIV-1 at 37 °C for 1 h, washed three times, and lysed, and the total amount of viral p24 core protein associated with the cells was determined by ELISA. The effects of anti-human CD4 mAb (NU-TH/I), anti-V3 loop mAb (0.5), and heparin on the binding of HIV-1 to MOLT-4 cells at a concentration of 10 µg/ml were firstly examined. At this concentration, we confirmed that these anti-HIV-1 reagents inhibited infection of MOLT-4 cells with HIV-1 (IIIB strain) by more than 95% (data not shown). Even when MOLT-4 cells were treated with NU-TH/I before inoculation of virus, 1670 pg of p24^Gag (58.8% of control) was still detected to be bound to the cells (Fig. 8A). To distinguish whether this p24^Gag was derived from the extracellular viruses bound to the surface of the cells or from intracellular viruses that entered the cells, the cells were treated with trypsin to remove the former fraction of viruses (Fig. 8B). The result showed that viral p24^Gag protein was hardly detectable after trypsin treatment, indicating that it was derived from the viruses bound to the surface of NU-TH/I-treated MOLT-4 cells. In agreement with a previous report (41), our findings suggested that HIV-1 was associated with not only CD4, but also with sites other than CD4 on the surface of MOLT-4 cells.

In contrast to anti-CD4 mAb (NU-TH/I), incubation of viruses with 0.5 or heparin before inoculation of viruses blocked the binding of HIV-1 to MOLT-4 cells almost completely (Fig. 8A), suggesting that the V3 loop contributed for the binding of HIV-1 to MOLT-4 cells. Interestingly, the binding of HIV-1 to MOLT-4 cells was also inhibited to a similar extent by GPR1ntP-(1-27) at a concentration of 20 µM: PCR assay showed the same concentration of GPR1ntP-(1-27) similarly inhibited the entry of HIV-1 (IIIB strain) to MOLT-4 cells (Fig. 4, C and D). A mutant peptide substituting all tyrosine residues in GPR1ntP-(1-27) to alanine, GPR1ntP-(Y/A), did not affect the binding of HIV-1 to cells at the same concentration. As expected, intracellular viral p24^Gag was hardly detectable (Fig. 8B) when viruses were treated with 0.5, heparin, or GPR1ntP-(1-27) prior to inoculation. GPR1ntP-(Y/A) again had hardly any effect on HIV-1 entry. Thus, these findings strongly suggested that the inhibition of binding of HIV-1 to MOLT-4 cells by GPR1ntP-(1-27) might be due to its interaction with the V3 loop exposed on the surface of intact virions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we found that the 27-amino acid-long synthetic peptide derived from the NH₂-ECR of GPR1 inhibits HIV-1 infection of human glioma-derived indicator cells (NP-2/CD4/GPCRs), a T-cell line, and more importantly, primary PBLs, which are natural targets for HIV-1 infection. The inhibitory effect of GPR1ntP-(1-27) was observed not only in GUN-1Ser strain, which utilizes GPR1 as a co-receptor, but also in diverse HIV-1 strains, including X4 (IIIB), R5X4 (GUN-1WT, GUN-4, and GUN-14), and R5 (BaL and SF162) (Figs. 2 and 3 and Table II). Among these HIV-1 strains tested, R5 viruses were relatively resistant to GPR1ntP-(1-27) as compared with the other strains.

We found that GPR1ntP-(1-27) binds to rgp120 (Fig. 7, A and B), and this interaction is inhibited by heparin (Fig. 7D), which has been reported to bind to the V3 loop of gp120 (48-50). Moreover, interaction between anti-V3 loop mAb (0.5) with rgp120 was inhibited by GPR1ntP-(1-27) (Fig. 7E). Because it has been reported that 0.5 recognizes 24 amino acids (308-331) of the V3 loop (31) and does not block binding of gp120 to CD4 (54), our data strongly suggested that the V3 loop plays an important role for binding of GPR1ntP-(1-27) to rgp120.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Inhibition of HIV-1 infectivity to T-cell line and primary PBLs by GPR1ntP-(1-27). A, detection of reverse-transcribed HIV-1 (IIIB strain) DNA by PCR. Human T-cell line, MOLT-4 cells, were inoculated with serially diluted HIV-1, lysed 20 h after inoculation, and examined by PCR using the primers specific for the HIV-1 gag region (lanes 1-3). Amplification of -globin was performed as a control. B, the intensity of signal in each lane was measured by a densitometer. Then the relative intensity of gag signal was determined by dividing the intensity of viral gag DNA by that of -globin DNA and plotted. C, dose-dependent inhibition of HIV-1 infection by GPR1ntP-(1-27). MOLT-4 cells were infected with HIV-1 in the presence of one of the synthetic peptides derived from the NH2-ECR of GPCRs (lanes 1-8). Formation of HIV-1 DNA in MOLT-4 cells was detected by PCR as described above. MOLT-4 cells were infected with heat-inactivated (56 °C, 30 min) HIV-1 (lane 9). Lane 10 is a mock-infected control. PCR amplification was performed without template DNA (lane 11). -Globin DNA was amplified as a control to confirm the efficiency of the amplification of each sample. D, the intensity of DNA signal in each lane was measured and the relative intensity of gag signal was determined as described above. E, inhibition of HIV-1 infection to primary human PBLs by GPR1ntP-(1-27). Primary PBLs prepared from two independent donors (PBL#1 and PBL#2) were infected with HIV-1 (IIIB strain) in the presence or absence of GPR1ntP-(1-27). Formation of reverse-transcribed HIV-1 gag DNA in PBLs was detected by PCR as described above. F, the intensity of DNA band in each lane was measured, and the relative intensity of gag signal was determined as described above. Three independent experiments gave similar results.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Inhibition of syncytium formation by GPR1ntP-(1-27). HIV-1-negative C8166 (2.5 x 104) cells were incubated with MOLT-4 (5 x 103) cells chronically infected with HIV-1 (IIIB strain) in the presence or absence of one of the synthetic peptides for 17 h at 37 °C in 96-well plates in 200 µl of culture medium. At that time, the number of syncytia was counted by microscopic examination. The inhibitory effect of each peptide (%) was calculated in comparison with the average number of syncytia (200) of triplicate control wells (no peptide added). Experiments were repeated three times to confirm results.

View larger version (29K): [in this window] [in a new window]   FIG. 6. Binding of GPR1ntP-(1-27) to the highly purified HIV-1 virions. A, culture supernatants of MOLT-4/IIIB cells were concentrated to pellet virions through a 20% sucrose cushion (100,000 x g for 2 h). The virus suspension was then subjected to a 20-60% sucrose density gradient sedimentation (100,000 x g for 16 h). Virus-containing fractions were concentrated, resuspended in TNE buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA), and incubated with the GPR1ntP-(1-27) or X4ntP-(1-28) for 1 h at 37 °C. The virus, virus-peptide mixture or peptide (indicated as input) was then subjected to 20-60% sucrose density gradient sedimentation (100,000 x g for 16 h). Fractions (700 µl) were collected from the top of the gradient. The dot blotting was carried out to detect the presence of the HIV-1 p24Gag, GPR1ntP-(1-27), and X4ntP-(1-28) in each fraction using -p24Gag, -GPR1, and -CXCR4, respectively. The exposure time for -p24Gag dot blots (the first and second dot blots) were 1 min, and that for -GPR1 dot blots (the third and fourth dot blots), and for -CXCR4 dot blot (the fifth dot blot) were 10 min. B, the density of each fraction was measured and plotted (fractions #5-#11) (closed circles). The intensity of signal in each dot spot (#5-#11) (p24Gag of the second dot blot (open squares) and GPR1ntP-(1-27) of the fourth dot blot (closed squares)) was measured by a densitometer, and relative spot signals were plotted.

It has also been reported that anti-V3 loop mAbs strongly bind to the intact virus particles, indicating that the V3 loop is already exposed on the surface of virions before interaction with CD4 receptor (52, 53). Indeed, we found that GPR1ntP-(1-27) binds to the highly purified virions (IIIB strain) even in the absence of sCD4 (Fig. 6), suggesting that GPR1ntP-(1-27) directly binds to the V3 loop exposed on the surface of intact HIV-1 virions under the physiological conditions. In contrast to the mAbs against the V3 loop, it has also been reported that none of four mAbs against the CD4-binding domain (CD4-bd) bind to intact virions, including IIIB strain, indicating that the CD4-bd is not exposed on the surface of intact virions (52).

It has been reported that there are CD4-dependent and CD4-independent sites for HIV-1 binding on the surface of CD4⁺ cells (41). The presence of cell surface molecule(s) other than CD4 on MOLT-4 cells responsible for HIV-1 binding was also suggested by our data, i.e. detection of viral p24^Gag bound to MOLT-4 cells even when the cells had been treated with anti-CD4 mAb before inoculation of virus (Fig. 8). In contrast to anti-CD4 mAb, anti-V3 loop mAb, heparin, and GPR1ntP-(1-27), expected to bind to the V3 loop exposed on the virions, almost completely inhibited the binding of HIV-1 to MOLT-4 cells (Fig. 8). Because CD4-bd on the surface of HIV-1 virions has not been reported to be exposed as described above, our results suggested that the binding of virus to the cells before interaction with CD4 is mediated by the V3 loop exposed on the surface of virions and that this initial CD4-independent virus binding step is an important target for GPR1ntP-(1-27) to inhibit HIV-1 infection.

Although GPR1 serves as a specific co-receptor for the HIV-1 variants, GPR1ntP-(1-27) blocked infection of diverse HIV-1 strains with different co-receptor usage. It may be explained in two ways. Firstly, the V3 loop is known to have the highest density of positively charged amino acids among all regions of gp120: it contains five to nine basic residues, and their distribution pattern within the V3 loop is well conserved among different strains (51, 55). Because GPR1ntP-(1-27) shows the highest ratio of acidic amino acids (net negative charge is -10) among the peptides used in this study (Table I), electrostatic interaction might be responsible for GPR1ntP-(1-27) to bind to basic residues within V3 loop, and to inhibit HIV-1 infection. Because the charge of amino acids in the V3 loop of R5 viruses is known to be more acidic than that of X4 or R5X4 viruses (51, 55), the relative resistance of R5 viruses (BaL and SF162) to GPR1ntP-(1-27) might be explained by the electrostatic repulsion between GPR1ntP-(1-27) and the V3 loop of the R5 viruses. Secondly, as the presence of a similarity in the conformation or antigenicity of the V3 loop has been suggested (52, 56), GPR1ntP-(1-27) would bind to this conserved conformation. Namely, an mAb, 447-52D, has been reported to bind to the V3 loop exposed on the surface of different HIV-1 strains even in the absence of the linear epitope (52, 56). Thus, recognition of the conserved conformation in the V3 loop by GPR1ntP-(1-27) might be important for its inhibitory activity to diverse HIV-1 strains.

The exact stoichiometry of GPR1ntP-(1-27) binding to target gp120 molecules in HIV-1 virions remains to be determined. It has been reported that gp120 is shed from infected cells and/or from virions soon after budding (57, 58). Schneider et al. (59) reported that virus-free culture fluid contains at least 100-fold more gp120 than virions pelleted from the same volume of culture supernatant. Thus, not only virion-associated gp120 but also free gp120 might also bind to GPR1ntP-(1-27) and affect its anti-HIV-1 activity.

Although GPR1ntP-(15-27) (YSYDLDYYSLESC) binds to rgp120 less efficiently than GPR1ntP-(1-27) (Fig. 7F), an inhibitory effect of GPR1ntP-(15-27) is much lower than GPR1ntP-(1-27) (Table III). Thus, there is a discrepancy between the binding ability to rgp120 and the anti-HIV-1 activity of GPR1ntP-(15-27). This is probably due to the inefficient binding of GPR1ntP-(15-27) to intact virions, because the conformation of monomer rgp120 is different from that of gp120 on the surface of HIV-1 virions. Namely, three gp120 molecules are associated noncovalently with the ectodomain of the gp41 envelope glycoprotein trimer to form oligomers (60).

View larger version (22K): [in this window] [in a new window]   FIG. 7. Specific interaction of GPR1ntP-(1-27) with rgp120 through its V3 loop. A, binding of rgp120 or sCD4 to GPR1ntP-(1-27). Microtiter wells were coated with different concentrations of GPR1ntP-(1-27) (0.015-5 µM), and incubated with rgp120 (0.016 µM), or sCD4 (0.016 µM). Bound rgp120 and sCD4 were detected using the sera from AIDS patients and anti-sCD4 antibody, respectively, and shown as absorbance values at 450 nm (optical density at 450 nm (A450)). Each datum point is the mean of values obtained from triplicate samples, and experiment was repeated on at least three times. B, inhibition of rgp120 binding to GPR1ntP-(1-27) by -GPR1 antibody. Microtiter wells were coated with GPR1ntP-(1-27) (1.0 µM), treated with -GPR1 or normal rabbit IgG (NRG), and incubated with rgp120 (0.016 µM). Bound rgp120 was detected as described above. 100 and 0% binding correspond to OD450 values of 0.52 and 0.07, respectively. Data are the mean of values obtained from triplicate samples, and experiment was repeated on at least three times. C, effect of sCD4 on the binding of GPR1ntP-(1-27) to rgp120. Microtiter wells were coated with GPR1ntP-(1-27) (0.5 µM), then rgp120 (0.0016 µM), which had been incubated with or without sCD4 (0.016 µM) (37 °C, 1 h) was added, and bound rgp120 was detected as described above. Data are means and standard deviations from triplicate wells. D, inhibition of gp120-GPR1ntP-(1-27) interactions, but not that of gp120-sCD4 by heparin. Microtiter wells were coated with either GPR1ntP-(1-27) (1.0 µM) or sCD4 (0.016 µM), then rgp120 (0.016 µM) which had been incubated with or without heparin (37 °C, 1 h) was added, and bound rgp120 was detected as described above. 100 and 0% binding of rgp120 to GPR1ntP-(1-27) correspond to A450 values of 0.54 and 0.06, respectively. 100 and 0% binding of rgp120 to sCD4 correspond to A450 values of 0.65 and 0.09, respectively. Data are the mean of values obtained from triplicate samples, and experiment was repeated on at least three times.E, inhibition of the binding of V3 loop-specific, neutralizing mAb, 0.5, to rgp120 by GPR1ntP-(1-27). Microtiter wells were coated with rgp120 (0.016 µM) and incubated with different concentrations of GPR1ntP-(1-27) or X4ntP-(1-28) (5-100 µM). Then 0.5 (ascites, 1:500 dilution) was added into each well, and the captured antibody was detected as described above. 100 and 0% binding of 0.5 to rgp120 correspond to A450 values of 0.36 and 0.05, respectively. Data are the mean values from triplicate samples, and three independent experiments gave similar results. F, binding of rgp120 to the fragment and mutant GPR1 peptides. Microtiter wells were coated with different concentrations of the peptides (0.015-5 µM), and incubated with rgp120 (0.016 µM). Bound rgp120 was detected using the sera from AIDS patients and shown as absorbance values at 450 nm. Data are the mean values from triplicate samples, and experiments were repeated three times to confirm results.

View larger version (23K): [in this window] [in a new window]   FIG. 8. Inhibition of HIV-1 binding to MOLT-4 cells by GPR1ntP-(1-27). A, detection of viral p24Gag associated with the cells, including both extracellular and intracellular portions after inoculation of HIV-1 (IIIB strain) to the cells. MOLT-4 cells were preincubated (1 h, 37 °C) with anti-CD4 mAb, NU-TH/I (10 µg/ml) before inoculation of HIV-1. HIV-1 was incubated (37 °C, 1 h) with anti-V3 loop mAb (0.5, 10 µg/ml), heparin (10 µg/ml), GPR1ntP-(1-27) (20 µM), GPR1ntP-(Y/A) (20 µM), or control buffer before addition to the cells. After the cells were incubated with HIV-1 for 1 h at 37 °C, cell lysates were made to determine p24 core protein levels by ELISA. The control value for HIV-1 was 2840 pg of p24Gag per 107 cells. B, detection of viral p24Gag present in the intracellular portion after HIV-1 binding to MOLT-4 cells. After MOLT-4 cells were incubated with HIV-1 for 1 h at 37 °C, the cells were treated with trypsin to remove viral particles still attached to the cell surface. The control value for HIV-1 was 1670 pg of p24Gag per 107 cells. Under these experimental conditions, normal mouse IgG1 with the same isotype as NU-TH/I and 0.5 had no effect at 10 µg/ml. Data are the mean values from triplicate samples, and experiments were repeated three times to confirm results.

The entry stages, including initial attachment in HIV-1 replication cycle, are effective targets for the development of new antiretroviral therapies, because de novo infection of HIV-1 to humans can be prevented. This has already been shown by the clinical application of several antiretroviral agents. For instance, PRO 542 is a fusion protein of the gp120-binding domain of CD4 with immunoglobulin constant domains, and is expected to block the interaction between gp120 and the CD4 receptor (63); or T20 is a peptide (36 amino acids) that inhibits the HIV-1 fusion step through binding to the viral envelope protein gp41 (64, 65). With a view to the antiviral potency of GPR1ntP-(1-27), the direct use of this peptide as a therapeutic agent will remain uncertain until studies of toxicity and pharmacokinetics are carried out. A critical concern with peptide-based antiviral therapy is its potential to elicit an antibody response to a peptide and make it ineffective (66). Because the amino acid sequence of GPR1ntP-(1-27) is derived from human, the antigenicity of GPR1ntP-(1-27) may be little or not at all in treated patients. The elucidation of the in vitro antiviral mechanism of GPR1ntP-(1-27) shown in this study will disclose valuable insights in the development of a new class of GPCR-based and peptide-based HIV-1 inhibitors.

	FOOTNOTES

 
^* This work was supported in part by grants-in-aid for Scientific Research and the 21st Century Centers of Excellence (COE) Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. 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.

Present address: Dept. of Hematology and Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

^|| To whom correspondence should be addressed: Tel.: 81-27-220-8000; Fax: 81-27-220-8006; E-mail: hoshino{at}med.gunma-u.ac.jp.

¹ The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; GPCR, G protein-coupled receptor; BT-cell, primary human brain-derived fibroblast-like cell; NH₂-ECR, NH₂-terminal extracellular region; FCS, fetal calf serum; PBL, peripheral blood lymphocyte; mAb, monoclonal antibody; sCD4, soluble CD4; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; bd, binding domain.

	ACKNOWLEDGMENTS

 
We thank T. Nakamura for excellent technical assistance. We are grateful to Drs. T. Kumanishi (Niigata University, Niigata, Japan) and S. Matsushita (Kumamoto University, Kumamoto, Japan) for kindly providing us with NP-2 glioma cells and an anti-V3 loop MAb, 0.5, respectively. We also thank Dr. H. Holmes (AIDS Reagent Project of the United Kingdom Medical Research Council) for supplying rgp120, sCD4, and anti-sCD4 antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., and Montagnier, L. (1983) Science 220, 868-871[Abstract/Free Full Text]
2. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske, J., Safai, B., White, G., Foster, P., and Markham, P. D. (1984) Science 224, 500-503[Abstract/Free Full Text]
3. D'Souza, M. P., and Harden, V. A. (1996) Nat. Med. 12, 1293-1300
4. Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1996) Science 272, 1955-1958[Abstract]
5. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996) Cell 85, 1135-1148[CrossRef][Medline] [Order article via Infotrieve]
6. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., and Landau, N. R. (1996) Nature 381, 661-666[CrossRef][Medline] [Order article via Infotrieve]
7. Doranz, B. J., Rucker, J., Yi, Y., Smyth, R. J., Samson, M., Peiper, S. C., Parmentier, M., Collman, R. G., and Doms, R. W. (1996) Cell 85, 1149-1158[CrossRef][Medline] [Order article via Infotrieve]
8. Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., Nagashima, K. A., Cayanan, C., Maddon, P. J., Koup, R. A., Moore, J. P., and Paxton, W. A. (1996) Nature 381, 667-673[CrossRef][Medline] [Order article via Infotrieve]
9. Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996) Science. 272, 872-877[Abstract]
10. Takeuchi, Y., Akutsu, M., Murayama, K., Shimizu, N., and Hoshino, H. (1991) J. Virol. 65, 1710-1718[Abstract/Free Full Text]
11. Shimizu, N., Kobayashi, M., Liu, H. Y., Kido, H., and Hoshino, H. (1995) FEBS Lett. 358, 48-52[CrossRef][Medline] [Order article via Infotrieve]
12. Shimizu, N. S., Shimizu, N. G., Takeuchi, Y., and Hoshino, H. (1994) J. Virol. 68, 6130-6135[Abstract/Free Full Text]
13. Shimizu, N., Soda, Y., Kanbe, K., Liu, H. Y., Jinno, A., Kitamura, T., and Hoshino, H. (1999) J. Virol. 73, 5231-5239[Abstract/Free Full Text]
14. Shimizu, N., Soda, Y., Kanbe, K., Liu, H. Y., Mukai, R., Kitamura, T., and Hoshino, H. (2000) J. Virol. 74, 619-626[Abstract/Free Full Text]
15. Liu, H. Y., Soda, Y., Shimizu, N., Haraguchi, Y., Jinno, A., Takeuchi, Y., and Hoshino, H. (2000) Virology 278, 276-288[CrossRef][Medline] [Order article via Infotrieve]
16. Dragic, T., Trkola, A., Lin, S. W., Nagashima, K. A., Kajumo, F., Zhao, L., Olson, W. C., Wu, L., Mackay, C. R., Allaway, G. P., Sakmar, T. P., Moore, J. P., and Maddon, P. J. (1998) J. Virol. 72, 279-285[Abstract/Free Full Text]
17. Rabut, G. E., Konner, J. A., Kajumo, F., Moore, J. P., and Dragic, T. (1998) J. Virol. 72, 3464-3468[Abstract/Free Full Text]
18. Doranz, B. J., Orsini, M. J., Turner, J. D., Hoffman, T. L., Berson, J. F., Hoxie, J. A., Peiper, S. C., Brass, L. F., and Doms, R. W. (1999) J. Virol. 73, 2752-2761[Abstract/Free Full Text]
19. Genoud, S., Kajumo, F., Guo, Y., Thompson, D., and Dragic, T. (1999) J. Virol. 73, 1645-1648[Abstract/Free Full Text]
20. Brelot, A., Heveker, N., Montes, M., and Alizon, M. (2000) J. Biol. Chem. 275, 23736-23744[Abstract/Free Full Text]
21. Dragic, T. (2001) J. Gen. Virol. 82, 1807-1814[Free Full Text]
22. Jinno, A., Shimizu, N., Soda, Y., Haraguchi, Y., Kitamura, T., and Hoshino, H. (1998) Biochem. Biophys. Res. Commun. 243, 497-502[CrossRef][Medline] [Order article via Infotrieve]
23. Soda, Y., Shimizu, N., Jinno, A., Liu, H. Y., Kanbe, K., Kitamura, T., and Hoshino, H. (1999) Biochem. Biophys. Res. Commun. 258, 313-321[CrossRef][Medline] [Order article via Infotrieve]
24. Salahuddin, S. Z., Markham, P. D., Wong-Staal, F., Franchini, G., Kalyanaraman, V. S., and Gallo, R. C. (1983) Virology 129, 51-64[CrossRef][Medline] [Order article via Infotrieve]
25. Srivastava, B. I., and Minowada, J. (1983) Leuk. Res. 7, 331-338[CrossRef][Medline] [Order article via Infotrieve]
26. Nagumo, T., and Hoshino, H. (1988) Jpn. J. Cancer Res. 79, 9-11[Medline] [Order article via Infotrieve]
27. Takeuchi, Y., Inagaki, M., Kobayashi, N., and Hoshino, H. (1987) Jpn. J. Cancer Res. 78, 11-15
28. Popovic, M., Sarngadharan, M. G., Read, E., and Gallo, R. C. (1984) Science 224, 497-500[Abstract/Free Full Text]
29. Gartner, S., Markovits, P., Markovitz, D. M., Kaplan, M. H., Gallo, R. C., and Popovic, M. (1986) Science 233, 215-219[Abstract/Free Full Text]
30. Simmons, G., Wilkinson, D., Reeves, J. D., Dittmar, M. T., Beddows, S., Weber, J., Carnegie, G., Desselberger U., Gray, P. W., Weiss, R. A., and Clapham, P. R. (1996) J. Virol. 70, 8355-8360[Abstract]
31. Matsushita, S., Robert-Guroff, M., Rusche, J., Koito, A., Hattori, T., Hoshino, H., Javaherian, K., Takatsuki, K., and Putney, S. (1988) J. Virol. 62, 2107-2114[Abstract/Free Full Text]
32. Oravecz, T., and Norcross, M. A. (1993) in Leukocyte Typing V: White Cell Differentiation Antigens (Schlossman, S. F., et al., eds) pp. 476-478, Oxford University Press, Oxford
33. Chesebro, B., and Wehrly, K. (1988) J. Virol. 62, 3779-3788[Abstract/Free Full Text]
34. Pincus, S. H., Wehrly, K., and Chesebro, B. (1991) BioTechniques 10, 336-342[Medline] [Order article via Infotrieve]
35. Frumkin, L. R., Patterson, B. K., Leverenz, J. B., Agy, M. B., Wolinsky, S. M., Morton, W. R., and Corey, L. (1995) J. Gen. Virol. 76, 2467-2476[Abstract/Free Full Text]
36. Kozak, S. L., Platt, E. J., Madani, N., Ferro, F. E., Jr., Peden, K., and Kabat, D. (1997) J. Virol. 71, 873-882[Abstract]
37. Platt, E. J., Wehrly, K., Kuhmann, S. E., Chesebro, B., and Kabat, D. (1998) J. Virol. 72, 2855-2864[Abstract/Free Full Text]
38. Mckeating, J. (1995) in The Practical Approach Series, HIV Volume 1 (Karn, J., ed) pp. 117-126, Oxford University Press, New York
39. Quiros, E., Garcia, F., Maroto, M. C., Bernal, M. C., Cabezas, T., and Piedrola, G. (1995) J. Med. Microbiol. 42, 411-414[Abstract/Free Full Text]
40. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich H. A. (1988) Science 239, 487-491[Abstract/Free Full Text]
41. Valenzuela, A., Blanco, J., Krust, B., Franco, R., and Hovanessian, A. G. (1997) J. Virol. 71, 8289-8298[Abstract]
42. Clapham, P. R., Blanc, D., and Weiss, R. A. (1991) Virology 181, 703-715[CrossRef][Medline] [Order article via Infotrieve]
43. Bess, J. W., Jr., Powell, P. J., Issaq, H. J., Schumack, L. J., Grimes, M. K., Henderson, L. E., and Arthur, L. O. (1992) J. Virol. 66, 840-847[Abstract/Free Full Text]
44. Dettenhofer, M., and Yu, X.-F. (1999) J. Virol. 73, 1460-1467[Abstract/Free Full Text]
45. Van Opstal, O., Fabry, L., Francotte, M., Thiriart, C., and Bruck, C. (1995) in The Practical Approach Series, HIV Volume 2 (Karn, J., ed) pp. 105-122, Oxford University Press, New York
46. Cormier, E. G., Persuh, M., Thompson, D. A. D., Lin, S. W., Sakmer, T. P., Olson, W. C., and Dragic, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5762-5767[Abstract/Free Full Text]
47. Farzan, M., Vasilieva, N., Schnitzler, C. E., Chung, S., Robinson, J., Gerard, N. P., Gerard, C., Choe, H., and Sodroski, J. (2000) J. Biol. Chem. 275, 33516-33521[Abstract/Free Full Text]
48. Batinic, D., and Robey, F. A. (1992) J. Biol. Chem. 267, 6664-6671[Abstract/Free Full Text]
49. Rider, C. C., Coombe, D. R., Harrop, H. A., Hounsell, E. F., Bauer, C., Feeney, J., Mulloy, B., Mahmood, N., Hay, A., and Parish, C. R. (1994) Biochemistry 33, 6974-6980[CrossRef][Medline] [Order article via Infotrieve]
50. Harrop, H. A., and Rider, C. C. (1998) Glycobiology 8, 131-137[Abstract/Free Full Text]
51. Moulard, M., Lortat-Jacob, H., Mondor, I., Roca, G., Wyatt, R., Sodroski, J., Zhao, L., Olson, W., Kwong, P. D., and Sattentau, Q. J. (2000) J. Virol. 74, 1948-1960[Abstract/Free Full Text]
52. Nyambi, P. N., Gorny, M. K., Bastiani, L., van der Groen, G., Williams, C., and Zolla-Pazner, S. (1998) J. Virol. 72, 9384-9391[Abstract/Free Full Text]
53. Gorny, M. K., Revesz, K., Williams, C., Volsky, B., Louder, M. K., Anyangwe, C. A., Krachmarov, C., Kayman, S. C., Pinter, A., Nadas, A., Nyambi, P. N., Mascola, J. R., and Zolla-Pazner, S. (2004) J. Virol. 78, 2394-2404[Abstract/Free Full Text]
54. Skinner, M. A., Langlois, A. J., McDanal, C. B., McDougal, J. S., Bolognesi, D. P., and Matthews, T. J. (1988) J. Virol. 62, 4195-4200[Abstract/Free Full Text]
55. Hwang, S. S., Boyle, T. J., Lyerly, H. K., and Cullen, B. R. (1991), Science 253, 71-74[Abstract/Free Full Text]
56. Cecilia, D., Vineet, N., KewalRamani, O'Leary, J., Volsky, B., Nyambi, P., Burda, S., Xu, S., Littman, D R., and Zolla-Pazner, S. (1998) J. Virol. 1998 72, 6988-6996[Abstract/Free Full Text]
57. Layne, S. P., Merges M. J., Dembo, M., Spouge, J. L., Conley S. R., Moore J. P., Raina, J. L., Renz, H., Gelderblom, H. R., and Nara, P. L. (1992), Virology 189, 695-714[CrossRef][Medline] [Order article via Infotrieve]
58. Hart, T. K., Klinkner, A. M., Ventre J., and Bugelski, P. J. (1993) J. Histochem. Cytochem. 41, 265-271[Abstract]
59. Schneider, J., Kaaden, O., Copeland, T. D., Oroszlan, S., and Hunsmann, G. (1986) J. Gen. Virol. 67, 2533-2538[Abstract/Free Full Text]
60. Hunter, E. (1997) in Retroviruses (Coffin, J. M., Hughes, S. H., and Varmus, H. E., eds) pp. 71-120, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
61. Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N. P., Gerard, C., Sodroski, J., and Choe, H. (1999) Cell 96, 667-676[CrossRef][Medline] [Order article via Infotrieve]
62. Ikeda, K., Konishi, K., Saito, M., Hoshino, H., and Tanaka, K. (2001) Bioorg. Med. Chem. Lett. 11, 2607-2609[CrossRef][Medline] [Order article via Infotrieve]
63. Jacobson, J. M., Lowy, I., Fletcher, C. V., O'Neill, T. J., Tran, D. N., Ketas, T. J., Trkola, A., Klotman, M. E., Maddon, P. J., Olson, W. C., and Israel, R. J. (2000) J. Infect. Dis. 182, 326-329[CrossRef][Medline] [Order article via Infotrieve]
64. Kilby, J. M., Hopkins, S., Venetta, T. M., DiMassimo, B., Cloud, G. A., Lee, J. Y., Alldredge, L., Hunter, E., Lambert, D., Bolognesi, D., Matthews, T., Johnson, M. R., Nowak, M. A., Shaw, G. M., and Saag, M. S. (1998) Nat. Med. 4, 1302-1307[CrossRef][Medline] [Order article via Infotrieve]
65. Munoz-Barroso, I., Durell, S., Sakaguchi, K., Appella, E., and Blumenthal, R. (1998) J. Cell Biol. 140, 315-323[Abstract/Free Full Text]
66. LaBranche, C. C., Galasso, G., Moore, J. P., Bolognesi, D. P., Hirsch, M. S., and Hammer, S. M. (2001) Antiviral Res. 50, 95-115[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				N. Shimizu, A. Tanaka, A. Oue, T. Mori, C. Apichartpiyakul, and H. Hoshino A short amino acid sequence containing tyrosine in the N-terminal region of G protein-coupled receptors is critical for their potential use as co-receptors for human and simian immunodeficiency viruses J. Gen. Virol., December 1, 2008; 89(12): 3126 - 3136. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 280/35/30924    most recent M500195200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jinno-Oue, A. Articles by Hoshino, H. Search for Related Content PubMed PubMed Citation Articles by Jinno-Oue, A. Articles by Hoshino, H. Social Bookmarking                      What's this?