1 Department of Industrial Chemistry and 3 High Technology Research Center, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016; 2 Department of Virology, Tohoku University, School of Medicine, 2-1 Seiryo 113-8519, Japan
Received 5 November 2002; returned 16 December 2002; revised 17 January 2003; accepted 20 January 2003
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Abstract |
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Methods: The unmodified and modified oligonucleotides were chemically synthesized. The anti-HIV activities of test compounds on HIV-1 infection were determined by protection against HIV-1-induced cytopathic effects. The mechanism of action of the oligonucleotides was determined by virus binding and detection [anti-CD4 monoclonal antibody (MAb) and anti-V3 MAb] assays.
Results: Gm3Um4Gm3-s was highly nuclease resistant, had significantly higher anti-HIV-1 activity than dG3T4G3-s, dG10-s and Gm10-s, and blocked the interaction between gp120 and CD4.
Conclusion: The anti-HIV-1 activity of this oligonucleotide was increased when the phosphodiester and 2'-hydroxyl groups on the oligonucleotide backbones were replaced with a phosphorothioate and 2'-O-methyl backbone; thus Gm3Um4Gm3-s may inhibit HIV-1 infection, at least in part, by blocking the interaction between gp120 and CD4.
Keywords: HIV-1, phosphorothioate group, 2'-O-methyl nucleoside, dimeric hairpin guanosine-quadruplex, nuclease resistant
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Introduction |
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We now describe the design of a highly nuclease-resistant, dimeric hairpin guanosine-quadruplex, [Gm3Um4Gm3-s], containing 2'-O-methyl groups on the nucleosides and sulphur groups on the internucleotidic bonds, and its anti-HIV-1 activity in cultured cells. This oligonucleotide might have increased nuclease resistance and anti-HIV-1 activity.
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Materials and methods |
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Phosphorothioate (S-oligo) and phosphodiester (P-O) oligonucleotides were synthesized by Kurabo BC (Tokyo, Japan). Their sequences are shown in Figure 1.
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The oligonucleotides (A260=0.2) were incubated with 200 µL of culture medium containing 10% fetal bovine serum (FBS) for 48 h at 37°C. Aliquots were taken at time-points from 0 to 48 h, and analysed by PAGE (20% polyacrylamide containing 7 M urea). Densitometric analyses of silver nitrate-stained gel were carried out on an LAS-1000 densitometer (Fuji Photo Film Co., Ltd, Tokyo, Japan).
Cells and virus
The human T lymphotropic virus type I (HTLV-I)-positive human T cell line, MT-4, and the HTLV-I-non-infected T cell line, MOLT-4, were grown and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin 0.1 U/L and streptomycin 100 mg/L. A strain of HIV-1, HTLV-IIIB, was obtained from the culture supernatant of chronically HIV-1-infected MOLT-4 cells (MOLT-4/HTLV-IIIB cells) and stored in a small volume at 80°C until use. The titre of the virus stocks was determined by 50% tissue culture infectious doses (TCID50).
Cells were maintained in RPMI 1640 culture medium containing 10% heat-inactivated FBS. HIV-1 strain HTLV-IIIB stocks were obtained from co-cultured HIV-1-producing cells and uninfected cells. The TCID50 of each virus stock was determined by endpoint titrations of five-fold dilutions in triplicate on MT-4 cells.
Anti-HIV assay
The anti-HIV activities of test compounds in a fresh HIV infection were determined by an HIV-induced cytopathic effects (CPE)-protection assay. MT-4 cells (3 x 105 cells/mL) were placed in 96-well microtitre plates and incubated in the presence of nine five-fold dilutions of the test compounds. Following 30 min incubation at 37°C, cells were inoculated with HIV-1 strain HTLV-IIIB at a multiplicity of infection (MOI) of 0.01 pfu/cell. Experiments were carried out in triplicate. After 5 days of culture at 37°C in a CO2 incubator, cell viability was quantified using a colorimetric assay to monitor the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to a blue formazan product. Absorbance was read in a microcomputer-controlled photometer (Titertec MultiscanR; Labsystem Oy, Helsinki, Finland) at two wavelengths (540 and 690 nm). The absorbance measured at 690 nm was automatically subtracted from that at 540 nm, to eliminate the effects of non-specific absorption. All data represent the mean values of triplicate wells. These values were then translated into percentages per well, cytotoxicity and antiviral protection.32,33
Virus binding assay
The procedure used to detect the binding of HIV-1 particles to the cell surface was described previously.34 Briefly, MT-4 cells (1.0 x 106 cells/mL) were exposed to HIV-1 (which was concentrated 100-fold from the supernatant of MOLT-4/HTLV-IIIB cultures) in the absence or presence of the oligonucleotides (1 µM) in 100 µL of PBS. After incubation at 4°C for 1 h, unbound virus particles were removed by washing the cells three times in PBS. Virus replication was monitored at the cellular level by analysing the culture supernatants with a p24 ELISA (Abbott GmhH Diagnostika, Wiesbaden-Delkenhein, Germany). The binding inhibitory activity ratio (BI) was calculated as follows:
BI (%)=[1 (%MFVS %MFCS)/(%MFV %MFC)] x 100
where MF = mean fluorescence; VS = HIV-infected cells treated with test oligomer; CS = control cells (not exposed to HIV) treated with test oligonucleotides; V = HIV-infected cells without test oligomer; C = control cells (not exposed to HIV and not treated with test oligonucleotides).
FACS analysis of anti-CD4 MAb binding inhibition in MT-4 cells treated with oligonucleotides
MT-4 cultures (1.0 x 106 cells/mL) were treated with oligonucleotides (10 µM). After 24 h, the cells were washed three times with PBS and stained for 30 min with or without the anti-CD4 monoclonal antibody (MAb) OKT-4. The cells were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG) at 4°C. After 30 min, the cells were rinsed in PBS2% FBS, fixed in 1% paraformaldehyde and analysed on a FACSCalibur system (Becton Dickinson Immunocytometry System, San Jose, CA, USA).
FACS analysis of binding inhibition of gp120 with the anti-V3 MAb by oligonucleotides
MT-4 cells (3.6 x 105 cells/mL) were placed into 48-well microtitre plates and incubated with 440 µL of medium containing 10 µM oligonucleotides or control (no oligonucleotides). Following 30 min incubation at 37°C, the cells were inoculated with MOLT-4/HTLV-IIIB (4.0 x 104 cells/mL). After 24 h, the cells were washed twice with PBS and treated with 100 µL of the anti-V3 loop MAb conjugated with FITC at 37°C. After 30 min, the cells were rinsed in PBS2% FBS, fixed in 1% paraformaldehyde and analysed on a FACSCalibur system.
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Results |
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One problem with oligonucleotides is their sensitivity to degradation by nucleases present in serum, especially 3'-exonucleases. The nuclease sensitivities of Gm3Um4Gm3-s and dG3T4G3-s were studied in FBS (Figure 2). For comparison, Gm3Um4Gm3, dG3T4G3 and ran-dG6T4 were used as controls. Unmodified dG3T4G3 was completely hydrolysed within 4 h. The ran-dG6T4, without the quadruplex struc- ture, however, had less nuclease resistance than dG3T4G3. On the other hand, the Gm3Um4Gm3-s and dG3T4G3-s oligonucleotides resisted serum digestion even after 48 h of incubation, whereas Gm3Um4Gm3, with the same sequence, was hydrolysed by 8 h. The stability of the oligonucleotides was increased by the 2'-O-methyl and sulphur groups and by the dimeric hairpin guanosine-quadruplex (basket-type structure). In particular, the Gm3Um4Gm3-s oligonucleotide exhibited higher nuclease resistance than dG3T4G3-s.
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Recently, we reported that dG3T4G3-s had both anti-HIV-1 activity and high nuclease stability. The structureactivity relationship of the dimeric hairpin guanosine-quadruplex (basket-type structure), [dG3T4G3-s], and the four-stranded guanosine-quadruplex, dG10-s, indicated that the stability of the guanosine-quadruplex structure affects anti-HIV-1 activity.31 In the present study, to clarify the anti-HIV activities of the Gm3Um4Gm3-s oligonucleotide, we evaluated its ability to inhibit virus-induced CPE in MT-4 cells. Control oligonucleotides, e.g. dG3T4G3-s, dG3T4G3, Gm3Um4Gm3, dG10-s, dG10, Gm10-s and Gm10, were prepared for comparison. Gm3Um4Gm3-s inhibited virus replication in HIV-1-infected MT-4 cells (Table 1). The EC50 value of Gm3Um4Gm3-s was 0.08 µM, and there was almost no cytotoxicity even at 10 µM. However, the EC50 of dG3T4G3-s was 1.01 µM, and the anti-HIV-1 activity of dG3T4G3-s was 13-fold lower than that of Gm3Um4Gm3-s. The homooligomers dG10-s and Gm10-s protected against HIV-1-induced CPE with EC50 values of 2.0 and 0.89 µM, respectively. In addition, there was no effect of 10 µM dG3T4G3, dG10 and Gm10, with phosphodiester linkages in the internucleotidic bonds (Table 1). However, Gm3Um4Gm3 with phosphodiester linkages inhibited HIV-1-induced CPE with EC50 values of 0.91 µM, a substantially lower inhibitory effect than that of Gm3Um4Gm3-s. The Gm3Um4Gm3 oligonucleotide, with phosphodiester linkages, had inhibitory effects similar to those of dG3T4G3-s. Considering the structure and the modificationactivity relationship of the dimeric hairpin guanosine-quadruplex (basket-type structure), [Gm3Um4Gm3-s], and the four-stranded guanosine-quadruplex, Gm10-s, these results indicate that the stability of the guanosine-quadruplex structure and the modification of the 2'-hydroxyl group of nucleosides affects anti-HIV-1 activity.
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To clarify the mechanism of action by Gm3Um4Gm3-s, we tested whether Gm3Um4Gm3-s inhibited the binding of HIV-1 particles to MT-4 cells, as assessed by a p24 antigen assay.34 Gm3Um4Gm3-s (1.0 µM) inhibited both HIV-1 binding and entry (Figure 3). The oligonucleotide dG3T4G3-s, and the homooligonucleotides dG10-s and Gm10-s, however, did not inhibit HIV-1 binding as effectively as Gm3Um4Gm3-s at concentrations of 1.0 µM. Thus, Gm3Um4Gm3-s affects the binding of HIV-1 particles to MT-4 cells.
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In addition to being the primary receptor for HIV-1, CD4 is also required for HIV-1 entry into target cells.35,36 In the present study, we examined the effects of oligonucleotide (Gm3Um4Gm3-s, Gm3Um4Gm3, dG3T4G3-s and dG3T4G3) treatment on the binding of anti-CD4 MAb, OKT-4, to MT4 cells. The ability of the oligonucleotides (10 µM each) to inhibit the interactions of the primary receptor CD4 with an anti-CD4 MAb OKT-4 was determined using the detection assay (antibody) described in Materials and methods. Excess oligonucleotides were removed by washing, and the binding of the anti-CD4 MAb OKT-4 was monitored using anti-mouse immunoglobulin G (IgG) coupled to FITC. The results indicated that Gm3Um4Gm3-s, Gm3Um4Gm3, dG3T4G3-s and dG3T4G3 did not inhibit binding of the anti-CD4 MAb (Figure 4). That is to say, Gm3Um4Gm3-s could not bind to the primary receptor for HIV-1, CD4.
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Stein et al.17 reported that the oligonucleotide SdC28 binds to the third variable loop domain of HIV-1 gp120 (V3 loop). The degree of interaction was dependent on the length of the oligonucleotide studied, with a rapid decrease in binding affinity observed for compounds shorter than 18 nucleotides. Short G-rich oligonucleotides also interact with the V3 loop of the HIV-1, however, and have anti-HIV-1 activity.1826,29 To compare the characteristics of the oligonucleotides, including Gm3Um4Gm3-s, Gm3Um4Gm3, dG3T4G3-s and dG3T4G3 interactions with the V3 loop on HIV-1 gp120, the next series of experiments examined oligonucleotide-mediated inhibition of the binding of an FITC-coupled MAb to the V3 loop of HIV-1 gp120. MT-4 cells were seeded onto flat-bottomed 48-well plates with 10 µM oligonucleotides. Following a 30 min incubation at 37°C, the cells were inoculated with MOLT-4/HTLV-IIIB (4.0 x 104 cells/mL). After 24 h, the cells were washed with PBS and then reacted with anti-V3 gp120 MAb. The cells were then washed with PBS and analysed by FACS. The results (Figure 5) indicated that 10 µM Gm3Um4Gm3-s, Gm3Um4Gm3, dG3T4G3-s and dG3T4G3 (10 µM) inhibited binding of the V3 loop-gp120 MAb. These results indicate that the Gm3Um4Gm3-s interaction with the V3 domain on gp120 contributes to the ability of this agent to inhibit HIV infection. In vivo results indicate that Gm3Um4Gm3-s blocked the interaction between gp120 and CD4.
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Discussion |
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Mechanism of action studies revealed that Gm3Um4Gm3-s has some antiviral properties, which suggests an activity mechanism similar to that of the known blockers of virus adsorption or virus-mediated cell fusion, such as dextran sulphate.35 Furthermore, the inhibition of viral entry into cells is accomplished by interfering with the gp120/CD4 interaction. We demonstrated that Gm3Um4Gm3-s inhibited this interaction by binding to the V3 loop domain of HIV-1-gp120. Gm3Um4Gm3-s did not bind to the primary receptor for HIV-1, CD4.
Gm3Um4Gm3-s has anti-HIV-1 activity and high nuclease stability. The structureactivity relationship of the dimeric hairpin guanosine-quadruplex (basket-type structure), [Gm3Um4Gm3-s], and the four-stranded guanosine-quadruplex, Gm10-s, indicated that the stability of the guanosine-quadruplex structure affects the anti-HIV-1 activity. Furthermore, additional studies suggested that interference with virus internalization is the key mechanism of action for Gm3Um4Gm3-s. HIV-1 inhibition by this methodology has important therapeutic potential, and these oligonucleotides hold promise for the development of more selective, non-toxic therapies.
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Acknowledgements |
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Footnotes |
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References |
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