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Journal of Virology, March 2002, p. 3015-3022, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.3015-3022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Activity In Vitro by a New Self-Stabilized Oligonucleotide with Guanosine-Thymidine Quadruplex Motifs

Jun-ichiro Suzuki,1 Naoko Miyano-Kurosaki,2 Tomoyuki Kuwasaki,1 Hiroaki Takeuchi,1,3 Gota Kawai,1,2 and Hiroshi Takaku1,2*

Department of Industrial Chemistry,1 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-machi, Aoba-ku, Sendai 980-8575, Japan3

Received 2 August 2001/ Accepted 3 December 2001


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ABSTRACT
 
An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.


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TEXT
 
Antisense oligonucleotides and their derivatives have been shown to be specific inhibitors of gene expression. They are considered to be a potential new generation of drugs, perhaps capable of inhibiting various pathogens and of regulating specific gene expression by inhibiting the translation of mRNA molecules in a highly specific manner. Antisense oligonucleotides may prove to be more effective than existing treatments for certain disorders and have been reported to have an inhibitory effect against HIV-1 (20, 32, 44). Antisense oligonucleotides with phosphorothioate backbones exhibit several advantages over the other forms, including relatively high nuclease resistance and the capacity to induce the degradation of the target sequence by RNase H (26, 33, 45). However, phosphorothioate oligonucleotides hybridize more weakly with the complementary nucleic acids than unmodified oligonucleotides and are eventually degraded, primarily from the 3' end. Antisense phosphorothioate oligonucleotides have also been shown to block the proliferation of HIV-1 in acutely infected cells in a non-sequence-specific manner (24), probably by inhibition of RT (4, 25) and/or the viral entry process (21, 36). On the other hand, Majumdar et al. have shown that the homocytidine phosphorothioate oligonucleotide SdC28 is a potent inhibitor of HIV-1 RT with respect to template primer binding (23). Stein et al. have also proposed that SdC28 specifically interacts with the positively charged V3 loop of HIV-1 gp120 (37). More recently, a few workers have described the interactions of short, G-rich oligonucleotides, which also interfere with the gp120-CD4 interaction or HIV integrase activity, and were found to have anti-HIV-1 activity (1, 5, 7, 14, 18, 22, 27, 38, 46). Physical characterizations of these oligonucleotides have demonstrated that they form tetramers stabilized by G quartets (16, 17, 19, 28, 39). This G quartet motif leads to remarkable anti-HIV-1 activity.

In this paper, we describe the design of a new dimeric hairpin guanosine-thymidine quadruplex (basket type structure), dG3T4G3-s, containing phosphorothioate groups, and its anti-HIV-1 activity in PBMCs (Fig. 1). This oligonucleotide may have increased nuclease resistance. We also describe the specific inhibition of entry of T-tropic HIV-1 into target cells by using dG3T4G3-s.



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  		FIG. 1. Self-stabilized- and control oligonucleotide sequences synthesized for anti-HIV-1 efficacy studies. The internucleoside backbone was composed of either the standard phosphodiester or chemically modified phosphorothioate linkages.

Abbreviations. The abbreviations used in this paper are as follows: HIV-1, human immunodeficiency virus type-1; RT, reverse transcriptase; dG3T4G3-s, G/T phosphorothioate oligonucleotide; NMM, N-methyl mesoporphyrin IX; PBMCs, peripheral blood mononuclear cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; FI, fusion index; M-tropic, macrophage-tropic; T-tropic, T cell line-tropic; CCR5, CC chemokine receptor 5; CXCR4, CXC chemokine receptor 4; V3 loop, third variable loop of gp120, t1/2, half-life; MOTL-4#8/HTLV-IIIB cells, persistently HIV-1 infected MOLT-4#8 cells; CPE, cytopathic effect; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; MAb, monoclonal antibody; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter.

Structure of DG3T4G3-s Selective detection of quadruplex DNA in the presence of complex mixtures of DNA, such as single- and double stranded DNA, is not possible by any current technique. Recently, Arthanari et al. reported that NMM dyes are shifted to longer wavelengths in the presence of quadruplex, but not duplex, DNA (2). Binding of NMM to the 12-mer (dG4T4G4) quadruplex DNA was examined and compared to that of the duplex DNA. There were essentially no absorption changes in the presence of duplex calf thymus DNA, while significant absorption changes occurred in the presence of the dG4T4G4 quadruplex DNA, which forms the dimeric hairpin guanosine quadruplex (basket type structure), dG4T4G4 (2, 19, 39).

All of the fluorescence and UV experiments were carried out with the samples in a solution containing 20 mM HEPES, 140 mM NaCl, and 5 mM KCl at pH 7.0. NMM (Fig. 2B) was obtained from Frontier Scientific Corp., Logan, Utah. Excitation and emission wavelengths of 399 and 614 nm, respectively, were used for NMM. All of the fluorescence experiments were carried out using a Shimadzu UV-220 spectrophotometer. Fluorescence titrations started with 600 µl of a 10-5 M NMM solution, to which oligonucleotides (DNA/NMM ratio, 50:1) were added (2). After each addition of oligonucleotides, samples were annealed by heating to 80°C, followed by slow cooling to room temperature. Fluorescence measurements were repeated three times for each sample, and the intensities were averaged and corrected by running a blank before each series of experiments. Four quadruplex DNAs (dG3T4G3, dG3T4G3-s, dG10, and dG10-s) were used for binding studies (Fig. 2). The binding of NMM to these four types of quadruplex DNAs was examined and compared to that of the control oligonucleotide, dT10. The absorption spectra of NMM in the presence and absence of the oligonucleotides are shown in Fig. 2A. These results show that the absorption spectrum of NMM is not changed by the presence of dT10-s, while the wavelength of maximal absorption shifts ~20 nm to longer wavelengths in the presence of the four quadruplex DNAs (dG3T4G3, dG3T4G3-s, dG10, and dG10-s). The absorption spectrum of NMM shifts to longer wavelengths with the oligonucleotides dG3T4G3 and dG3T4G3-s (basket type structure [Fig. 2C]) than with the homooligonucleotides dG10 and dG10-s (four-stranded guanosine quadruplex). These results suggest that all four types of quadruplex DNAs significantly changed the absorption spectrum of NMM, while the control oligonucleotide, dT10-s, did not. The dimeric hairpin guanosine quadruplex, dG3T4G3-s, has increased stability compared to the four-stranded guanosine quadruplex, dG10-s (2, 19, 39).




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  		FIG. 2. (A) Absorption spectra of NMM in the absence and presence of a 50:1 oligomer-to-drug ratio are shown for the oligonucleotides dG3T4G3, dG3T4G3-s, dG10, dG10-s, and dT10-s. The concentration of NMM was 10-5 M. (B) Structure of NMM. (C) Schematic model of possible hairpin dimer class of structure (19).

Furthermore, to test the nuclease sensitivities of the oligonucleotides, dG3T4G3 and dG3T4G3-s, containing phosphodiester and phosphorothioate groups in the internucleotide linkages, respectively, were studied in fetal bovine serum (data not shown). The dG3T4G3-s oligonucleotide with the phosphorothioate groups was remarkably stable in serum, with about 95% remaining intact after 48 h of incubation. The unmodified oligonucleotide with the same sequence, dG3T4G3, was less stable, with a t1/2 of 2 h. On the other hand, dG10-s yielded results similar to those of dG3T4G3-s. However, dG10 was less stable, with a t1/2 of 1 h. The stability of the quadruplex DNA structure is among the important factors in determining the efficacy of nuclease resistance.

Inhibition of syncytium formation We investigated inhibitory activity against multinuclear giant cell (syncytium) formation in cocultures of MOTL-4#8/HTLV-IIIB and uninfected MOLT-4#8 cells. MOLT-4#8 cells and MOLT-4#8/HTLV-IIIB cells were mixed in a ratio of 1:1 (final cell concentration, 1.8 x 105 cells/ml). The mixed cell suspension was then cocultured with various concentrations of the oligonucleotides at 37°C in a CO2 incubator. After 20 h of coculture, the number of viable cells was determined by the trypan blue dye exclusion method, and the FI was calculated as 1 - [cell number in test well (MOLT-4#8 cells + MOLT-4#8/HTLV-IIIB cells)]/[cell number in control well (MOLT-4#8 cells)]. The FI values obtained for each oligonucleotide concentration can be expressed as a fraction of the control value, leading to the definition of percent fusion inhibition as [1 - (FIT/FIC)] x 100, where FIT is the FI of the test sample and FIC is that of the control sample (43).

In this experiment, we selected the modified oligonucleotides, dG3T4G3-s, dG10-s, and dT10-s, because the corresponding unmodified oligonucleotides, dG3T4G3, dG10, and dT10, did not inhibit virus-induced CPE on acutely infected MT-4 cells at a concentration of 10 µM (data not shown). The dG3T4G3-s oligonucleotide inhibited giant cell formation by 75% at a concentration of 10 µM compared to the untreated control (Fig. 3). However, dG10-s and dT10-s did not show any inhibitory effects at a concentration of 10 µM. This effect was similar to that observed with dextran sulfate, which has been shown to inhibit the absorption of HIV-1 onto the cell surface in the earliest step of viral infection (3). Furthermore, the nuclease resistance of the oligonucleotides and the stability of the guanosine quadruplex structure affect the enhanced anti-HIV-1 activity. These results suggest that dG3T4G3-s might interfere with HIV-1 absorption onto the cells.



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  		FIG. 3. Inhibition of HIV-1-induced syncytium formation in coculture of MOLT-4#8 cells and MOLT-4#8/HTLV-IIIB cells by the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s. The number of viable cells was determined at 20 h after coculture in the presence of various concentrations of the oligonucleotides. Percent fusion inhibition was calculated as described in the text.

Inhibition of virus binding To clarify the mechanism of action of dG3T4G3-s, we tested whether dG3T4G3-s inhibited the binding of HIV-1 particles to MT-4 cells, as assessed by the p24 antigen assay (30). MT-4 cells (1.0 x 106/ml) were exposed to an HIV-1 preparation (which had been concentrated 100-fold from the supernatant of MOLT-4#8/HTLV-IIIB cultures) in the absence or presence of the oligonucleotides (10 µM) in 100 µl of PBS. After incubation at 4°C for 1 h, cells were washed three times in PBS to remove unbound virus particles. Virus replication was monitored at the cellular level in the culture supernatants by a p24 ELISA (Cellular Products Inc.). The binding inhibitory activity ratio was calculated as [1-(%MFVS - %MFCS)/(%MFV - %MFC)] x 100, where MF stands for mean fluorescence, VS stands for HIV-infected cells treated with test oligomer, CS stands for control cells (not exposed to HIV) treated with test oligonucleotides, V stands for HIV-infected cells without test oligomer, and C stands for control cells (not exposed to HIV and not treated with test oligonucleotides). As shown in Fig. 4, dG3T4G3-s inhibited both HIV-1 binding and entry at a concentration of 10 µM. However, the homooligonucleotides, dG10-s and dT10-s, did not show any inhibition of HIV-1 binding at a concentration of 10 µM. Thus, dG3T4G3-s affects the binding of HIV-1 particles to MT-4 cells.



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  		FIG. 4. Effects of the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s on HIV-1 binding to MT4 cells. MT-4 cells (1.0 x 10-6/ml) were exposed to an HIV-1 preparation (the supernatant of MOLT-4#8/HTLV-IIIB cultures) in the absence or presence of the oligonucleotides (10 µM). After incubation at 4°C for 1 h, cells were washed with PBS to remove unbound virus particles. Virus replication was monitored at the cellular level in the culture supernatants by a p24 ELISA (Cellular Products Inc.).

DG3T4G3-s blocks T-tropic HIV-1 infection HIV-1 enters cells by binding to the cell surface CD4 and coreceptor molecules (8, 11-13). Although the list of possible coreceptors is continuously expanding, the major coreceptors are CCR5 and CXCR4, which facilitate the entry of the M-tropic (R5) and T-tropic (X4) viruses, respectively. The M-tropic virus represents the most prevalent phenotype isolated from individuals shortly after seroconversion and during the asymptomatic period of the disease. It replicates in PBMCs but neither forms syncytia in culture nor infects CD4+-transformed T-cell lines (10, 29, 31). The T-tropic virus replicates in PBMCs and induces syncytia, which typically emerge later in the infection in association with the decline in CD4+ T-cell levels and the progression to AIDS (40-42). On occasion, a highly cytopathic, dualtropic strain is isolated (9, 35). In this study, we evaluated the ability of dG3T4G3-s to inhibit the infection of activated PBMCs using the M-tropic JR-CSF isolate and the T-tropic NL4-3 isolate. It has been shown that specific sequences in the envelope glycoprotein that determine tropism for the host cell reside in the V3 loop of HIV-1 gp120 (6, 15, 34).

PBMCs (3 x 105 cells/ml) were incubated with the M-tropic JR-CSF and T-tropic NL4-3 isolates (at a multiplicity of infection of 0.01) for 1.5 h to allow absorption. We also used the pNL4-3-Luc/VSV-G pseudotype virus with a district viral envelope, VSV-G. The pNL4-3-Luc vector has the luciferase gene and the other parts of the HIV-1 genome, except env and nef, and VSV-G were cotransfected into COS cells. After 3 days, we collected the supernatant, containing the envelope pseudotype virus. Cells were then washed to remove the virus from the medium, and the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s (at 10 µM) were added with fresh medium. Virus production in the culture supernatant was monitored by the HIV-1 p24 antigen assay (Fig. 5). Control-infected cells (no oligonucleotide added) exhibited maximal HIV-1 replication. Treatment of M-tropic JR-CSF-infected PBMCs with dG3T4G3-s (10 µM) inhibited HIV-1 replication to a slight extent, compared to that in the untreated control, after 3 days (Fig. 5A). Furthermore, pNL4-3-Luc/VSV-G-infected PBMCs treated with dG3T4G3-s (10 µM) also expressed high levels of p24 products (Fig. 5B). In contrast, treatment of T-tropic NL4-3-infected PBMCs with dG3T4G3-s (10 µM) greatly inhibited HIV replication compared to that in the untreated control (Fig. 5C). In this assay, dG3T4G3-s inhibited NL4-3 replication in a dose-dependent manner (Fig. 5C). On the other hand, neither the homooligonucleotide dG10-s (10 µM) nor the control oligonucleotide dT10-s (10 µM) showed any inhibitory effect on HIV-1 replication for the T-tropic isolate, the M-tropic isolate, or the pNL4-3-Luc/VSV-G pseudotype virus. The anti-HIV-1 activity of dG3T4G3-s is influenced by specific sequences in the envelope glycoprotein of the virus. That is to say, the dG3T4G3-s oligonucleotide sequence specifically interacted with the envelope glycoprotein of the virus to inhibit HIV infection. These results suggest that dG3T4G3-s specifically blocks T-tropic HIV-1 entry into target cells.



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  		FIG. 5. Inhibitory effects of the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s on replication of the different tropic viruses (T- and M-tropic HIV-1) and the pNL4-3-Luc/VSV-G pseudotype virus in PBMCs. PBMCs (3 x 105 cells/ml) were infected with either JR-CSF (A), pNL4-3-Luc/VSV-G (B), or NL4-3 (C) at a multiplicity of infection of 0.01. After 1.5 h of infection, cells were washed and treated with the oligonucleotides at a 10 µM concentration. Virus replication was monitored at the cellular level by analyzing the culture supernatants by a p24 ELISA (Cellular Products Inc.).

Mechanism of HIV-1 infection inhibition To understand the molecular mechanism by which oligonucleotides inhibit HIV-1 adsorption, we studied the effects of oligonucleotide treatment on the primary receptor for HIV-1, CD4, the coreceptors (CXCR4 and CCR5), and the V3 loop domain of HIV-1gp120. In addition to the primary receptor for HIV-1, CD4, the coreceptors (CXCR4 and CCR5) are required for HIV-1 entry into target cells (8, 11-13).

To determine whether the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s interact with the primary receptor for HIV-1, CD4, and the coreceptors (CXCR4 and CCR5), we studied the effect of oligonucleotide treatment on the binding of a panel of anti-CD4, anti-CXCR4, and anti-CCR5 antibodies to PBMCs. The abilities of the oligonucleotides (each at 10 µM) to inhibit the interactions of the primary receptor, CD4, and the coreceptors, CXCR4 and CCR5, with the anti-CD4 MAb OKT-4, the anti-CXCR4 MAb 12G5, and the anti-CCR5 MAb 2D7, were determined as follows. Phytohemagglutinin-prestimulated PBMCs (2.0 x 106 cells/ml) were treated with oligonucleotides (10 µM). After 24 h, the cells were washed (three times) with PBS and then stained for 30 min with the anti-CXCR4 MAb 12G5, the anti-CCR5 MAb 2D7, or the anti-CD4 MAb OKT-4. Cells were then stained with FITC-conjugated anti-mouse immunoglobulin G at 4°C. After 30 min, the cells were rinsed in PBS-2% fetal bovine serum, fixed in 1% paraformaldehyde, and analyzed on a FACSCalibur system (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).

Treatments of the PBMCs with the oligonucleotides dG3T4G3-s, dG10-s, and dT10-s did not affect the binding of the anti-CD4 MAb, the anti-CCR5 MAb, or the anti-CXCR4 MAb. These results suggest that the oligonucleotides did not bind to the primary receptor for HIV-1, CD4, or to the coreceptors (CXCR4 and CCR5) (Fig. 6). That is to say, the anti-HIV-1 activity of dG3T4G3-s is not attributable to an interaction between the oligonucleotide and the primary receptor for HIV-1, CD4, or the coreceptors (CXCR4 and CCR5).




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  		FIG. 6. FACS analysis of the interaction of the primary receptor, CD4, and the coreceptors (CCR5 and CXCR4) with oligonucleotides. PBMCs (2.0 x 106/ml) were treated with oligonucleotides (10 µM). After 24 h, these cells were incubated with MAbs against CD4 (A through C), CCR5 (D through F), or CXCR4 (G through I), followed by incubation with FITC-conjugated anti-mouse immunoglobulin G. After staining, the cells were analyzed on a FACSCalibur system.

Stein et al. reported that the oligonucleotide SdC28 was able to bind to the V3 loop domain of HIV-1 gp120 (V3 loop) (37). 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. However, the short G-rich oligonucleotides also interact with the V3 loop of HIV-1 and have anti-HIV-1 activity (1, 5, 7, 14, 18, 22, 27, 38, 46). To compare the characteristics of the oligonucleotides, including the dG3T4G3-s, dG10-s, and dT10-s interactions with the V3 loop or the CD4-binding sites on HIV-1 gp120, another series of experiments studied the oligonucleotide-mediated inhibition of the binding of an FITC-coupled anti-V3 HIV-1 gp120 MAb to the V3 loop of HIV-1 gp 120 or of an FITC-coupled anti-CD4 MAb to the CD4 binding site on HIV-1 gp120. MOLT-4#8 cells (3.6 x 105/ml) and MOLT-4#8/HTLV-IIIB cells (4.0 x 104/ml) were seeded in flat-bottom 48-well microtiter plates with 440 µl of medium containing 10 µM oligonucleotides. After 24 h, the syncytium-forming cells were washed twice with PBS and were treated with 100 µl of the anti-V3 loop MAb or the anti-CD4 binding site MAb conjugated with FITC at 37°C. After 30 min, the cells were rinsed in PBS-2% fetal bovine serum, fixed in 1% paraformaldehyde, and analyzed on a FACSCalibur system (Becton Dickinson Immunocytometry Systems). The binding of the anti-CD4 MAb to CD4 on HIV-1 gp120 was not affected by dG3T4G3-s, dG10-s, or dT10-s treatment (Fig. 7A through C); however, dG10-s and dT10-s minimally affected the binding of the V3 loop HIV-1 gp120 MAb to the V3 loop HIV-1 gp120 at a 10 µM concentration of oligonucleotide (Fig. 7E and F). In contrast, dG3T4G3-s treatment drastically decreased the binding of the anti-V3 HIV-1 gp120 MAb to the V3 loop of HIV-1 gp 120 (Fig. 7D). These results suggest that the dG3T4G3-s interaction with the V3 domain on HIV-1 gp120 may contribute to the ability of this agent to inhibit HIV infection. Furthermore, we have clarified the importance of the specific sequence in the envelope of the virus and the pseudotype virus by using the different tropic viruses to inhibit the absorption of HIV-1 onto the cells (Fig. 5).



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  		FIG. 7. FACS analysis of the inhibition of HIV-1 gp120-cellular CD4 interactions with oligonucleotides. PBMCs (2.0 x 106 cells/ml) were treated with oligonucleotides (10 µM). After 24 h, these cells were incubated with the FITC-conjugated anti-CD4 binding site MAb (A through C) or the anti-V3 loop MAb (D through F). After staining, the cells were analyzed on a FACSCalibur system.

The dG3T4G3-s oligonucleotide had anti-HIV-1 activity and high nuclease stability. The structure-activity relationships of the dimeric hairpin guanosine-thymidine quadruplex (basket type structure), dG3T4G3-s, and the four-stranded guanosine quadruplex, dG10-s, showed that the stability of the guanosine quadruplex structure affects anti-HIV-1 activity. Furthermore, additional studies have suggested that interference with virus internalization is the key mechanism of action for dG3T4G3-s. It is quite possible that dG3T4G3-s specifically inhibits the entry of T-tropic HIV-1. Inhibition of HIV-1 by this methodology has important therapeutic potential and holds some promise for more selective, nontoxic therapy in the future.


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ACKNOWLEDGMENTS
 
This work was supported in part by a Grant-in-Aid for High Technology Research from the Ministry of Education, Science, Sports, and Culture, Japan; a grant from the Japan Society for the Promotion of Science in the "Research for the Future" program (JSPS-RFTF97L00593); and a research grant from the Human Science Foundation (HIV-K-1031).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Industrial Chemistry and High Technology Research Center, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan. Phone: 81-474-78-0407. Fax: 81-474-71-8764. E-mail: takaku{at}ic.it-chiba.ac.jp. Back


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Journal of Virology, March 2002, p. 3015-3022, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.3015-3022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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