Inhibition of HIV-1 Replication by Triple-Helix-Forming Phosphorothioate Oligonucleotides Targeted to the Polypurine Tract

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233, 742–747 (1997)

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Inhibition of HIV-1 Replication by Triple-Helix-Forming Phosphorothioate Oligonucleotides Targeted to the Polypurine Tract Satoru Tsukahara,* Junji Suzuki,* Takashi Hiratou,* Kazuyuki Takai,* Yoshio Koyanagi,† Naoki Yamamoto,† and Hiroshi Takaku* *Department of Industrial Chemistry, Chiba Institute of Technology, Tsudanuma, Narashino, Chiba 275, Japan; and †Department of Microbiology, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo 113, Japan

Received March 21, 1997

We show the effects of triple-helix formation by assays of primer extension inhibition in vitro using two systems (two-strand-system (FTFOs) or threestrand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The FTFOs were more effective than the TFOs. We found that the FTFOs containing phosphorothioate groups at the 3 *- and 5*-ends, or inside the hairpin loop, exhibited higher inhibitory effects on cDNA synthesis and greater exonuclease resistance than the unmodified FTFOs and TFOs. The abilities of the FTFOs containing phosphorothioate groups at the antisense sequence sites to inhibit HIV-1 replications were examined. The FTFOs containing phosphorothioate groups at the antisense sequence sites inhibit the replication of HIV-1 more efficiently than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication. q 1997 Academic Press

Replication of HIV-1 proceeds by means of the reverse transcriptase (RT), which catalyzes the conversion of the single-stranded viral RNA genome into double-stranded DNA and allows integration into the cellular genome [1–4]. This process involves multiple steps. The RNase H activity of the RT catalyzes the hydrolysis of the viral RNA from an RNA/DNA hybrid molecule [3]. A polypurine tract (PPT) consisting of 16 nt, which is resistant to RNase H cleavage, serves as a primer for plus-strand DNA synthesis by the DNA-directed DNA polymerase activity of the RT. The PPT is a highly conserved region adjacent to the 3*-end (U3) of the viral RNA, and it has an essential function during reverse transcription [5,6]. When the homopolymeric PPT functions as a target sequence, antisense oligonucleotides as well as triple-helix-forming oligonucleotides (TFOs) [6-13] might be useful tools to interfere with retroviral replication. 0006-291X/97 $25.00

In this paper, we have tested the effect of inhibition of the RT activities by a three-strand-system (FTOs) and a two-strand-system (FTFOs) targeted to the PPT region. The FTFOs contained phosphorothioates at both the 3*-end and the 5*-end, or inside the hairpin loop (Fig. 1). The three-strand-system comprises an RNA/DNA hybrid and triplex-forming-oligonucleotides (TFOs), and the two-strand-system comprises a Watson-Crick and Hoogsteen base pairing sequence on a single strand connected by one hairpin loop (T)5. These are referred to as foldback triplex-forming-oligonucleotides (FTFOs) [14-20]. The formation of the pyr/pur/ pyr triple-helix, which is pH-dependent and unstable under physiological conditions, was avoided by the substitution of C/ for G in the third Hoogsteen base-pairing strand. We found that the FTFOs inhibit the action of the RT most effectively and form a more stable triplex with the PPT-RNA in vitro than the TFOs. In particular, the FTFOs containing phosphorothioate groups at the 3*- and 5*-ends, or inside the hairpin loop, show enhanced inhibition of the RT activities and greater exonuclease resistance. We also describe the anti-HIV-1 activities of the modified FTFOs. MATERIALS AND METHODS Oligonucleotides The oligonucleotides were synthesized by the phosphoramidite method using an Applied Biosystems Model 392 DNA/RNA synthesizer on the 1 mM scale, and with controlled pore glass supports. RNA and DNA phosphoramidite units were purchased from PerSeptive Biosystems, Inc. 2*-Methoxyuridine and 2*-methoxycytidine were purchased from Yamasa Co.. Phosphoramidite units were prepared from the reaction of 5*-dimethoxytrityl-N-protected-2*-methoxyuridine and 2*-methoxycytidine with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite as a condensing unit. The support was treated with concentrated ammonia for 15 h at 557C. For the synthetic RNA oligomers, deprotection was performed by the procedure

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of Slim and Gait [21]. The deprotected oligomers were purified by reverse phase HPLC or by electrophoresis on 20% polyacrylamide/ 7M urea gels. The nucleoside compositions were determined after snake venom phosphodiesterase/bacterial alkaline phosphatase hydrolyses.

Thermal Denaturation Profiles Thermal transitions were recorded at 260 nm using a Shimadzu UV-2200 spectrometer. The insulated cell compartment was warmed from 57C to 907C, with increments of 17C and equilibration for 1 min after attaining each temperature, using the temperature controller SPR-8 (Shimadzu). Samples were heated in masked 1 cm path length quartz cuvettes fitted with Teflon stoppers. Each thermal denaturation was performed in 25 mM Tris/acetate buffer (pH 6.5), 0.4 mM spermine, 10 mM MgCl2 , and 50mM NaCl, containing 1 mM of each strand. The mixture of duplex and single strands was kept at 907C for 5 min, and was then cooled to 227C. At temperatures below 157C, N2 gas was continuously passed through the sample compartment to prevent the formation of condensate.

Exonuclease Stabilities of FTOs and FTFOs The oligonucleotides (0.2 A260) were incubated with 200 ml of culture medium containing 10% fetal bovine serum for 24 h at 377C. Aliquots were taken at 0, 3, 6, 9, 12, and 24 h and were analyzed by PAGE (20% polyacrylamide containing 7 M urea). Densitomeric analysis of gels stained with silver nitrate was performed on a Millipore BioImage 60 S.

In Vitro Transcription Plasmid pSV2neo JRCSF-B, containing the polypurine tract of the HIV-1 genome, was digested by Xho I and Nhe I to yield a dsDNA fragment (0.47 kb). The 23 base T7 promoter sequence was incorporated into the dsDNA by a PCR strategy. The PCR product was transcribed with T7 RNA polymerase according to the manufacturer’s instructions (Ambion).

Inhibition of Reverse Transcription by TFOs and FTFOs RNA/DNA hybrid formation. In vitro transcribed RNA containing the PPT-target sequence (430 nt) (0.1 mM), a 32P-labeled primer (5*-TCAGGGAAGTAGCCTTGTGT-3*, complementary to HIV-1 mRNA, nt 252-271, 0.1 mM), and an antisense oligonucleotide (5*-TCCCCCCTTTTCTTTT-3*, H-20, 1 mM) were diluted in a buffer consisting of 25 mM Tris-acetate (pH6.8), 50 mM NaCl, 10 mM MgCl2, 1 mM b-mercaptoethanol, and 0.4 mM spermine, incubated for 3 min at 907C, and then cooled slowly to 377C. TFOs were added in a 10-100 fold excess to the RNA template. HIV-1 RT and dNTPs were then added to the samples. After an incubation for 1 hour at 377C, the samples were analyzed on a 10% polyacrylamide-TBE-urea gel. The reactions were quantified by radioanalytic imaging with a Bioimage analyzer BSA 2000 (Fujifilm). Triple-helix formation on a single-stranded RNA target. In vitro transcribed RNA containing the PPT-target sequence (430 nt) (0.1 mM) and a 32P-labeled primer (5*-TCAGGGAAGTAGCCTTGTGT-3*), complementary to HIV-1 mRNA, nt 252-271, 0.1 mM), and FTFOs (1 mM or 10 mM) were diluted in a buffer consisting of 25 mM Trisacetate (pH6.8), 50 mM NaCl, 10 mM MgCl2, 1 mM b-mercaptoethanol, and 0.4 mM spermine. The mixture was incubated for 3 min at 907C and was then slowly cooled to 377C. HIV-1 RT and dNTPs were then added to the samples. After an incubation for 1hour at 377C, the samples were analyzed on a 10% polyacrylamide-TBE-urea gel. The reactions were quantified by radioanalytic imaging with a Bioimage analyzer BSA 2000 (Fujifilm).

Cell and Virus The human T lymphotropic virus type I (HTLV-I)-non-infected T cell line, MOLT-4, was grown and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100IU/mL penicillin, and 100 mg/mL of streptomycin. 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 was stored in a small volume at 0807C until use. The titer of the virus stocks was determined by 50% tissue culture infections doses (TCID50).

Anti-HIV Assay The CD4/ T-cell line, MOLT-4 (3 1 106 ml01), was infected with HTLV-IIIB at a multiplicity of infection (MOI) of 0.01. After a 2 h infection the cells were washed and treated with the synthetic oligonucleotides at a 1 mM concentration in the culture medium. After 2 days the 6medium was removed and fresh medium containing the oligonucleotides at either a 1 mM or 2.5 mM concentration was added. Virus replication was monitored at the cellular level by syncytia formation and in the culture supernatants by a p24 ELISA (Du Pont). At the time points indicated, an aliquot of culture supernatant was removed for p24 antigen analysis and was replaced by fresh medium. Once a week, viable cells were counted and passaged at 3 1 106 cells per ml.

RESULTS AND DISCUSSION First, we tested direct inhibition of reverse transcription by the TFOs and the FTFOs. In order to test the inhibition of cDNA synthesis by the TFOs, primer extension assays were performed with a primer binding downstream of the PPT. An RNA 430 nucleotides in length, transcribed in vitro from plasmid pSV2neo JRCS-B and covering the PPT region of the HIV-1 genome, was used as the template. We determined whether the three-strand system or the two-strand system could interfere with the enzyme activities of the RT. Various TFOs and FTFOs were designed (Fig. 1) and analyzed by primer extension assays. The control primer extension reaction was performed with the antisense oligonucleotide (5*-TCCCCCCTTTTCTTTT-3*, H-20, 1 mM) corresponding to the Watson-Crick parts of the PPT region. Primer extension inhibition was assessed with a 5*-end labeled primer DNA (5*-TCAGGGAAGTAGCCTTGTGT-3*, complementary to HIV-1 mRNA, nt 252-271) and was analyzed by gel electrophoresis and autoradiography. Figure 2A shows the results of the TFOs. Four of the TFOs (the purine motif (oligonucleotides bind antiparallel to the purine strand of the RNA/DNA duplex, T/A/T or A/A/T and G/G/C), D5-GA-anti, the pyrimidine motif (oligonucleotides bind parallel to the purine, T/A/T and C//G/C), D1para-TC, the purine-pyrimidine motif, D2-TG-para, and D3-GT-anti) tested at a 1 mM concentration in the primer extension reaction gave RT protection of only 2-16%, as compared with the antisense oligonucleotide. On the other hand, the two other TFOs (the pyrimidine motif R1-UC para, and the pyrimidine-purine motif D4AC-anti) at the highest concentration (10 mM) dis-

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FIG. 1. The sequences of the three-strand (FTOs) and two-strand (FTFOs) oligonucleotide systems used in this work. The terms anti and para refer to the antiparallel and parallel orientations of the third strand oligonucleotides. The nucleotide sequences of the RNA/ DNA hybrid substrate, consisting of the HIV-1 mRNA (430 nt), and the 20 mer oligonucleotide (H 20) complementary to the PTT-region, are shown above. Abbreviations: D, DNA; R, RNA, Rm, 2*-methoxynucleosides; S, phosphorothioate group.

played inhibitory effects of the same order as the antisense oligonucleotide (1 mM). Furthermore, the inhibition of cDNA synthesis by the TFOs occurred at two positions in the PPT region and at positions up to 100 nucleotides distant. Spectroscopic analyses were also performed to determine the triplex to duplex transition temperatures. The Tms of the triplex to duplex transitions are listed (Fig. 2A and 2B, bottom). In the case of the TFOs, lower Tm curves were obtained with D13 and 5. These results are consistent with the gel-retardation analysis (data not shown) and the primer extension assays. However, the three strand system (TFOs) is not very suitable to triple-helix forming oligonucleotides. In a subsequent experiment, we examined how to prevent cDNA elongation by the FTFOs, instead of the FTOs, under the same conditions as described above. Figure 2B shows the results of the FTFOs. In the sepectroscopic analysis, the stability of the triple-helix forming oligonucleotide is also due to the oligonucleotide

loop size (Fig. 2B bottom). However, the DD-loop-T4 36 mer yielded the same band corresponding to the triplex, as compared with the DD-loop-T5 37 mer. All of the FTFOs (DD-loop-T4 36 mer, DD-loop-T5 37 mer, DRloop-T5 37 mer, DRm-loop-T5 37 mer, DD-loop-Ts5 37 mer, DsDs-loop-T5 37 mer, and DsDs-loop-Ts5 37 mer) showed higher inhibitory effects (18-47% at a 1 mM concentration) on cDNA synthesis as compared with the antisense oligonucleotide (1 mM). The relative stabilities of the triplexes formed by the two strand system (FTFOs), found in the primer extension assays (Fig. 2B), are consistent with the results from gel-retardation (data not shown) and spectroscopic analyses (Fig. 2B bottom). We have also designed FTFOs with different combinations of DNA and RNA (DR-loop-T5 37 mer) or with 2*-O-methyl RNA (DRm-loop-T5 37 mer) in the Hoogsteen domains, which bind to the RNA target. However, both modified RNA FTFOs inhibited at two positions in the PPT region and at positions up to 100 nucleotides distant (Fig. 2B). This result suggests that the binding affinity of either the R or the Rm third strand to the RD double helical oligomers must be less than that of the DRD complex [22,23]. Replacement of the 2*-proton by a 2*-hydroxyl leads to a distortion of the third strand conformation. An RNA third strand should be less favored than a DNA third strand, when reverse Hoogsteen hydrogen bonding interactions are involved in triple-helix formation with third strands containing G and A or G and T/U. Our results presented below are in agreement with these predictions. Furthermore, we have synthesized phosphorothioated FTFOs (DD-loop-Ts5 37 mer, DsDs-loop-Ts5 37 mer, and DsDs-loop-Ts5 37 mer). Phosphorothioate internucleotidic bonds have been among the most widely examined oligonucleotide backbone modifications in pharmacological studies [24-28]. These phosphorothioated FTFOs were observed to form triple-helix structures by spectroscopic analysis (Fig. 2B, bottom). However, the oligomers with phosphorothioate internucleotidic bonds that exist as a mixture of diastereoisomers have slightly reduced abilities to form triple-helices than the unmodified FTFOs (DD-loop-T5) (Fig. 2B, bottom) [29,30]. In the case of primer extension assays, the phosphorothioated FTFOs (DD-loop-Ts5 37 mer, DsDs-loop-T5 37 mer, and DsDs-loop-Ts5 37 mer) completely blocked cDNA synthesis, whereas the unmodified FTFOs (DD-loop-T4 36 mer, DD-loop-T5 37 mer, DR-loop-T5 37 mer, and DRm-loop-T5 37 mer) inhibited at two positions in the PPT region and at positions up to 100 nucleotides distant. In particular, we could detect higher inhibitory effects on cDNA synthesis for the phosphorothioated FTFOs (DsDs-loop-T5 37 mer and DsDs-loop-Ts5 37 mer) at the 1 mM concentration. However, in the case of the oligonucleotide with phosphorothioates at the 3* and 5*-ends and the loop site, DsDs-loop-Ts5 37 mer, sequence-specific cDNA synthesis inhibition was not detectable at the higher concen-

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FIG. 2. Inhibition of primer extension by the three-strand (FTOs) and two-strand (FTFOs) systems. (A) Primer extension assay in the presence of FTOs. HIV-1 mRNA (430 nt), transcribed in vitro, was hybridized with a complementary antisense oligonucleotide (20 mer) and was primed with a radioactively-labeled oligonucleotide primer (20 mer). The FTOs were added and incubated. Lanes 1 and 2 contain the FTOs as indicated, at 1 mM and 10 mM concentrations. Lane a shows a primer extension with hybrid HIV-1 mRNA (430 nt) and a radioactively-labeled oligonucleotide primer (20 mer), which binds downstream of the PPT. Lane b, control primer extension reactions with the antisense oligonucleotide (H 20) at 1 mM concentration. M, molecular size markers. Tm values are listed at the bottom. (B) Primer extension assay in the presence of FTFOs. HIV-1 mRNA (430 nt), transcribed in vitro, was primed with a radioactively-labeled oligonucleotide primer (20 mer). The FTFOs were added and incubated. Lanes 1 and 2 contain the FTFOs as indicated, at 1 mM and 10 mM concentrations. Lane a shows a primer extension with the hybrid HIV-1 mRNA (430 nt) and a radioactively-labeled oligonucleotide primer (20 mer), which binds downstream of the PPT. Lane b, control primer extension reactions with the antisense oligonucleotide (H 20) at a 1 mM concentration. M, molecular size markers. Tm values are listed at the bottom.

tration. At the highest concentration (10 mM), direct enzyme inhibition is observed. The oligonucleotides containing more than 14 phosphorothioate groups are better substrates than the phosphodiester oligonucleotides at the highest concentration [31,32]. We looked for similar reverse transcription inhibition by binding to the reverse transcriptase rather than to the mRNA [33,34]. We found that the two strand system inhibited the reverse transcription of HIV-1 more efficiently and formed a more stable triplex with the PPT-RNA than the three strand system, in an n vitro assay of primer extension inhibition. Next, the exonuclease resistances of the two-strandsystem modified oligonucleotides (DRm-loop-T5 37 mer, DD-loop-Ts5 37 mer, DsDs-loop-T5 37 mer, and DsDs-loop-Ts5 37 mer) were examined in the medium containing 10% fetal bovine serum (FBS) used for antiHIV assays (Fig. 3) [35]. For comparison, the unmodified oligonucleotide, DD-Loop-T5 37 mer was chosen as a control. The DsDs-loop-T5 37 mer and the DsDs-loopTs5 37 mer were stable (95%) after 24 h of incubation, whereas the DD-loop-Ts5 37 mer was slightly degraded

(10%) after 9 h and was completely degraded (100%) after 24 h. The DD-Loop-T5 37 mer and the DRm-loopT5 37 mer were completely degraded (100%) after 3 h. An increased resistance to nuclease degradation has been observed in 3*- and 5*-end phosphorothioate oligonucleotide derivatives incubated with 3*-exonucleases. This stabilization should help us to design much more efficient third strand oligonucleotides, which could be used as tools in cellular biology. Finally, in order to clarify the anti-HIV activities of the FTFOs in infected cells, the FTFOs containing phosphorothioate groups at the antisense sequence sites (5*-TTTTCTTTTCCCCCCTTTTTTTsCsCsCsCsCsCsTsTsTsTsCsTsTsTsT-3*, DsD-loopT5 37 mer) were chosen. Control oligonucleotides were prepared for comparison, such as the antisense oligonucleotide (5*-TsCsCsCsCsCsCsTsTsTsTsCsTsTsTsT-3*, Ds 16mer) and one with a base content identical to the FTFOs sequences but in a scrambled order (5*-TCTTCTTCTTCTTCTCTCTCTCsTsCsTsTsCsTsTsCsTsTsCsTsTsCsT-3*, DsDloopT5-ran 37 mer). The MOLT-4 cells were incubated with HIV-1III-B for 2h to allow absorption. The cells

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FIG. 3. Stabilities of FTFOs (DD-loop-T5 37 mer, DRm-loop-T5 37 mer, DD-loop-Ts5 37 mer, RNA/DsDs-loop-T5 37 mer, and DsDsloop-Ts5 37 mer) in the presence of 10% calf serum at 377C for 0-24 h.

were then washed to remove the virus from the medium, and the modified FTFOs were added with fresh medium. After 2 days new medium supplemented with the oligonucleotides was added. The virus production in the culture supernatant was monitored by the HIV1 p24 assay (Fig. 4). In this assay system, the phosphorothioate-modified oligonucleotides cannot interfere with retroviral binding at the CD4-receptor because the cells were incubated with infectious HIV-1 supernatant for 2 h before the application of oligonucleotides. The control infected cells (no oligomer added) exhibited maximal HIV-1 replication at day 11. The level of p24 antigen on this day in the supernatant of the culture treated with the random oligomer, DsD-loopT5-ran 37 mer (2.5 mM), was 50% of that of the untreated control cells. The level of p24 antigen in the cells treated with the antisense oligomer, Ds-16 mer (2,5 mM), was 38% of that of the untreated control cells. However, in the cells treated with the DsD-loopT5 37 mer (2.5 mM), p24 expression was inhibited by 100% as compared to the untreated control. After 18 days, the random oligomer (DsD-loopT5-ran 37 mer) and the antisense oligomer (Ds 16mer) treated cells expressed high levels of p24. In contrast, the FTFOs (DsD-loopT5 37 mer) inhibited virus replication by 80% as compared to the untreated control. Furthermore, no cytotoxicities were observed

even at 20 mM concentrations of these oligonucleotides. In addition, we could detect the greatest inhibitory effects on HIV-1 replication with the DsD-loopT5 37 mer at the 2.5 mM concentration. On the other hand, we could not detect any inhibitory effects with the twostrand-system modified oligonucleotides (DD-loop-Ts5 37 mer, DsDs-loop-T5 37 mer, and DsDs-loop-Ts5 37 mer) at the 2.5 mM concentration (data not shown). These results suggest that the FTFOs containing phosphorothioate groups at the antisense sequence sites inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication. These results support the notion that the twostranded composition of a triple-helix is thermodynamically and kinetically superior to the triple-helix system [6-14] and the antisense oligonucleotides. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e, the triplex actually formed at the PPT and blocked the RT. In particular, the phosphorothioated FTFOs may be useful in nucleic acid based anti-viral therapies with triple-helix approaches. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, No. 07277103, from the Ministry of Education, Science and Culture, Japan, and by the Scientific Promotion Found from Japan Private School Promotion Foundation.

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FIG. 4. Antiviral activity of the phosphorothioated FTFOs and antisense oligonucleotide at 2.5 mM. After 2 h, the virus was removed from MOLT-4 cells newly infected with HIV-1IIIB and the cells were treated with the synthetic oligonucleotides. The second treatment was performed 2 days later. Supernatants were collected and p24 expression was determined by the p24 antigen assay.

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