In an attempt to develop a lead for the application of 2–5A-antisense to the targeted destruction of human immunodeficiency virus (HIV) RNA, specific target sequences within the HIV mRNAs were identified by analysis of the theoretical secondary structure. 2-5A-antisense chimeras were chosen against a total of 11 different sequences: three in the gag mRNA, three in the rev mRNA and five in the tat mRNA. 2-5A-antisense chimera synthesis was accomplished using solid-phase phosphoramidite chemistry. These chimeras were evaluated for their activity in a cell-free assay system using purified recombinant human RNase L to effect cleavage of 32P-labelled RNA transcripts of plasmids derived from HIV NL4-3. This screening revealed that of the three 2-5A-antisense chimeras targeted against gag mRNA, only one had significant HIV RNA cleavage activity, approximately10-fold-reduced compared to the parent 2-5A tetramer and comparable to that reported for the prototypical 2-5A-anti-PKR chimera, targeted against PKR mRNA. The cleavage activity of this chimera was specific, since a scrambled antisense domain chimera and a chimera without the key 5′-monophosphate moiety were both inactive. The 10 other 2-5A-antisense chimeras against tat and rev had significantly less activity. These results imply that HIV gag RNA, like PKR RNA and a model HIV tat-oligoA-vif RNA, can be cleaved using the 2-5A-antisense approach. The results further imply that not all regions of a potential RNA target are accessible to the 2-5A-antisense approach.
BoiziauCKurfurstRCazenaveCRoigVThuongNT & ToulmeJ-J (1988) Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism. Nucleic Acids Research16:3341–3358.
2.
CagnonLCucchiariniMLefebvreJ-C & DoglioA (1995) Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology9:349–358.
3.
ChastinM & TinocoIJr (1993) RNA structure as related to antisense drugs. In Antisense Research and Applications, pp. 55–66. Edited by CrookeST & LebleuB. Boca Raton: CRC Press.
4.
CirinoNMLiGXiaoWTorrencePF & SilvermanRH (1997) Targeting RNA for decay in respiratory syncytial virus infected cells with 2”5′ oligoadenylate antisense. Proceedings of the National Academy of Sciences, USA94:1937–1942.
5.
DashPLotanIKnappMKandelER & GoeletP (1987) Selective elimination of mRNA in vivo: complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity. Proceedings of the National Academy of Sciences, USA84:7896–7900.
6.
De ClercqE (1995a) Antiviral therapy for human immunodeficiency virus infections. Clinical Microbiology Reviews8:200–209.
7.
De ClercqE (1995b) Toward improved anti-HIV chemotherapy: therapeutic strategies for intervention with HIV infections. Journal of Medicinal Chemistry38:2491–2517.
8.
EckerDJ (1993) Strategies for invasion of RNA secondary structure. In Antisense Research and Applications, pp. 387–400. Edited by CrookeST & LebleuB. Boca Raton: CRC Press.
9.
FreierS (1993) Hybridization: considerations affecting antisense drugs. In Antisense Research and Applications, pp. 67–82. Edited by CrookeST & LebleuB. Boca Raton: CRC Press.
10.
HomannMTzortzakakiSRittnerKSczakielG & TablerM (1993) Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Research21:2809–2814.
11.
JencksWP (1969) Catalysis in Chemistry and Enzymology, pp. 1–41. New York: McGraw-Hill.
12.
KinS-GSuzukiYNakashimaHYamamotoN & TakakuH (1991) Phosphorothioate analogues of oligodeoxyribonucleotides: synthesis and activity as inhibitors of replication of human immunodeficiency virus. Biochemical and Biophysical Research Communications179:1614–1619.
13.
KinchingtonDGalpinSJaroszewskiJWGhoshKSubasingheC & CohenJS (1992) A comparison of gag, pol and rev antisense oligodeoxy nucleotides as inhibitors of HIV-1. Antiviral Research17:53–62.
LiGXiaoW & TorrencePF (1997) Synthesis and properties of second generation 2-5A-antisense chimeras with enhanced resistance to exonucleases. Journal of Medicinal Chemistry40:2959–2966.
16.
LisziewiczJSunDKlotmanMAgrawalSZamecnikP & GalloRC (1992) Specific inhibition of human immunodeficiency virus type 1 replication by antisense oligonucleotides: an in vitro model for treatment. Proceedings of the National Academy of Sciences, USA89:11209–11213.
17.
LisziewiczJSunDMetelevVZamecnikPGalloRC & AgrawalS (1993) Long-term treatment of human immunodeficiency virus-infected cells with antisense oligonucleotide phosphorothioates. Proceedings of the National Academy of Sciences, USA90:3860–3864.
18.
LundOSNielsenJO & HansenJ-E (1995) Inhibition of HIV-1 in vitro by C-5 propyne phosphorothioate antisense to rev Antiviral Research28:81–91.
19.
MaitraRKLiGXiaoWDongBTorrencePF & SilvermanRH (1995) Catalytic cleavage of an RNA target by 2–5A antisense and RNase L. Journal of Biological Chemistry270:15071–15705.
20.
MaranAMaitraRKKumarADongBXiaoWLiGWilliamsBRGTorrencePF & SilvermanRH (1994) Blockage of NF-κB signaling by selective ablation of an mRNA target by 2–5A antisense chimeras. Science265:789–792.
21.
MatsukuraMShinozukaKZonGMitsuyaHReitzMCohenJS & BroderS (1987) Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus. Proceedings of the National Academy of Sciences, USA84:7706–7710.
MilliganJFMateucciMD & MartinJC (1993) Current concepts in antisense drug design. Journal of Medicinal Chemistry36:1923–1937.
24.
RaynerBMatsukaraMMorvanFCohenJS & ImbachJ-L (1989) In vitro anti-HIV activity of phosphorothioate α-anomeric oligodeoxy nucleotides. CR Acad Sci III310:61–64.
25.
SarinPSAgrawalSCiveiraMPGoodchildJIkeuchiT & ZamecnikPC (1988) Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates. Proceedings of the National Academy of Sciences, USA85:7448–7451.
26.
SczakielGPawlitaM & KleinheinzA (1990) Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribed in sense and antisense orientation from the 5′-leader/gag region. Biochemical and Biophysical Research Communications169:643–651.
27.
ShibaharaSMukaiSMorisawaHNakashimaHKobayashiS & YamamotoN (1989) Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives. Nucleic Acids Research17:239–252.
28.
SilvermanRH & KrauseD (1987) Analysis of antiviral mechanisms: interferon regulated 2′,5′-oligoadenylate and protein kinase systems. In Lymphokines and Interferons: A Practical Approach, pp. 149–193. Edited by ClemensMJMorrisAG and GearingAJH. Oxford: IRL Press.
29.
StephensonML & ZamecnikPC (1978) Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxynucleotide. Proceedings of the National Academy of Sciences, USA75:285–288.
30.
TorrencePFMaitraRKLesiakKKhamneiSZhouA & SilvermanRH (1993) Targeting RNA for degradation with a (2′-5′)oligoadenylate-antisense chimera. Proceedings of the National Academy of Sciences, USA90:1300–1304.
31.
TorrencePFXiaoWLiGLesiakKKhamneiSMaranAMaitraRDongB & SilvermanRH (1994) 2′,5′-Oligoadenylate antisense chimeras for targeted ablation of RNA. In Carbohydrate Modifications in Antisense Research, pp. 119–132. Edited by SanghviYS & CookPD. Washington DC: American Chemical Society.
32.
TorrencePFXiaoWLiGCramerHPlayerMR & SilvermanRH (1997) Recruiting the 2–5A system for antisense therapeutics. Antisense Nucleic Acid Drug Development7:203–206.
33.
XiaoWLiGLesiakKDongBSilvermanRH & TorrencePF (1994) Synthesis of a 5′-thiophosphate analogue of 2-5A, a phosphatase resistant activator of the 2-5A-dependent ribonuclease. Bioorganic and Medicinal Chemistry Letters4:2609–2614.
34.
XiaoWPlayerMRLiGZhangKLesiakK & TorrencePF (1996) Synthesis and characterization of composite nucleic acids containing 2′,5′-oligoriboadenylate linked to antisense DNA. Antisense Research and Development6:247–258.
35.
ZamecnikPC & StephensonML (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proceedings of the National Academy of Sciences, USA75:280–284.
36.
ZhangRYanJShahinianHAminGLuZLiuTSaagMSJiangZTemsamaniJMartinRRSchechterPJAgrawalS & DiasioRB (1995) Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM91) in HIV-infected subjects. Clinical Pharmacology and Therapeutics58:44–53.
37.
ZukerM (1989) Computer predicition of RNA structure. Methods in Enzymology180:262–288.
