ABSTRACT
The PPT is highly conserved among the known HIV-1 strains, and is a possible target for triplex formation. We show triple-helix formation by a two-strand-system (FTFOs, DsDGloopT5-37) targeted to the polypurine tract (PPT) of HIV-1. In HIV-1 infected MOLT-4 cells, the FTFOs containing phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phos-phorothioate oligonucleotides indicating sequence-specific inhibition of HIV-1 replication for 62 days. However, AZT, treated cells expressed high levels of p 24 products after 46 days.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Oligonucleotides, Antisense/pharmacology , Purines/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Thionucleotides/pharmacology , CD4-Positive T-Lymphocytes/virology , Cell Line , Fluorometry , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/genetics , HIV-1/metabolism , Humans , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , RNA, Viral/metabolism , Thionucleotides/genetics , Thionucleotides/metabolism , Virus Replication/drug effects , Zidovudine/pharmacologyABSTRACT
The polypurine tract (PPT) is highly conserved among the known human immunodeficiency virus (HIV)-1 strains, and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand-system (FTFOs) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In the observation of the FITC-DsDGloopT5-37 with MOLT-4 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm and nucleus. However, weak fluorescence was observed within the cytoplasm and nucleus of MOLT-4 cells treated with the antisense phosphorothioate oligonucleotides (S-ODN-gag-AUG). In HIV-1 infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.
Subject(s)
Anti-HIV Agents/chemistry , HIV-1/chemistry , Oligonucleotides, Antisense/chemistry , Purines/chemistry , Reverse Transcriptase Inhibitors/chemistry , Thionucleotides/chemistry , Amino Acid Sequence , Anti-HIV Agents/pharmacology , Base Sequence , Cell Line , Chromatography, High Pressure Liquid , HIV-1/drug effects , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Oligonucleotides, Antisense/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Thionucleotides/pharmacologyABSTRACT
Reverse transcription of HIV-1 into double-stranded DNA involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by reverse transcriptase (RT). The PPT is a possible target for triple-helix formation. We show the effects of triple-helix formation by assays of RNase H cleavage inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RNase H activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RNase H. The FTFOs containing the phosphorothioate groups at the antisense strand showed greater 3'-exonuclease resistance. In HIV-1 infected MT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phosphorothioate oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Oligonucleotides/pharmacology , Purines/metabolism , RNA, Viral/drug effects , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Base Sequence , Cell Line , Humans , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolismABSTRACT
A phosphorothioate oligonucleotide composed of deoxyguanosine and thymidine was identified as an inhibitor of HIV-1 infection at an early stage of the HIV-1 replication cycle in vitro. The phosphodiester and phosphorothioate chimeric oligonucleotides were found to be potent inhibitors of several steps of HIV-1 infection: the interaction with CD4 and the chemokine receptor, the reverse transcriptase activity, and the integrase activity. To elucidate the mechanism of the anti-HIV-1 function of these oligonucleotides in terms of their structure, we focussed on their G-serial sequences and investigated the characteristics of their solution structures. These oligonucleotides were proved to be able to adopt G-quadruplex structures by UV and CD measurements. We presume that the anti-HIV-1 activities of these oligonucleotides are consequently attributable to G-quadruplex formation.
Subject(s)
Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , HIV-1/drug effects , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacology , Base Sequence , HIV-1/physiology , Humans , In Vitro Techniques , Nucleic Acid Conformation , Spectrophotometry , Thionucleotides/chemistry , Thionucleotides/pharmacology , Virus Replication/drug effectsABSTRACT
Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.
Subject(s)
HIV-1/drug effects , Oligonucleotides, Antisense/pharmacology , Oligonucleotides/chemistry , Oligonucleotides/pharmacology , Virus Replication/drug effects , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Base Sequence , HIV Reverse Transcriptase/antagonists & inhibitors , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides, Antisense/chemistry , Purines/chemistry , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Thionucleotides/chemistry , Thionucleotides/pharmacologyABSTRACT
We show the effects of triple-helix formation by assays of primer extension inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-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.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-1/physiology , Oligonucleotides, Antisense/pharmacology , Thionucleotides/pharmacology , Virus Replication/drug effects , Anti-HIV Agents/chemistry , Base Sequence , Cell Line , DNA Primers/genetics , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , DNA, Viral/biosynthesis , DNA, Viral/genetics , HIV-1/genetics , Humans , Nucleic Acid Conformation , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Thionucleotides/chemistry , Thionucleotides/genetics , Virus Replication/geneticsABSTRACT
Replication of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). The polypurine tract (PPT) serves as a primer for plus-strand DNA synthesis and is highly conserved among HIV-1. The PPT region is a possible target for triple-helix formation. Here, we show the effects of triple-helix formation by analyses of melting temperature and gel shift using a foldback triplex-forming-oligonucleotides (FTFOs). We found that the FTFOs containing phosphorothioate groups at the 3'- and 5'-ends, or inside the hairpin loop, exhibited greater exonuclease resistance than the unmodified FTFOs. Several triplex oligonucleotides have thermal stability. The abilities of the FTFOs (DsDG-37) containing the guanosine in place of the cytidine in the third Hoogsteen base-pairing strand to inhibit HIV-1 replications were examined. The FTFOs (DsDG-37) inhibit the replication of HIV-1 more efficiently than the FTFOs (DsD-37) indicating sequence-specific inhibition of HIV-1 replication.
