ABSTRACT
BACKGROUND: We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection. METHODS AND RESULTS: Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5' untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out. GENERAL SIGNIFICANCE: Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.
Subject(s)
Gene Expression Regulation, Viral , Oligodeoxyribonucleotides/genetics , Oligonucleotides, Antisense/genetics , RNA, Small Interfering/genetics , Base Sequence , Cell Line , Cell Line, Transformed , Flow Cytometry , Gene Silencing , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , HIV-1/physiology , Humans , Hydrolysis , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Poly U/genetics , Poly U/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease H/genetics , Ribonuclease H/metabolismABSTRACT
BACKGROUND: The retroviral RNase H is essential for viral replication. This component has not yet been extensively studied for antiviral therapy. It can be activated by an oligodeoxynucleotide (ODN) resulting in self-destruction of the virions. OBJECTIVE: To examine antiviral potential of ODN in clinical samples using plasma of HIV-1-infected patients. DESIGN: Plasma of 19 HIV-1-infected patients from Zurich and 10 HIV-1 isolates from Africa and drug-resistant strains were processed for ex-vivo treatment. METHODS: Cell-free virions were treated with ODN in the plasma and HIV RNA was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, infectivity of the treated virions was tested on primary human peripheral blood mononuclear cells. RESULTS: Cell-free virions in plasma contained significantly less intact HIV RNA upon treatment with ODN (P = 0.0004), and their infectivity was decreased 52-fold (P = 0.0004). In 39% of the Zurich samples, infectivity was reduced more than 10-fold, in 33% more than 100-fold, and in 28% more than 1000-fold. Also, the isolates from Africa exhibited a 63-fold reduction in infectivity (P = 0.0069) with 80% of the isolates responding more than 10-fold, 40% more than 100-fold, and 10% more than 1000-fold. CONCLUSION: Significant reduction of plasma HIV RNA levels and infectivity of treated virions was achieved on the basis of induced self-destruction of HIV observed with clinical samples. Reduction of viral load ex vivo was designed as model for potential effects in vivo. Premature activation rather than inhibition of a viral enzyme could be a model strategy for future antiretroviral control.
