RESUMEN
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.
Asunto(s)
Regulación Viral de la Expresión Génica , Oligodesoxirribonucleótidos/genética , Oligonucleótidos Antisentido/genética , ARN Interferente Pequeño/genética , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Citometría de Flujo , Silenciador del Gen , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/fisiología , Humanos , Hidrólisis , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Poli U/genética , Poli U/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa H/genética , Ribonucleasa H/metabolismoRESUMEN
Human immunodeficiency virus (HIV) has been shown to undergo self-destruction upon treatment of cell-free virions with partially double-stranded oligodeoxynucleotides targeting the polypurine tract (PPT) of the viral RNA in the virus particle. The ODN forms a local hybrid with the PPT activating the viral RNase H to prematurely cleave the genomic RNA. Here we are describing the self-destruction of a recombinant lentivirus harboring the PPT of HIV in a mouse vagina model. We showed a decrease in viral RNA levels in cell-free virus particles and a reduction reverse transcribed complementary DNA (cDNA) in virus-infected human and primary murine cells by incubation with ODNs. In the vagina simultaneous, prophylactic or therapeutic ODN treatments led to a significant reduction in viral RNA levels. Our finding may have some relevance for the design of other viral self-destruction approaches. It may lead to a microbicide for reduction of sexual and mother-to-child transmission.
