Abstract
The 5’-untranslated region (5’UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5’ UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5’UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.
Highlights
The human immunodeficiency virus (HIV-1) is the etiological agent of AIDS [1]
In silico studies based on biochemical data support the notion that the long distance interaction (LDI) is the most abundant conformer [6], while ex vivo structure probing assays indicate the prevalence of the branched multiple hairpins (BMH) conformer, i.e. the dimerizing-competent form [7]
We report here the characterization of a series of chimeric molecules composed of a trans-cleaving ribozyme, either a hairpin or a hammerhead, targeting two different sites within the HIV-5’-untranslated region (5’UTR) and an anti-dimerization initiation site (DIS) specific molecule, either a DIS sense molecule that may act as a decoy element and an antisense DIS molecule (Figure 1B)
Summary
The human immunodeficiency virus (HIV-1) is the etiological agent of AIDS [1]. It primarily targets CD4+ cells like T cells and macrophages, leading to the suppression of their immune function. RNA contains several conserved structural domains which play essential roles in viral replication and infection cycles. These functional RNA domains exhibit the highest sequence and structural conservations of the viral genome, making them very attractive therapeutic targets. In silico studies based on biochemical data support the notion that the LDI is the most abundant conformer [6], while ex vivo structure probing assays indicate the prevalence of the BMH conformer, i.e. the dimerizing-competent form [7]. We report here the characterization of a series of chimeric molecules composed of a trans-cleaving ribozyme, either a hairpin or a hammerhead, targeting two different sites within the HIV-5’UTR and an anti-DIS specific molecule, either a DIS sense molecule that may act as a decoy element and an antisense DIS molecule (Figure 1B). Grey boxes indicate the substrate recognition arms of the ribozyme domains. (f) Schematic representation of the chimeras used in the ex vivo inhibition assays, inhibitor RNAs are flanked by U6 snRNA end hairpin loops
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