Abstract
Background: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. Methods: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. Results: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. Conclusions: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes.
Highlights
The hypothesis of an all-RNA based world inspired the recreation of the natural molecular selection and evolution processes in a test tube, with the aim of isolating nucleic acids with diverse activities
It had been previously observed that a small set of aptamers targeting the Hepatitis C virus (HCV) cis-acting replication element (CRE) region interfered with viral replication in cell culture [32,34], pointing to the CRE as a potential antiviral target
Total RNA was extracted 18 h post-transfection and the relative amount of the subgenomic HCV RNA was monitored by quantitative RT-PCR, as described in the Experimental
Summary
The hypothesis of an all-RNA based world inspired the recreation of the natural molecular selection and evolution processes in a test tube, with the aim of isolating nucleic acids with diverse activities. We had previously reported the isolation of a collection of RNA aptamers targeting the CRE region of the HCV genome [32] Some of these selected compounds were shown to interact with the 5BSL3.2 domain and to promote an inhibition of the viral replication of ~50% in cell culture [33]. The selected aptamers contain sequence motifs targeting the highly conserved 5BSL3.2 domain and/or the stem-loop harboring the translation stop codon They efficiently bind to the CRE region and promote changes in its functionality, either at the translational level or at the recruitment of the viral RNA-polymerase. Numbers refer to the nucleotide positions of the HCV Con isolate (GenBank accession number AJ238799)
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