Application of RNA aptamers to the control of the hepatitis C virus-CRE region function

Hepatitis C virus is an enveloped, ssRNA virus, which infects 3% of the world population. No fully efficient therapy for treating hepatitis C exists. This is mainly due to the quasispecies structure of the RNA genome population, which favors the emergence of resistant viral variants. Despite the high variability rate, significant sequence and, more importantly, structure conservation can be found in the so-called functional genomic RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are potential therapeutic targets. This study validates the use of RNA-based inhibitors (aptamers) as molecular tools to control the activity of the cis-acting replication element (CRE) within the HCV genome. The CRE is an essential partner for viral replication. Also this structural domain is involved in the regulation of the protein synthesis. A set of forty-four RNA aptamers was assayed for the ability to interfere with the viral RNA synthesis in a subgenomic replicon system. Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with specific and well-defined functional RNA domains of the CRE, yielding a decrease in the HCV genomic RNA levels higher than 90%. Concomitantly, one of them also promoted a significant increase in viral translation (>50%), likely by its interaction with the nucleotides surrounding the viral stop translation codon. The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE, thus explaining their antiviral activity. Present findings confirm the potential of the CRE as an anti-HCV drugs target and support the use of aptamers as molecular tools for challenging the functionality of RNA domains in viral genomes.


IRES 3'UTR Abstract:
Hepatitis C virus is an enveloped, ssRNA virus, which infects 3% of the world population. No fully efficient therapy for treating hepatitis C exists. This is mainly due to the quasispecies structure of the RNA genome population, which favors the emergence of resistant viral variants. Despite the high variability rate, significant sequence and, more importantly, structure conservation can be g y g q p y found in the so-called functional genomic RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are potential therapeutic targets. This study validates the use of RNA-based inhibitors (aptamers) as molecular tools to control the activity of the cis-acting replication element (CRE) within the HCV genome. The CRE is an essential partner g ( ) g for viral replication. Also this structural domain is involved in the regulation of the protein synthesis. A set of forty-four RNA aptamers was assayed for the ability to interfere with the viral RNA synthesis in a subgenomic replicon system. Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with specific and well-defined functional RNA domains of the CRE, yielding a decrease in the HCV genomic RNA levels higher than 90%. Concomitantly, one of them also promoted a significant increase in viral translation (>50%), likely by its interaction with the nucleotides surrounding the viral stop translation codon. The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE, thus explaining their antiviral activity. Present findings confirm the potential of the CRE as an anti-HCV drugs target and support the use of aptamers as molecular tools for challenging the functionality of RNA domains in viral genomes.

In vitro selection
Exponential enrichment) process, which consists on iterative cycles of synthesis, binding, positive selection and amplification steps over a randomized oligonucleotide pool. The resulting population is enriched in those molecules able to bind to the desired target molecule. The highly dynamic folding of nucleic acids is the key to understand the specific and efficient interaction of aptamers to their cognate target, thus demonstrating the versatility and flexibility of nucleic acids Introduction of nucleic acids.

FUNCTIONAL RNA DOMAINS WITHIN THE HCV GENOME AND LONG-DISTANT RNA-RNA CONTACTS
The isolation of aptamers directed against different protein targets of the hepatitis C virus (HCV) has been largely The isolation of aptamers directed against different protein targets of the hepatitis C virus (HCV) has been largely described. The HCV genome is a (+)ssRNA molecule encoding a single open reading frame (ORF) flanked by untranslated regions (UTRs), which are essential for viral replication, translation and infectivity. Conserved functional RNA domains have also been identified within the coding region, such as the cis-acting replication element (CRE), which is defined by three stem-loops, 5BSL3. g y y p y to interfere with the CRE functionality: replication, translation and binding to the viral RNA-dependent RNA polymerase (NS5B). All of them beared sequence motifs (the so-called consensus motifs, indicated as groups 1-5) complementary to different conserved elements whithin the HCV CRE.  Aptamers-mediated inhibition is directly related to their biochemical features, such as their threedimensional folding and binding affinity to their target.

THE SELECTED APTAMERS FOLD INTO SINGLE STEM-LOOP EXPOSING THE CONSENSUS MOTIF
In silico structural analysis of the RNA molecules P6-  o t at goa e use o t s st ategy epo ted a common secondary structure for the tested inhibitors, in which the constant sequences, corresponding to the ones used as primer binding site (PBS) during the selection process, appeared as single-stranded tails flanking the stem loop containing the selected 6 03 flanking the stem-loop containing the selected consensus motifs. These nucleotide motifs locate, at least partially, exposed in the apical loop. This folding gives the idea that the functional unit in the aptamer molecules is restricted to the stem-loop, which is used to efficiently interact with the target site in the CRE, in a similar way to that previously described for other regulatory RNA molecules.

EFFICIENT BINDING OF THE SELECTED APTAMERS TO THE CRE REGION
Binding affinity was analyzed by incubating a constant concentration of each 32 P-internally labeled aptamer (~2 nM) with increasing amounts of the unlabeled CRE. The titration curve showed differential interaction efficiency for the different aptamers under study. The results suggest that the selected aptamers P6-96, P6-103, and P7-49 may exert their anti-HCV activity by directly interacting with the CRE region. The role of P7-49 as IRES-mediated translation inductor could be derived from its intearction with other essential domains of the CRE. By using the folding softwares RNAcofold and RNAup, it was detected a major interacting site in the apical loop of the 5BSL3.4 domain, involving the stop codon. This newly predicted targeted motif differs slightly from that previously defined as that of the group five from the initial sequence and clustering analysis. By modifying the conformation of the environment surrounding the stop codon, the ribosomal recycling could be enhanced to improve the IRESdependent translation rate. Work in this area is currently being accomplished in our laboratory, since this idea opens new dependent translation rate. Work in this area is currently being accomplished in our laboratory, since this idea opens new fields to evaluate the real role of the stem-loop containing the stop codon in the viral cycle, and for that purpose, P7-49 could be used as a novel molecular tool.