Abstract
The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277–343. Based on their antiviral activity, we mapped a druggable region (nts 313–343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5′ or 3′ direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.
Highlights
Hepatitis C virus (HCV) infection, which often evades innate and adaptive immune responses, leads to chronic liver diseases including cirrhosis and hepatocellular carcinoma
A total of 25 small interfering RNA (siRNA) were initially tiled with overlapping sequences that were shifted by two nucleotides over the internal ribosome entry site (IRES) SL region spanning nts 277–343 (Fig 1A, siRNA tiled region is shown in thick gray line; see siRNA sequences in Table 1 and Fig 1B)
Sequence-based prediction of siRNA-accessible targets in HCV IRES is challenging because of its complicated RNA structure and association with multiple host proteins [8, 20, 21, 47, 48], which can even dynamically change the structure of this cis-acting RNA element required for viral translation [23]
Summary
Hepatitis C virus (HCV) infection, which often evades innate and adaptive immune responses, leads to chronic liver diseases including cirrhosis and hepatocellular carcinoma. The ORF is flanked by the 50 and 30 untranslated regions (UTRs), which contain conserved cis-acting RNAs essential for viral genome replication and translation and are attractive targets for designing nucleic acids-based anti-HCV agents [6,7,8]. Multiple proteins are recruited to IRES to stimulate translation and to enhance viral replication These include so called IRES trans-acting factors (ITAFs) such as La protein, polypyrimidine tract-binding protein, poly (rC)-binding protein 2, and eukaryotic initiation factor 3, several heterogeneous nuclear RNA proteins [20] as well as 40S ribosomal subunit [8, 21] and miR-122 [13, 22]. We report the clinical application potential of the IRES-targeting siRNA therapy for the treatment of HCV infection
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