Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X‐RNA

Abstract

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication

“…In addition, PNAs bearing oligolysine tails exhibit enhanced cellular uptake, without compromising sequence selectivity 16, 17, 31, 32. However, in spite of numerous studies aiming at elucidating the mechanisms of CPP-PNA conjugate entry and uptake in cultured cells, data on in vivo activity of such conjugates are scarce and essentially limited to mouse models 18, 33, 34…”

A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

“…These conjugated PNA molecules have been successfully developed as antibacterial agents, 10 as well as antiviral agents against HIV, HCV, and DHBV. 11,12,13 In previous studies, PNAs used were usually designed to target a single site that has a certain function in the lifecycle of the pathogenic bacteria and viruses, which elevates the risk of drug resistance to a therapeutic agent. Therefore, PNA molecules with dual or more targets in pathogenic bacteria or viruses are expected to reduce this possibility or even to improve their bioactivity.…”

Mangosteen xanthones suppress hepatitis C virus genome replication

“…In vitro HCV RNA polymerase assay Recombinant HCV NS5B protein with an N-terminal hexahistidine tag was expressed in Escherichia coli and purified, as described previously [27]. In vitro RNA polymerase activity assays were performed with 50 ng of recombinant HCV NS5B lacking the C-terminal 21 hydrophobic amino acids (NS5BD21), as described previously [27][28][29] After electrophoresis, gels were dried and then exposed to X-ray film for autoradiography. The amount of radio-labeled RNA was quantified using a Phosphorimager.…”