Article

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

Department of Biotechnology and Translational Research Center for Protein Function Control, Yonsei University, Seoul, Korea.
Journal of Viral Hepatitis (Impact Factor: 3.91). 07/2011; 18(7):e298-306. DOI: 10.1111/j.1365-2893.2010.01416.x
Source: PubMed

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.

Download full-text

Full-text

Available from: Seong-Jun Kim
  • [Show abstract] [Hide abstract]
    ABSTRACT: Encephalitis refers to an acute, usually diffuse, inflammatory process affecting the brain. The clinical hallmark of acute encephalitis is the triad of fever, headache, and altered mental status. The most common and important cause of encephalitis is the infection by a virus although other organisms can cause the disease. This article is a general overview of the most common viral encephalitides, divided into two families, Flavivirus and Alphavirus, and provides details about virus and RNA interference. More detailed descriptions of each viral family are provided below.
    No preview · Article · Dec 2011 · Central Nervous System Agents in Medicinal Chemistry(Formerly Current Medicinal Chemistry - Central Nervous System Agents)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.
    No preview · Article · Apr 2012 · Biochemical and Biophysical Research Communications
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT:   The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3' end of the region coding for viral RNA-dependent RNA polymerase (NS5B) has been characterized as a functional cis-acting replication element (CRE). This study reports the ability of two RNA aptamers, P-58 and P-78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure-probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem-loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti-HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.
    Full-text · Article · Feb 2013 · Journal of Viral Hepatitis
Show more

We use cookies to give you the best possible experience on ResearchGate. Read our cookies policy to learn more.