Comparative analysis of intracellular inhibition of hepatitis C virus replication by small interfering RNAs

Chang, Byeongyong; Lee, Chang Ho; Lee, Joo Han; Lee, Seong-Wook

doi:10.1007/s10529-010-0298-5

Cited by 7 publications

(3 citation statements)

References 17 publications

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“…As expected, upon downregulation of NS5B gene significant decrease in viral RNA and protein expression was observed (Figures 1(g) , 1(h) , and 3 ). Several groups have directed siRNAs against different regions of viral genome including NS5B and demonstrated different levels of inhibition of viral replication and protein expression [ 62 , 63 ]. During HCV life cycle several cellular factors bind to the viral RNA at different regions in order to help with translation and replication or to protect viral RNA [ 26 , 37 – 39 , 64 ].…”

Section: Discussionmentioning

confidence: 99%

Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Mandal

Ganta

Chaubey

2016

Hepatitis Research and Treatment

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Hepatitis C virus is major cause of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Presently available direct-acting antiviral drugs have improved success rate; however, high cost limits their utilization, especially in developing countries like India. In the present study, we evaluated anti-HCV potential of several siRNAs targeted against the HCV RNA-dependent RNA polymerase NS5B and cellular factors, La autoantigen, PSMA7, and human VAMP-associated protein to intercept different steps of viral life cycle. The target genes were downregulated individually as well as in combinations and their impact on viral replication was evaluated. Individual downregulation of La autoantigen, PSMA7, hVAP-A, and NS5B resulted in inhibition of HCV replication by about 67.2%, 50.7%, 39%, and 52%, respectively. However, antiviral effect was more pronounced when multiple genes were downregulated simultaneously. Combinations of siRNAs against La autoantigen with NS5B or hVAP-A resulted in greater inhibition in HCV replication. Our findings indicate that siRNA is a potential therapeutic tool for inhibiting HCV replication and simultaneously targeting multiple viral steps with the combination of siRNAs is more effective than silencing a single target.

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Section: Discussionmentioning

confidence: 99%

Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Mandal

Ganta

Chaubey

2016

Hepatitis Research and Treatment

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“…Total RNA was isolated from each RNA-treated cell and reverse transcribed using random primers (Promega). qRT-PCR was performed using the Rotor-Gene (Corbett) and SYBR green PCR method to analyze the innate immune gene expression with primers as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

Lee

Kim

et al. 2013

J Virol

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This study identified specific and avid RNA aptamers consisting of 2=-hydroxyl-or 2=-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2=-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2=-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2=-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2=-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach. Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). Although HCV infection causes worldwide health problems, efficient and specific antiviral therapy has not yet been developed.HCV belongs to the genus Hepacivirus in the family Flaviviridae. The enveloped virus contains a single, positive-stranded RNA genome about 9.6 kb in length encoding a single polyprotein of about 3,010 amino acids (3). This polyprotein precursor is co-or posttranslationally processed by cellular and viral proteases to yield functional structural and nonstructural proteins (4). The structural proteins include the core protein, C, and the envelope glycoproteins E1 and E2. The nonstructural (NS) proteins comprise the protease NS2, the multifunctional protein NS3 consisting of serine protease and helicase, the serine protease cofactor NS4A, the proteins NS4B and NS5A, and the RNA-dependent RNA polymerase NS5B, which are components of a complex responsible for HCV replication (5). NS5B is the central catalytic enzyme in HCV RNA replication. A number of drugs targeting HCV NS5B replicase are under development. However, the rapid appearance of drug-resistant escape mutant viruses has been reported (6).RNA aptamers are small structured single-stranded RNAs which have emerged as attractive and feasible alternatives to small-molecule and antibody-based therapy. Aptamers can be evolved by systematic evolution of ligands by exponential enrichment (SELEX), an iterative selection method...

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“…Many studies have focused on the effect of siRNA against single gene targets (Chang et al 2010;Kanda et al 2007) but monotherapy based on a single RNAi target is likely to fail due to development of viral resistance. Thus, it is necessary to develop an effective and combinatorial strategy against HCV.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of hepatitis C virus replication by single and dual small interfering RNA using an HCV-infected cell model

Xing

et al. 2011

Biotechnol Lett

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Dual siRNA against different regions of gene in hepatitis C virus (HCV) synergistically inhibited replication of HCV RNA. An HCV-infected cell model was established, and HCV RNA and core protein were detected by RT-PCR and Western blot, respectively. Four HCV-specific siRNAs (siCore, siNS3, siNS4B, siNS5B) were designed and transfected into HCV-infected Huh7.5.1 cells. The antiviral efficacies of the siRNAs were compared using real time PCR and agarose gel electrophoresis. HCV replication in infected cells was inhibited by IFNα-2b in a dose-dependent manner. Synergistic inhibition effects were achieved with combination treatment of any two of the siRNAs (siCore, siNS3 and siNS5B) at low doses (0.1 and 10 nM), as compared to single siRNA treatment (P < 0.05). Furthermore, CCK-8 assay showed no toxicity of the siRNAs to Huh7.5.1 cells. These findings indicate a promising new therapeutic approach for treatment of HCV.

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Comparative analysis of intracellular inhibition of hepatitis C virus replication by small interfering RNAs

Cited by 7 publications

References 17 publications

Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

Inhibition of hepatitis C virus replication by single and dual small interfering RNA using an HCV-infected cell model

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