期刊
VIROLOGY
卷 481, 期 -, 页码 223-234出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.virol.2015.02.023
关键词
Hepatitis; Group I; Intron; Apoptosis; IRES
类别
资金
- University of Notre Dame Strategic Research Initiative
- NIH/NIAID [RO1AI048561]
The HCV-IRES sequence is vital for both protein translation and genome replication and serves as a potential target for anti-HCV therapy. We constructed a series of anti-HCV group I introns (alpha HCV-GrpIs) to attack conserved target sites within the HCV IRES. These alpha HCV-GrpIs were designed to mediate a trans-splicing reaction that replaces the viral RNA genome downstream of the 5' splice site with a 3' exon that encodes an apoptosis-inducing gene. Pro-active forms of the apoptosis inducing genes BID, Caspase 3, Caspase 8, or tBax were modified by incorporation of the HCV NS5A/5B cleavage sequence in place of their respective endogenous cleavage sites to ensure that only HCV infected cells would undergo apoptosis following splicing and expression. Huh7.5 cells transfected with each intron were challenged at MOI 0.1 with HCV-Jc1FLAG2 which expresses a Gaussia Luciferase (GLuc) marker. Virus-containing supernatants were then assayed for GLuc expression as a measure of viral replication inhibition. Cellular extracts were analyzed for the presence of correct splice products by RT-PCR and DNA sequencing. We also measured levels of Caspase 3 activity as a means of quantifying apoptotic cell death. Each of these ccHCV-GrpI introns was able to correctly splice their 3' apoptotic exons onto the virus RNA genome at the targeted Uracil, and resulted in greater than 80% suppression of the GLuc marker. A more pronounced suppression effect was observed with TCID50 virus titrations, which demonstrated that these alpha HCV-GrpIs were able to suppress viral replication by more than 2 logs, or greater than 99%. Robust activation of the apoptotic factor within the challenged cells was evidenced by a significant increase of Caspase 3 activity upon viral infection compared to non-challenged cells. This novel genetic intervention tool may prove beneficial in certain HCV subjects. (C) 2015 Elsevier Inc. All rights reserved.
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