Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells.
Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells.
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靶向丙型肝炎病毒 IRES 的反剪接 I 型内含子在 Huh7.5 细胞中介导病毒感染后的细胞死亡。
DOI：
10.1016/j.virol.2015.02.023
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
FraserJr,MalcolmJ
中科院分区：
文献类型：
--
作者：
Nawtaisong,Pruksa;Fraser,MarkE;Carter,JamesR;FraserJr,MalcolmJ
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 (αHCV-GrpIs) to attack conserved target sites within the HCV IRES. These αHCV-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 αHCV-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 TCID50virus titrations, which demonstrated that these αHCV-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.
影响因子：
37.8
作者：
R. F. Wilson;Donald E. Laugh;In;P. Ackell;W. Chilian;Myrl Holida;C. J. Hartley;Mark L. Armstrong;MELVtN L. Marcus;Carl W. White
通讯作者：
R. F. Wilson;Donald E. Laugh;In;P. Ackell;W. Chilian;Myrl Holida;C. J. Hartley;Mark L. Armstrong;MELVtN L. Marcus;Carl W. White
测量人体心肌血流量的方法。
影响因子：
9.1
作者：
C. White;R. Wilson;M. Marcus
通讯作者：
M. Marcus
冠状动脉疾病和近期心肌血运重建患者的电影血管造影和尸检结果之间的差异
影响因子：
37.8
作者：
C. Grondin;I. Dyrda;A. Pasternac;L. Campeau;M. Bourassa;J. Lésperance
通讯作者：
J. Lésperance