Nawtaisong Pruksa, Fraser Mark E, Carter James R, Fraser Malcolm J
Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States.
Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States.
Virology. 2015 Jul;481:223-34. doi: 10.1016/j.virol.2015.02.023. Epub 2015 Apr 7.
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 TCID₅₀ virus 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.
丙型肝炎病毒内部核糖体进入位点（HCV-IRES）序列对于蛋白质翻译和基因组复制都至关重要，并且是抗丙型肝炎病毒治疗的潜在靶点。我们构建了一系列抗丙型肝炎病毒I组内含子（αHCV-GrpIs），以攻击HCV IRES内的保守靶位点。这些αHCV-GrpIs旨在介导一种反式剪接反应，该反应将5'剪接位点下游的病毒RNA基因组替换为编码凋亡诱导基因的3'外显子。通过掺入HCV NS5A/5B切割序列来取代其各自的内源性切割位点，对凋亡诱导基因BID、半胱天冬酶3、半胱天冬酶8或tBax的活性形式进行了修饰，以确保只有被HCV感染的细胞在剪接和表达后才会发生凋亡。用每种内含子转染的Huh7.5细胞以0.1的感染复数（MOI）用表达高斯荧光素酶（GLuc）标记物的HCV-Jc1FLAG2进行攻击。然后测定含病毒的上清液中的GLuc表达，作为病毒复制抑制的指标。通过逆转录聚合酶链反应（RT-PCR）和DNA测序分析细胞提取物中是否存在正确的剪接产物。我们还测量了半胱天冬酶3的活性水平，作为量化凋亡细胞死亡的一种手段。这些αHCV-GrpI内含子中的每一个都能够在靶向尿嘧啶处将其3'凋亡外显子正确剪接到病毒RNA基因组上，并导致GLuc标记物的抑制率超过80%。在半数组织培养感染剂量（TCID₅₀）病毒滴定中观察到更明显的抑制作用，这表明这些αHCV-GrpIs能够将病毒复制抑制超过2个对数，即超过99%。与未受攻击的细胞相比，病毒感染后半胱天冬酶3活性显著增加，证明了受攻击细胞内凋亡因子的强烈激活。这种新型的基因干预工具可能对某些丙型肝炎患者有益。
