The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA.
The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA.
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DOI：
10.1093/nar/gkt068
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发表时间：
2013-04-01
影响因子：
14.9
通讯作者：
Kay MA
中科院分区：
文献类型：
--
作者：
Lisowski L;Elazar M;Chu K;Glenn JS;Kay MA
丙型肝炎病毒（HCV）和其他正链RNA病毒通常需要产生少量阴性基因组（比阳性基因组低20 - 100倍）进行复制，这使得数量较少的抗基因组成为基于RNA干扰（RNAi）治疗的有吸引力的靶标。由于双链短发夹RNA/小干扰RNA （shRNA/ sirna）与基因组和抗基因组病毒RNA链具有互补性，并且这两种shRNA链都有可能成为靶向复合物的一部分，因此临床前RNAi研究无法区分感染细胞中实际靶向的病毒链。在这里，我们解决了阴性HCV基因组是否对RNAi具有生物可及性的问题。我们首先筛选了针对HCV基因组中最保守区域的最活跃的shRNA分子，然后将其用于生成非对称抗HCV shRNA，这些shRNA可产生特异性针对HCV基因组或抗基因组序列的生物活性RNAi。使用这种简单但功能强大且有效的筛选shRNA链选择性的方法，我们证明了HCV的抗基因组链在HCV复制过程中不是一个可行的RNAi靶标。这些发现为HCV生物学提供了新的见解，并对设计更有效和更安全的抗病毒RNAi策略寻求针对HCV和其他具有类似复制策略的病毒具有重要意义。
Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.
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