Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes.
Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes.
复制标题
通过慢病毒介导的针对水泡性口炎病毒核糖核复合体基因的短发夹 RNA 表达下调病毒复制。
DOI：
10.1016/j.antiviral.2012.05.007
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发表时间：
2012
影响因子：
7.6
通讯作者：
Long,CharlesR
中科院分区：
文献类型：
--
作者：
Ramirez-Carvajal,Lisbeth;Long,CharlesR
水泡性口炎病毒（VSV）是研究非节段负链RNA（NSNR）病毒的原型病毒，对畜牧业产生巨大的经济影响。在这项研究中，我们评估了独特的短发夹RNA（shRNA）靶向基因，形成核糖核蛋白（RNP）复合物的VSV血清型印第安纳州（VSIV）的抗病毒潜力。我们使用慢病毒载体构建稳定表达靶向VSIV的RNP基因的七种shRNA之一的细胞系，即核衣壳（N），磷蛋白（P）或聚合酶（L）。我们报道了两个靶向N的5′或3′端的N-shRNA序列，它们显著降低了Vero细胞中N、P和L病毒的转录物（p<0.001），降低了病毒蛋白的表达，并减少了病毒颗粒的脱落（p<0.01）。当我们分析来自两个田间分离株的N-shRNA靶区域的序列多样性时，我们检测到种子区域外的单个碱基替换。我们还报道了其他五种靶向病毒RNA组分的shRNA序列，这些序列显著降低了N、P和L病毒转录物（p<0.001），但未能有效削弱病毒复制。测试的shRNA效率的差异不是由于VSIV基因组中靶区域内的错配。尽管单个shRNA对病毒转录物的部分沉默削弱但不阻断VSIV复制，但将本文鉴定的shRNA组合到多个shRNA载体中可能导致病毒复制的抑制。这些数据有助于基于RNAi的技术的持续发展，以对抗病毒性疾病。
Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5′ or 3′ end of N that significantly reduced N, P, and L viral transcripts (p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells (p<0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts (p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.