Inhibition of flavivirus infections by antisense oligorners specifically suppressing viral translation and RNA replication
Inhibition of flavivirus infections by antisense oligorners specifically suppressing viral translation and RNA replication
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DOI：
10.1128/jvi.79.8.4599-4609.2005
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发表时间：
2005-04-01
影响因子：
5.4
通讯作者：
Shi, PY
中科院分区：
文献类型：
--
作者：
Deas, TS;Binduga-Gajewska, I;Shi, PY
黄病毒基因组内的RNA元件是抗病毒治疗的潜在靶点。一组磷酰二胺吗啉代寡聚物（PMO），其序列与位于西尼罗河（WN）病毒基因组5 '-和3'-末端的RNA元件互补，被设计为退火到重要的顺式作用元件，并可能抑制WN感染。一种新的精氨酸丰富的肽缀合到每个PMO有效的细胞传递。这些PMO在与含有WN病毒复制子的细胞系孵育后表现出不同程度的抗病毒活性。其中，靶向5 '末端20个核苷酸（5'末端）或靶向参与潜在基因组环化相互作用的3 '末端元件（3' CSI）的PMO表现出最大的效力。当用5 ′末端或3 ′ CSI PMO处理感染WN病毒流行株的细胞时，在5 μ M浓度下病毒滴度降低约5至6个对数，而没有明显的细胞毒性。3 'CSI PMO还抑制除WN病毒以外的蚊媒黄病毒，并且抗病毒效力与特定病毒的靶向3' CSI序列的保守性相关。作用模式分析表明，5 '端和3' CSI PMO通过两种不同的机制抑制病毒感染。5 '端PMO抑制病毒翻译，而3' CSI PMO不显著影响病毒翻译，但抑制RNA复制。结果表明，反义PMO介导的阻断黄病毒基因组的顺式作用元件可以潜在地发展成抗黄病毒疗法。此外，我们报告说，虽然全长WN病毒含有荧光素酶报告基因（基因组的3'非翻译区工程）是不稳定的，这种报告病毒的早期通过可以用来筛选抑制剂对病毒生命周期的任何步骤。
RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5'- and 3'-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5'-terminal 20 nucleotides (5'End) or targeting the 3'-terminal element involved in a potential genome cyclizing interaction (3'CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5'End or 3'CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 mu M concentration without apparent cytotoxicity. The 3'CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3'CSI sequences of specific viruses. Mode-of-action analyses showed that the 5'End and 3'CSI PMOs suppressed viral infection through two distinct mechanisms. The 5'End PMO inhibited viral translation, whereas the 3'CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3' untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.