Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.
Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.
复制标题
DOI：
10.1371/journal.ppat.1005321
复制
发表时间：
2015-12
期刊：
影响因子：
6.7
通讯作者：
Roy P
中科院分区：
文献类型：
--
作者：
Fajardo T Jr;Sung PY;Roy P
蓝舌病病毒（BTV）可引起重要经济牲畜的出血性疾病。BTV基因组被组织成10个离散的双链RNA分子（S1-S10），其被认为在病毒衣壳组装期间遵循从最小到最大片段的顺序包装途径。为了证实和扩展这些研究，我们研究了RNA分选和包装机制与一个新的实验方法，使用抑制性寡核苷酸。BTV片段的3 '非翻译区中存在的推定包装信号被许多核酸酶抗性寡核糖核苷酸（ORN）靶向，并评估它们对细胞培养物中病毒复制的影响。与BTV RNA的3' UTR互补的ORN显著抑制病毒复制而不影响蛋白质合成。当将相同的ORN加入到一种新的RNA-RNA相互作用测定中时，发现其抑制复合物的形成，该测定测量了不同RNA片段之间的超分子复合物的形成。靶向BTV片段10（最小的RNA片段）3 'UTR的ORN被证明是最有效的，靶序列的缺失或取代突变会减少RNA复合物，并消除使用反向遗传学回收活病毒。无细胞衣壳组装/RNA包装测定也证实了抑制性ORN可以干扰RNA包装，并且在推定的RNA包装序列内的进一步取代突变已经鉴定了相关的识别序列。区段之间3 'UTR的交换进一步证明了RNA识别是区段特异性的，最有可能充当整个基因组区段的二级结构的一部分。我们的数据证实，该节段dsRNA病毒中的基因组包装通过由位于3'UTR中的特定序列的相互作用形成的超分子复合物的形成而发生。此外，用抑制性ORN抑制反式包装表明，相互作用是设计具有抗病毒活性的化合物的真正目标。蓝舌病病毒（BTV）是反刍动物的重要经济病原，属于基因组由多段双链RNA组成的病毒。为了使病毒合成有活力和感染性的后代，必须选择一组精确的10个新复制的BTV片段用于包装到每个新的病毒颗粒中。病毒如何能够从大量的细胞RNA中选择自己的基因组链尚不清楚。一种可能性是，BTV片段包含交互信号，该交互信号允许它们被分类和打包为一组。这些信号的正确识别对于通过抑制基因组分选和包装过程的抗病毒治疗的可能靶点具有基础和应用意义。在这里，我们发现一系列与BTV RNA上多个位点互补的短寡核苷酸（ORN）阻止了受感染细胞中活病毒的生长。病毒生长抑制阳性的ORN也阻止了基因组RNA在体外包装测定中被包装。此外，当这些相同的靶序列在病毒基因组中缺失或突变时，活病毒回收被消除。在片段之间交换末端序列未能回收病毒，证实此类变化对病毒活力有害。这些研究已经鉴定了dsRNA病毒中基因组包装和存活力的关键的特定区域和序列。由ORN的抑制活性靶向的特定基因组包装序列是真正的药物靶标，作为所有血清型中常见的机制，其可能代表病毒治疗剂开发的阿喀琉斯之踵。
Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. Bluetongue virus (BTV) is an economically important pathogen of ruminants that belongs to a group of viruses whose genome consists of multiple segments of double-stranded RNA. In order for the virus to synthesize viable and infectious progeny, a precise set of the 10 newly replicated BTV segments must be selected for packaging into each new virus particle. How the virus is able to select its own genomic strands from the vast array of cellular RNAs is not clearly understood. One possibility is that that BTV segments harbours an interaction signal that allows them to be sorted and packaged as a set. Correct identification of these signals has basic and applied implications for a possible target of antiviral therapeutics through inhibition of genome sorting and packaging process. Here we showed that a series of short oligonucleotides (ORNs) complementary to multiple sites on the BTV RNA prevented the growth of viable virus in infected cells. ORNs positive for inhibition in virus growth also prevented the genomic RNA to be packaged in an in vitro packaging assay. Moreover, when these same targeted sequences were deleted or mutated in viral genome, viable virus recovery was abolished. Exchanging the terminal sequences between segments failed to recover virus confirming that such changes are deleterious to virus viability. These studies have identified specific regions and sequences key to genome packaging in dsRNA viruses and viability. The specific genome packaging sequences targeted by inhibitory activities of ORNs are bona fide drug target which, as a mechanism common amongst all serotypes, may represent an Achilles’ heel for the development of virus therapeutics.