Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense
Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense
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DOI：
10.1046/j.1365-2443.1996.d01-261.x
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发表时间：
1996-06-01
期刊：
影响因子：
2.1
通讯作者：
Nagai, Y
中科院分区：
文献类型：
--
作者：
Kato, A;Sakai, Y;Nagai, Y
背景：单负病毒超家族（Mononegavirales）包括弹状病毒科（Rhabdoviridae）、副粘病毒科（Paramyxoviridae）和丝状病毒科（Filoviridae）。这些病毒具有单链负义RNA作为基因组，最近成功地从包括仙台病毒（一种原型副粘病毒）在内的单负性病毒的转染cDNA中回收感染性病毒，开启了它们的基因工程的可能性。然而，感染性病毒的回收只能通过启动感染循环的cDNA指导反基因组正义（+）RNA的合成，从基因组负义（-）RNA开始一直是不成功的，而且回收效率往往非常低。我们在这里描述了一个类似的系统，允许恢复仙台病毒在一个高的速度，来自这样的细胞，其中转染的cDNA和支持病毒核衣壳蛋白和RNA聚合酶合成的质粒由牛痘病毒驱动的噬菌体T7聚合酶共表达。我们的系统能够从cDNA中回收病毒，不仅指导（+）RNA，而且指导（-）RNA。此外，使用该系统，我们通过转染体外合成的（+）RNA或（-）RNA成功地回收了病毒。这种改进的病毒回收似乎是通过以最佳比例提供支持质粒和通过特异性抑制剂使牛痘病毒的细胞病变效应最小化来实现的。此外，可能关键的是，我们的cDNA被构建成产生病毒真实RNA，而不向5'末端添加T7启动子特异性核苷酸。该系统的直接应用被证明通过创建一个候选疫苗株与一个预定的减毒突变的切割激活位点的病毒融合glycoprotein.Conclusion：我们已经建立了方法，大大提高了仙台病毒的cDNA的回收率。从（-）RNA模板中回收病毒基本上没有绝对的障碍，甚至裸露形式的完整全长RNA链似乎也被适当地修饰成为功能性模板。
Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome, Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA, Starting with genomic negative sense (-) RNA has been unsuccessful, Furthermore, the recovery efficiency has often been extremely low.Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (-)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (-)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5' ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein.Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (-)RNA template, Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.