Kato A, Sakai Y, Shioda T, Kondo T, Nakanishi M, Nagai Y
Department of Viral Infection, University of Tokyo, Japan.
Genes Cells. 1996 Jun;1(6):569-79. doi: 10.1046/j.1365-2443.1996.d01-261.x.
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.
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.
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.
单股负链RNA病毒超家族（单股负链RNA病毒目）包括弹状病毒科、副粘病毒科和丝状病毒科三个科。这些病毒以单链负义RNA作为基因组。近期，从包括仙台病毒（一种典型的副粘病毒）在内的单股负链RNA病毒的转染cDNA中成功拯救出感染性病毒，这为其基因工程改造开辟了可能性。然而，仅通过用指导合成反基因组正链（+）RNA的cDNA启动感染周期才能拯救出感染性病毒。以基因组负链（-）RNA起始则未成功。此外，拯救效率通常极低。
我们在此描述了一种类似的系统，该系统可使仙台病毒以高比率从细胞中拯救出来，在这些细胞中，由痘苗病毒驱动的噬菌体T7聚合酶共表达转染的cDNA和支持病毒核衣壳蛋白及RNA聚合酶合成的质粒。我们的系统不仅能够从指导（+）RNA合成的cDNA中，还能从指导（-）RNA合成的cDNA中拯救出病毒。此外，使用该系统，我们通过转染体外合成的（+）RNA或（-）RNA成功拯救出了病毒。这种改进的病毒拯救似乎是通过以最佳比例提供支持质粒以及用特异性抑制剂将痘苗病毒的细胞病变效应降至最低来实现的。此外，至关重要的可能是我们构建的cDNA在5'端不添加T7启动子特异性核苷酸就能产生病毒真实RNA。通过在病毒融合糖蛋白的切割激活位点产生预定的减毒突变，创建了一种候选疫苗株，证明了该系统的直接应用。
我们已经建立了能极大提高从cDNA中拯救仙台病毒效率的方法。从（-）RNA模板拯救病毒基本上不存在绝对障碍。即使是裸露形式的完整全长RNA链似乎也能被正确包装成为功能性模板。
