CELLULAR EXPRESSION OF A FUNCTIONAL NODAVIRUS RNA REPLICON FROM VACCINIA VIRUS VECTORS
CELLULAR EXPRESSION OF A FUNCTIONAL NODAVIRUS RNA REPLICON FROM VACCINIA VIRUS VECTORS
复制标题
DOI：
10.1128/jvi.66.4.2335-2345.1992
复制
发表时间：
1992-04-01
影响因子：
5.4
通讯作者：
BALL, LA
中科院分区：
文献类型：
--
作者：
BALL, LA
RNA复制为RNA的扩增提供了一种强有力的手段，但迄今为止，它仅在RNA病毒中自然发生。在利用该过程扩增异源mRNA的尝试中，使用牛痘病毒-噬菌体T7 RNA聚合酶载体以功能形式表达了RNA复制酶及其相应的RNA模板。构建了质粒，其按5 '至3'顺序包含（i）噬菌体T7启动子;（ii）编码鸡舍病毒（FHV）的RNA复制酶（RNA 1）或外壳蛋白（RNA 2）的全长cDNA，（iii）编码卫星烟草环斑病毒的自切割核酶的cDNA序列，和（iv）T7转录终止子。在体外和体内，这种结构的环状质粒被T7 RNA聚合酶转录，产生大小与两种真正的FHV基因组RNA RNA RNA 1和RNA 2非常相似的RNA。在表达真实FHV RNA复制酶的幼仓鼠肾细胞中，RNA 2（外壳蛋白）转录物被准确复制。此外，RNA 1（复制酶）转录本指导合成的酶，不仅可以复制真正的病毒体衍生的FHV RNA，而且质粒衍生的转录本本身。在后一种条件下，RNA转录物的复制扩增随之发生，并导致编码蛋白质的高合成率。这种从DNA载体成功表达RNA复制的复杂生物学过程将极大地促进其机制的研究，并且是朝向利用RNA复制进行mRNA扩增的目标迈出的重要一步。
RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.