Bredenbeek P J, Frolov I, Rice C M, Schlesinger S
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, Missouri 63110-1093.
J Virol. 1993 Nov;67(11):6439-46. doi: 10.1128/JVI.67.11.6439-6446.1993.
Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.
自从从全长cDNA克隆中获得感染性RNA转录本以来，甲病毒基因组RNA已被改造以允许表达异源RNA和蛋白质。当病毒结构基因被异源编码序列取代时，可实现异源产物的最高表达水平。这种重组RNA是自我复制的（复制子），可以作为裸RNA引入细胞，但它们需要反式互补才能作为感染性病毒粒子包装并从细胞中释放出来。在本报告中，我们描述了一系列有缺陷的辛德毕斯病毒辅助RNA，可用于包装辛德毕斯病毒RNA复制子。有缺陷的辅助RNA包含复制所需的顺式作用序列以及驱动结构蛋白基因表达的亚基因组RNA启动子。在同时转染了复制子和有缺陷的辅助RNA的细胞中，从复制子RNA翻译的病毒非结构蛋白允许有缺陷的辅助RNA进行复制和转录，以产生病毒粒子结构蛋白。比较了一系列有缺陷的辅助RNA包装复制子RNA的能力以及它们被复制和包装的能力。一种有缺陷的辅助RNA不仅包装了复制子，而且自身也被衣壳化，在需要大量扩增的条件下将很有用。其他有缺陷的辅助RNA能够有效地包装复制子，但它们自身的包装效率很低。这些辅助RNA在不需要病毒结构蛋白表达或病毒传播的应用中应该会很有用。
