Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus
Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus
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DOI：
10.1128/jvi.75.10.4633-4640.2001
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发表时间：
2001-05-01
影响因子：
5.4
通讯作者：
Westaway, EG
中科院分区：
文献类型：
--
作者：
Khromykh, AA;Varnavski, AN;Westaway, EG
为了研究黄病毒RNA包装是否依赖于RNA复制，我们产生了两个基于DNA的昆津病毒构建体，pKUN 1和pKUN 1dGDD，允许通过细胞RNA聚合酶II的核转录分别连续生产复制型（野生型）和非复制型（缺失NS 5基因RNA聚合酶基序GDD）全长昆津病毒RNA。正如预期的那样，pKUN 1质粒DNA转染到BHK细胞中导致分泌的感染性Kunjin病毒粒子的回收。然而，将pKUN 1dGDD DNA转染到BHK细胞中，没有导致回收任何含有双核苷酸化dGDD RNA的分泌病毒颗粒，尽管通过北方印迹分析证明了这种RNA在细胞中的明显积累，并且通过检测正确加工的标记结构蛋白证明了其有效翻译（至少prM和E），使用抗E抗体的共免疫沉淀分析。相反，当在PKUN 1dGDD DNA转染的repBHK细胞中产生甚至更少量的dGDD RNA时（其中它通过互补复制），它被包装成分泌的病毒颗粒，因此，只有当它被复制时，缺陷的昆津病毒RNA才能被包装。我们用基因组长度的昆津病毒RNA的结果和用脊髓灰质炎病毒复制子RNA的结果（C，I。Nugent等，J，Virol，73：427-435，1999），两者都证明了RNA在包装之前复制的必要性，强烈表明存在一种共同的机制，用于使正链RNA病毒中准种中缺陷RNA的扩增和传递最小化。因此，这种机制可以帮助减轻RNA依赖性RNA聚合酶的高拷贝错误率。
In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in PKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles, Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C, I. Nugent et al,, J, Virol, 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses, This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.