Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: A functional role for viral replicase in RNA packaging
Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: A functional role for viral replicase in RNA packaging
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DOI：
10.1016/j.virol.2005.05.013
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发表时间：
2005-07-20
期刊：
影响因子：
3.7
通讯作者：
Rao, ALN
中科院分区：
文献类型：
--
作者：
Annamalai, P;Rao, ALN
为了开始阐明雀麦花叶病毒（BMV）复制和侵染之间的关系，我们使用基于T-DNA的农杆菌介导的瞬时表达（农杆菌渗入）系统在本氏烟草叶片中表达三种基因组RNA中的单个或所需对。分析了这些RNA进入由瞬时表达的外壳蛋白（CP）形成的病毒体的包装能力。我们发现，在没有功能性复制酶的情况下，组装的病毒体包含非复制病毒RNA（RNA 1或RNA 2或RNA 3或RNA 1 + RNA 3或RNA 2 + RNA 3）以及细胞RNA。相比之下，在功能性复制酶存在下组装的病毒体仅含有病毒RNA。为了进一步阐明功能性病毒复制酶在RNA包装中表现出的特异性，通过删除3'tRNA样结构（3' TLS）构建复制缺陷型RNAi和RNA 2。无TLS的RNAi和RNA 2与wt RNA 3的共表达导致亚基因组RNA 4的有效合成。从共表达无TLS的RNAi和RNA 2以及CP mRNA或wt RNA 3的叶回收的病毒体仅含有病毒RNA。这些结果表明BMV基因组RNA的包装不是复制依赖性的，而功能性病毒复制酶的表达在增加RNA包装的特异性中起着积极的作用。(C)2005年爱思唯尔公司All rights reserved.
To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 orRNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNAI and RNA2 were constructed by deleting the 3' tRNA-like structure (3'TLS). Co-expression of TLS-less RNAI and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNAI and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging. (C) 2005 Elsevier Inc. All rights reserved.