The multifunctional protein CI of potyviruses plays interlinked and distinct roles in viral genome replication and intercellular movement.
The multifunctional protein CI of potyviruses plays interlinked and distinct roles in viral genome replication and intercellular movement.
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马铃薯病毒属的多功能蛋白CI在病毒基因组复制和细胞间运动中发挥着相互关联且独特的作用
DOI：
10.1186/s12985-015-0369-2
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发表时间：
2015-09-15
期刊：
影响因子：
4.8
通讯作者：
Wang A
中科院分区：
文献类型：
--
作者：
Deng P;Wu Z;Wang A
多病毒的多功能圆柱包涵体（CI）蛋白具有ATP结合和RNA解旋酶活性。作为病毒复制复合体的一部分，它可能通过与RNA结合并解开RNA双链来协助病毒基因组复制。它还在病毒细胞间运动中发挥作用，可能通过在胞间连丝（PD）上形成锥形结构以及与外壳蛋白（CP）的相互作用。方法为了进一步了解CI在病毒感染过程中的作用，我们采用丙氨酸扫描诱变方法，对绿色荧光蛋白标记的芜菁花叶病毒（TuMV）感染性全长cDNA克隆进行CI突变。在聚集的带电残基上共进行了40次双取代。这些突变对病毒基因组扩增的影响是用原生质体接种试验确定的。所有突变体也被引入到底栖植物中，以评估它们的细胞间和长距离运动。随机选择三个细胞间运动消除的突变体，通过共聚焦显微镜确定其突变的CI蛋白是否靶向PD并与CP相互作用。结果20个CI突变体存在复制缺陷（5个消除，15个减少），1个产生比亲本病毒高的病毒基因组水平，其余19个保持与亲本病毒相同的复制水平。复制缺陷突变主要位于解旋酶结构域和c端区。所有15个复制减少突变体都表现出延迟或取消细胞间运动。20个复制能力突变体中有9个在单个细胞内含有感染。其中5个突变分布在n端100个氨基酸中。大多数复制缺陷或细胞间运动消除的突变体不能系统地感染植物。对随机选择的三个具有复制能力但细胞间运动被破坏的突变体的分析表明，突变的CI在PD位点不能形成规则的点状结构，也不能与cp相互作用。结论：TuMV CI的解旋酶结构域和c端区域对病毒基因组复制至关重要，而n端序列调节病毒的细胞间运动。TuMV - CI在复制和细胞间运动中起着相互联系和不同的作用。CI靶向PD并与CP相互作用的能力与其在病毒细胞间运动中的功能作用有关。
BackgroundThe multifunctional cylindrical inclusion (CI) protein of potyviruses contains ATP binding and RNA helicase activities. As part of the viral replication complex, it assists viral genome replication, possibly by binding to RNA and unwinding the RNA duplex. It also functions in viral cell-to-cell movement, likely via the formation of conical structures at plasmodesmata (PD) and the interaction with coat protein (CP).MethodsTo further understand the role of CI in the viral infection process, we employed the alanine-scanning mutagenesis approach to mutate CI in the infectious full-length cDNA clone ofTurnip mosaic virus(TuMV) tagged by green fluorescent protein. A total of 40 double-substitutions were made at the clustered charged residues. The effect of these mutations on viral genome amplification was determined using a protoplast inoculation assay. All the mutants were also introduced intoNicotiana benthamianaplants to assess their cell-to-cell and long-distance movement. Three cell-to-cell movement-abolished mutants were randomly selected to determine if their mutated CI protein targets PD and interacts with CP by confocal microscopy.ResultsTwenty CI mutants were replication-defective (5 abolished and 15 reduced), one produced an elevated level of viral genome in comparison with the parental virus, and the remaining 19 retained the same replication level as the parental virus. The replication-defective mutations were predominately located in the helicase domains and C-terminal region. All 15 replication-reduced mutants showed delayed or abolished cell-to-cell movement. Nine of 20 replication-competent mutants contained infection within single cells. Five of them distributed mutations within the N-terminal 100 amino acids. Most of replication-defective or cell-to-cell movement-abolished mutants failed to infect plants systemically. Analysis of three randomly selected replication-competent yet cell-to-cell movement-abolished mutants revealed that the mutated CI failed to form regular punctate structures at PD and/or to interact with CP.ConclusionsThe helicase domain and C-terminal region of TuMV CI are essential for viral genome replication, and the N-terminal sequence modulates viral cell-to-cell movement. TuMV CI plays both interlinked and distinct roles in replication and intercellular movement. The ability of CI to target PD and interact with CP is associated with its functional role in viral cell-to-cell movement.