Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement.
Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement.
复制标题
开发适用于分析病毒复制和细胞间运动的水稻条纹细病毒微型复制子反向遗传学系统
DOI：
10.3389/fmicb.2021.655256
复制
发表时间：
2021
影响因子：
5.2
通讯作者：
Li Z
中科院分区：
文献类型：
--
作者：
Zhang X;Sun K;Liang Y;Wang S;Wu K;Li Z
水稻条纹病毒是一种细小病毒，具有4个负义/双链基因组片段，是影响亚洲许多国家水稻生产的最具破坏性的病毒病原之一。尽管进行了广泛的研究，但由于缺乏反向遗传学工具，我们对RSV感染周期和发病机制的了解已经严重受损。在这项研究中，我们设计了RSV微型复制子(MR)/微型基因组盒，用报告基因取代了负义RNA1或双链RNA2-4片段中的病毒开放阅读框。农业渗入烟草叶片后，只有当密码子优化的大病毒核糖核酸聚合酶蛋白(L)与核衣壳蛋白(N)共表达时，才能检测到MR报告基因的表达。MR活性也严重依赖于RNA沉默的共表达病毒抑制子，但RSV编码的NS3沉默抑制子的异位表达显著降低了报告基因的表达。我们还开发了具有细胞间运动能力的MR系统，运动蛋白在基于RNA4的MR顺式或反式双质粒中表达。最后，我们通过直接从互补正义RNA1和RNA3衍生物中表达N和L蛋白来构建多组分复制子系统，这增强了报告基因的表达，允许自主复制和细胞间移动，并减少了递送所需的质粒数量。总之，这项工作使RSV复制、转录和细胞间运动的反向遗传学分析成为可能，并为设计更复杂的重组系统提供了一个平台。
Rice stripe virus (RSV), a tenuivirus with four negative-sense/ambisense genome segments, is one of the most devastating viral pathogens affecting rice production in many Asian countries. Despite extensive research, our understanding of RSV infection cycles and pathogenesis has been severely impaired by the lack of reverse genetics tools. In this study, we have engineered RSV minireplicon (MR)/minigenome cassettes with reporter genes substituted for the viral open reading frames in the negative-sense RNA1 or the ambisense RNA2-4 segments. After delivery to Nicotiana benthamiana leaves via agroinfiltration, MR reporter gene expression was detected only when the codon-optimized large viral RNA polymerase protein (L) was coexpressed with the nucleocapsid (N) protein. MR activity was also critically dependent on the coexpressed viral suppressors of RNA silencing, but ectopic expression of the RSV-encoded NS3 silencing suppressor drastically decreased reporter gene expression. We also developed intercellular movement-competent MR systems with the movement protein expressed either in cis from an RNA4-based MR or in trans from a binary plasmid. Finally, we generated multicomponent replicon systems by expressing the N and L proteins directly from complementary-sense RNA1 and RNA3 derivatives, which enhanced reporter gene expression, permitted autonomous replication and intercellular movement, and reduced the number of plasmids required for delivery. In summary, this work enables reverse genetics analyses of RSV replication, transcription, and cell-to-cell movement and provides a platform for engineering more complex recombinant systems.
登录
查看更多内容
DOI：
10.5423/ppj.rw.10.2012.0158
发表时间：
2013-09
期刊：
The plant pathology journal
影响因子：
--
作者：
Cho WK;Lian S;Kim SM;Park SH;Kim KH
通讯作者：
Kim KH
影响因子：
5.4
作者：
Blakqori, G;Weber, F
通讯作者：
Weber, F
影响因子：
3.8
作者：
HAMAMATSU, C;TORIYAMA, S;ISHIHAMA, A
通讯作者：
ISHIHAMA, A
影响因子：
3.8
作者：
Blakqori, G;Kochs, G;Weber, F
通讯作者：
Weber, F
拯救植物细胞杆状病毒作为飞虱和谷物基因组研究的多功能表达平台
影响因子：
9.4
作者：
Gao, Qiang;Xu, Wen-Ya;Wang, Xian-Bing
通讯作者：
Wang, Xian-Bing