Zhang Tong, Xu Xiongbiao, Huang Changjun, Qian Yajuan, Li Zhenghe, Zhou Xueping
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, People's Republic of China.
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, People's Republic of China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China
J Virol. 2015 Dec 9;90(4):2077-89. doi: 10.1128/JVI.02290-15. Print 2016 Feb 15.
Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution.
Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses.
植物双生病毒单链基因组的滚环复制是由病毒复制相关蛋白（Rep）在复制起点与同源基因组的序列特异性DNA结合启动的。单组分菜豆金色花叶病毒属相关的β卫星可以被同源和一些非同源辅助病毒反式复制，但β卫星复制混杂性的分子基础仍未明确。早期研究表明，当中国番茄黄化曲叶病毒（TYLCCNV）或烟草曲茎病毒（TbCSV）与同源和非同源β卫星共同接种时，同源β卫星在感染后期比非同源β卫星占优势。在本研究中，我们构建了中国番茄黄化曲叶β卫星和烟草曲茎β卫星之间的相互嵌合β卫星，并在与TYLCCNV或TbCSV共同接种到植物上时，检测它们与野生型β卫星的竞争能力。我们绘制了β卫星起源保守滚环十字形结构上游的一个区域，该区域赋予同源Rep介导的相对于非同源卫星的复制优势。DNase I保护和体外结合试验进一步鉴定了一个称为Rep结合基序（RBM）的新序列元件，它特异性结合同源Rep蛋白，也能结合非同源Rep，尽管亲和力较低。此外，我们表明RBM-Rep结合亲和力与原生质体中β卫星的复制效率相关。我们的数据表明，尽管不存在Rep介导复制的严格特异性，但β卫星在共同进化过程中已适应其同源Rep以实现高效复制。
菜豆金色花叶病毒属是众多环状DNA病毒，在全球范围内导致作物毁灭性病害。单组分菜豆金色花叶病毒属病毒经常与β卫星相关联，β卫星对于诱导典型病害症状至关重要。两种不同的β卫星与一种辅助病毒共存在自然界中很少见。我们之前的研究表明，菜豆金色花叶病毒属病毒反式复制同源β卫星的水平高于非同源β卫星。然而，β卫星选择性复制的分子机制在很大程度上仍然未知。我们研究了菜豆金色花叶病毒属病毒复制相关蛋白与β卫星DNA之间的相互作用。我们发现复制相关蛋白特异性结合β卫星中的一个基序，对同源基序的亲和力高于非同源基序。这种对同源基序结合的偏好决定了β卫星的选择性复制。我们还证明了这个基序对于β卫星复制至关重要。这些发现为辅助双生病毒对β卫星混杂但选择性的复制提供了新的见解。
