Maio Francesca, Helderman Tieme A, Arroyo-Mateos Manuel, van der Wolf Miguel, Boeren Sjef, Prins Marcel, van den Burg Harrold A
Molecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, Netherlands.
Laboratory of Biochemistry, Wageningen University, Wageningen, Netherlands.
Front Plant Sci. 2020 Jul 15;11:1069. doi: 10.3389/fpls.2020.01069. eCollection 2020.
Geminiviruses are plant-infecting DNA viruses that reshape the intracellular environment of their host in order to create favorable conditions for viral replication and propagation. Viral manipulation is largely mediated interactions between viral and host proteins. Identification of this protein network helps us to understand how these viruses manipulate their host and therefore provides us potentially with novel leads for resistance against this class of pathogens, as genetic variation in the corresponding plant genes could subvert viral manipulation. Different studies have already yielded a list of host proteins that interact with one of the geminiviral proteins. Here, we use affinity purification followed by mass spectrometry (AP-MS) to further expand this list of interacting proteins, focusing on an important host (tomato) and the Replication initiator protein (Rep, AL1, C1) from (TYLCV). Rep is the only geminiviral protein proven to be essential for geminiviral replication and it forms an integral part of viral replisomes, a protein complex that consists of plant and viral proteins that allows for viral DNA replication. Using AP-MS, fifty-four 'high confidence' tomato proteins were identified that specifically co-purified with Rep. For two of them, an unknown EWS-like RNA-binding protein (called Geminivirus Rep interacting EWS-like protein 1 or GRIEP1) and an isoform of the THO complex subunit 4A (ALY1), we were able to confirm this interaction with Rep using a second method, bimolecular fluorescence complementation (BiFC). The THO subunit 4 is part of the THO/TREX (TRanscription-EXport) complex, which controls RNA splicing and nuclear export of mRNA to the cytoplasm and is also connected to plant disease resistance. This work represents the first step towards characterization of novel host factors with a putative role in the life cycle of TYLCV and possibly other geminiviruses.
双生病毒是感染植物的DNA病毒，它们重塑宿主的细胞内环境，以便为病毒复制和传播创造有利条件。病毒操纵在很大程度上是由病毒蛋白与宿主蛋白之间的相互作用介导的。识别这种蛋白质网络有助于我们了解这些病毒如何操纵它们的宿主，因此有可能为我们提供针对这类病原体的抗性新线索，因为相应植物基因的遗传变异可能会颠覆病毒操纵。不同的研究已经得出了与一种双生病毒蛋白相互作用的宿主蛋白列表。在这里，我们使用亲和纯化结合质谱分析（AP-MS）来进一步扩展这个相互作用蛋白列表，重点关注一种重要的宿主（番茄）和来自番茄黄化曲叶病毒（TYLCV）的复制起始蛋白（Rep，AL1，C1）。Rep是唯一被证明对双生病毒复制至关重要的双生病毒蛋白，它是病毒复制体的一个组成部分，病毒复制体是一种由植物和病毒蛋白组成的蛋白质复合物，允许病毒DNA复制。使用AP-MS，鉴定出了54种与Rep特异性共纯化的“高可信度”番茄蛋白。对于其中两种蛋白，一种未知的EWS样RNA结合蛋白（称为双生病毒Rep相互作用EWS样蛋白1或GRIEP1）和THO复合物亚基4A（ALY1）的一种异构体，我们能够使用另一种方法，即双分子荧光互补（BiFC），来证实它们与Rep的这种相互作用。THO亚基4是THO/TREX（转录-输出）复合物的一部分，该复合物控制RNA剪接以及mRNA从细胞核输出到细胞质，并且还与植物抗病性相关。这项工作代表了表征在TYLCV以及可能其他双生病毒的生命周期中具有假定作用的新型宿主因子的第一步。
