Formation of large viroplasms and virulence of <i>Cauliflower mosaic virus</i> in turnip plants depend on the N-terminal EKI sequence of viral protein TAV

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11–13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.花椰菜花叶病毒（Cauliflower mosaic virus, CaMV）的TAV蛋白（TransActivator/Viroplasmin，反式激活因子/病毒基质蛋白）在病毒侵染循环中发挥关键作用：它可激活病毒多顺反子RNA的翻译重新起始过程，并抑制RNA沉默。同时，该蛋白也是被称为病毒基质的细胞质电子致密包涵体（electron-dense inclusion bodies, EDIBs）的主要组成成分，此类包涵体在致病力强的CaMV Cabb B-JI毒株侵染的细胞中尤为显著。这类EDIBs被认为是病毒粒子工厂、CaMV胞内运动的载体以及蚜虫传毒的储存库。本研究针对体内的不同TAV突变体开展实验，证实三个物理分隔的结构域共同参与大型EDIBs的形成：N端EKI基序、参与翻译重新起始的MAV结构域序列，以及包含锌指结构的C端区域。令人意外的是，TAVm3突变体——即将第11-13位氨基酸的EKI基序替换为三个丙氨酸（AAA）——虽完全阻断了大型病毒基质的形成，但并未对CaMV造成致死性影响，仅大幅降低了其致病力，且未影响系统侵染速率。在病毒感染环境中表达TAVm3，会导致形成小型不规则形状的包涵体，引发轻度症状，并降低病毒DNA与粒子的积累水平，尽管此时可产生大量成熟衣壳蛋白。出乎意料的是，对于CaMV-TAVm3而言，对CaMV蚜虫传毒至关重要的含P2电子低密度包涵体（electron-light inclusion body, ELIB）的形成也受到破坏，这提示EKI三肽在CaMV植株间传播中存在间接作用。EKI基序在CaMV生物学中的这一重要功能贡献，可解释为何所有CaMV毒株的TAV序列中该基序均处于严格保守状态。