Leastro Mikhail Oliveira, Kitajima Elliot Watanabe, Pallas Vicente, Sánchez-Navarro Jesús A
Department of Stress Biology, Institute of Molecular and Cellular Biology of Plants, CSIC- Universitat Politècnica de València, Valencia, Spain.
Department of Phytopathology and Nematology, University of Sao Paulo, Luiz de Queiroz College of Agriculture, Piracicaba, Brazil.
PLoS Pathog. 2025 Aug 1;21(8):e1013388. doi: 10.1371/journal.ppat.1013388. eCollection 2025 Aug.
Kitaviridae, a newly recognized virus family, includes plant viruses infecting crops of great global importance, notably citrus. Despite its significant impact on citrus agricultural production, the molecular mechanisms underlying kitavirus infections remain largely unknown. Here, we engineered a recombinant citrus leprosis virus C (CiLV-C, genus Cilevirus) expressing green fluorescent protein (GFP) and demonstrated its feasibility for studying the biology of cilevirus. Genetic manipulation of rCiLV-C-GFP revealed that vRNA1 is essential for replication and can self-replicate independently, while vRNA2 is crucial for movement. The intergenic region between the polymerase and capsid protein (CP) acts as a promoter for CP gene expression. Frameshift and deletion analyses provided key insights into replication, movement, and morphogenesis. We reported that CP is critical for viral RNA accumulation, while movement protein (p32) facilitates viral spread. The putative glycoprotein (p61) is not structurally essential, as its deletion did not affect virion assembly, whereas the putative matrix protein (p24) is critical for morphogenesis, likely acting as a structural protein. Deletion of the RNA silencing suppressor (RSS, p15) and p15-p61 attenuated symptoms, implicating them as virulence factors. Additional analyses revealed that CP enhances vRNA accumulation through a mechanism independent of RSS. CP exhibits RNA-binding properties and interacts with eukaryotic initiation factor 4A (eIF4A), suggesting a role in translation. Overexpression of eIF4A increased CiLV-C RNA accumulation, while eIF4A knockdown reduced it, indicating that CP may recruit eIF4A to promote replication. Similar results were observed with turnip crinkle virus (TCV), and notably, the TCV CP efficiently restored RNA accumulation of a CP-defective CiLV-C, suggesting the existence of a conserved, CP-dependent, replication-related mechanism shared across distinct virus families. Our findings support the proposal of an initial model that elucidates the mechanism through which the CPs drive the production of high levels of vRNA manipulating host eIFs.
奇塔病毒科是一个新确认的病毒科，包括感染对全球具有重要意义的作物（尤其是柑橘）的植物病毒。尽管其对柑橘农业生产有重大影响，但奇塔病毒感染的分子机制在很大程度上仍不清楚。在此，我们构建了一种表达绿色荧光蛋白（GFP）的重组柑橘麻风病毒C（CiLV-C，西病毒属），并证明了其在研究西病毒生物学方面的可行性。对重组CiLV-C-GFP进行基因操作表明，病毒RNA1（vRNA1）对复制至关重要且能独立自我复制，而vRNA2对病毒移动至关重要。聚合酶与衣壳蛋白（CP）之间的基因间隔区作为CP基因表达的启动子。移码和缺失分析为复制、移动和形态发生提供了关键见解。我们报道CP对病毒RNA积累至关重要，而移动蛋白（p32）促进病毒传播。推测的糖蛋白（p61）在结构上并非必需，因为其缺失不影响病毒粒子组装，而推测的基质蛋白（p24）对形态发生至关重要，可能作为一种结构蛋白发挥作用。RNA沉默抑制因子（RSS，p15）和p15-p61的缺失减轻了症状，表明它们是毒力因子。进一步分析表明，CP通过一种独立于RSS的机制增强vRNA积累。CP具有RNA结合特性并与真核起始因子4A（eIF4A）相互作用，表明其在翻译中发挥作用。eIF4A的过表达增加了CiLV-C RNA积累，而eIF4A的敲低则降低了积累，这表明CP可能招募eIF4A来促进复制。在芜菁皱缩病毒（TCV）中也观察到了类似结果，值得注意的是，TCV CP有效地恢复了CP缺陷型CiLV-C的RNA积累，这表明在不同病毒科之间存在一种保守的、依赖CP的、与复制相关的机制。我们的研究结果支持了一个初始模型的提议，该模型阐明了CP驱动高水平vRNA产生并操纵宿主eIFs的机制。
