Duarte Christiane Eliza Motta, Machado João Paulo Batista, Gouveia-Mageste Bianca, Silva Fredy Davi Albuquerque, Fontes Elizabeth Pacheco Batista
National Institute of Science and Technology in Plant-Pest Interactions/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
Glycobiology and Cell Signaling Laboratory, Universidade do Estado de Minas Gerais, Passos, MG, Brazil.
Methods Mol Biol. 2025;2912:205-226. doi: 10.1007/978-1-0716-4454-6_18.
In eukaryotic cells, the subcellular localization of proteins is inherently linked to their function. Since viruses rely on the host cellular machinery to complete their life cycle, viral proteins are expected to employ the host transport machinery to reach various compartments. Several factors, including the multifunctional nature of viral proteins, the stage of virus infection, and interactions with both viral and host proteins, influence the final destination of viral proteins. For instance, NSP (nuclear shuttle protein) from bipartite begomoviruses and CP (coat protein) from monopartite begomoviruses typically exhibit nuclear localization, yet their subcellular distribution can vary depending on coexpression partners and stage of infection. Virtually all viral proteins display dynamic subcellular distribution patterns that change under their specific functions at different stages of the virus life cycle. Thus, identifying the subcellular distribution of viral proteins is essential for comprehending their multiple roles during infection. This chapter outlines a protocol for efficiently determining the subcellular localization of viral proteins during infection or when expressed with protein partners. The protocol essentially consists of three steps: (i) cloning the viral protein and protein partners fused to fluorescent tags, (ii) transiently expressing the tagged proteins in N. benthamiana leaves, and (iii) determining the subcellular localization of the tagged proteins using confocal microscopy.
在真核细胞中，蛋白质的亚细胞定位与其功能有着内在联系。由于病毒依赖宿主细胞机制来完成其生命周期，因此病毒蛋白有望利用宿主转运机制抵达各个区室。包括病毒蛋白的多功能性质、病毒感染阶段以及与病毒和宿主蛋白的相互作用在内的多种因素，都会影响病毒蛋白的最终定位。例如，双生病毒的核穿梭蛋白（NSP）和单生病毒的外壳蛋白（CP）通常表现出核定位，但它们的亚细胞分布会因共表达伙伴和感染阶段的不同而有所变化。实际上，所有病毒蛋白都呈现出动态的亚细胞分布模式，这些模式会在病毒生命周期的不同阶段根据其特定功能而发生变化。因此，确定病毒蛋白的亚细胞分布对于理解它们在感染过程中的多种作用至关重要。本章概述了一种在感染期间或与蛋白伙伴共表达时高效确定病毒蛋白亚细胞定位的方法。该方法主要包括三个步骤：（i）克隆与荧光标签融合的病毒蛋白和蛋白伙伴；（ii）在本氏烟草叶片中瞬时表达带标签的蛋白；（iii）使用共聚焦显微镜确定带标签蛋白的亚细胞定位。
