Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA.
J Virol. 2012 Jun;86(11):6210-21. doi: 10.1128/JVI.07184-11. Epub 2012 Mar 21.
Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.
基因组包装在人类病原体 RNA 病毒（脊髓灰质炎病毒）、昆虫（禽白细胞增生症病毒）和植物（雀麦花叶病毒）的复制中具有功能相关性。然而，其潜在机制尚未完全了解。我们之前观察到，在禽白细胞增生症病毒和雀麦花叶病毒中，与异位表达的衣壳蛋白（CP）不同，包装特异性是由从复制基因组 RNA 产生的 mRNA 翻译的 CP 进行 RNA 包裹导致的。因此，我们假设与复制酶的物理相互作用会增加 CP 对包装病毒 RNA 的特异性。我们通过使用禽白细胞增生症病毒-菸草系统来评估复制酶蛋白与 CP 之间的分子相互作用来检验这一假设。双分子荧光互补实验结合荧光细胞蛋白标记和共免疫沉淀实验表明，禽白细胞增生症病毒复制酶（蛋白 A）和 CP 在复制的线粒体部位发生物理相互作用，这种相互作用需要 CP 的 N 端起始 1 至 31 位氨基酸或 32 至 50 位氨基酸。与源自禽白细胞增生症病毒复制的 CP 的线粒体定位不同，异位表达在内质网（ER）上呈现出特征性的点状模式。当删除 CP 的 C 端疏水区时，这种模式被改变，CP 被重新定位到细胞质中。对 CP 突变体的包装表型分析，这些突变体要么在蛋白 A-CP 相互作用中存在缺陷，要么在 ER 定位中存在缺陷，表明蛋白 A-CP 相互作用与其亚细胞定位之间的同步对于赋予包装特异性是至关重要的。
