Visualization of microtubule-mediated transport of influenza viral progeny ribonucleoprotein
Visualization of microtubule-mediated transport of influenza viral progeny ribonucleoprotein
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DOI：
10.1016/j.micinf.2007.07.007
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发表时间：
2007-10-01
影响因子：
5.8
通讯作者：
Morikawa, Yuko
中科院分区：
文献类型：
--
作者：
Momose, Fumitaka;Kikuchi, Yuji;Morikawa, Yuko
我们利用流感病毒A/波多黎各/8/34(PR8)株的核蛋白(NP)制备了一种独特的单抗mAb61A5。截短和丙氨酸替代实验表明，mAb61A5识别17-123个残基的NP片段，其中由β1片断和α1和α2螺旋之间的连接区形成构象表位。MAb61A5与A/Aichi/2/68或A/鸭子/宾夕法尼亚/10128/84株的NP反应较差的部分原因是抗原表位或其附近的变异。有趣的是，免疫沉淀分析表明mAb61A5优先与病毒核糖核蛋白复合体相互作用，该复合体由RNA聚合酶、正负向RNA和NP组成，而不是外源添加的NP。应用mAb61A5的免疫荧光显微镜显示，在感染后期，细胞质内有点状染色。点状NPs聚集在微管组织中心，与微管共定位。用软霉素B阻断依赖CRM1的核输出的治疗未能产生点状NP。微管解聚剂诺康唑处理后，核糖核酸在细胞质中呈随机分布。这些结果表明，尽管微管网络不是形成点状结构所必需的，但它负责点状NP抗原的极化分布，很可能是病毒后代核糖核蛋白复合体。(C)2007年爱思唯尔·马森公司。版权所有。
We developed a unique monoclonal antibody, mAb61A5, using the nucleoprotein (NP) of influenza virus A/Puerto Rico/8/34 (PR8) strain. Truncation and alanine substitution experiments showed that mAb61A5 recognized the NP fragment with residues 17 to 123 in which a conformational epitope formed by the beta 1 sheet and the linker region between the alpha 1 and alpha 2 helices. Variations in the epitope or nearby can partly account for the poor mAb61A5 reactivity with the NP of A/Aichi/2/68 or A/duck/Pennsylvania/ 10128/84 strains. Interestingly, immunoprecipitation analysis revealed that mAb61A5 preferentially interacted with viral ribonucleoprotein complexes, composed of RNA polymerase, negative/positive sense RNA and NP, rather than exogenously added NP. Immunofluorescence microscopy using mAb61A5 showed a punctate staining in the cytoplasm during the late phase of infection. The punctate NPs accumulated at the microtubule organizing center and co-localized with microtubules. The treatment with leptomycin B to block a CRM1-dependent nuclear export failed to produce the punctate NP. The treatment with nocodazole, a microtubule-depolymerizing agent, showed random distribution of the punctate NP in the cytoplasm. These results suggest that microtubule networks, although were not required for the formation of punctate structures, were responsible for the polarized distribution of the punctate NP antigens, most likely viral progeny ribonucleoprotein complexes. (c) 2007 Elsevier Masson SAS. All rights reserved.