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Studies on the mechanism of nuclear-cytoplasmic transport -From the studies of the influenza virus assembly

核质转运机制研究——从流感病毒组装研究开始

基本信息

批准号：
05680589
负责人：
FIKUDA Ryuji
金额：
$ 1.34万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680589/
关键词：
Nuclear-cytoplasmic Transport Influenza Virus Nuclear Pore M1 Protein NA Protein Virus Assembly Reverse Genetics ウイルス粒子形成 Ml蛋白質 NSl蛋白質

项目摘要

We have realized that the studies of the influenza virus growth provide several interesting problems concerning nuclear-cytoplasmic transport. In the early stage of virus assembly, the virus genomic RNA produced in the nucleus immediately with virus core proteins, and the ribonucleoprotein complex (vRNP) thus formed is transported through the nuclear pore into the cytosol, and then into budding virus particles on the plasma membrane. In the first part of this study, nuclear-cytoplasmic transport of vRNPs was studied by analysing the distribution of vRNAs employing an in situ hybridization technique. The analyzes were performed using wild-type virus as well as a ts mutant virus, ts-51, which harbors a mutation in the segment 7, and has a defect in the late phase of virus growth. Nucleotide sequence analysis revealed a single amino acid change in the M1 protein. In the ts-51 virus-infected cells at a nonpermissive temperature, more than 95% of the vRNAs and the M1 protein remained in the nucleus, even at 6 hrpi and thereafter, when about 50% of them moved to the cytoplasm for the wild-type virus.These observations indicated that the M1 protein participated in the nuclear-cytoplasmic transport of the vRNAs. One hypothesis was that the M1 was associated with vRNPs in the nucleus forming possible M1-vRNP complexes, which were then transported into the cytosol. In the second part of this study, the virus assembly process in plasma membrane was investigated on the significance of the conserved sequence of the NA protein (its cytoplasmic domain and a successive sequence of the transmembrane domain) by the reverse genetic technique. It was indicated that both successive regions playd important roles in the formation of the infective virus particles.

我们已经认识到，流感病毒生长的研究提供了几个有趣的问题，有关核质运输。在病毒装配的早期阶段，在细胞核中产生的病毒基因组RNA立即与病毒核心蛋白结合，由此形成的核糖核蛋白复合物（vRNP）通过核孔转运到细胞质中，然后进入质膜上的出芽病毒颗粒。在本研究的第一部分，核质运输的vRNP进行了研究，采用原位杂交技术分析的vRNA的分布。使用野生型病毒以及ts突变病毒ts-51进行分析，ts-51在片段7中具有突变，并且在病毒生长的后期具有缺陷。核苷酸序列分析显示M1蛋白中的单个氨基酸变化。在非允许温度下，在ts-51病毒感染的细胞中，超过95%的vRNA和M1蛋白仍然留在细胞核中，即使在6 hrpi和之后，当野生型病毒的约50%的vRNA移动到细胞质时，这些观察表明M1蛋白参与了vRNA的核质转运。一种假设是M1与细胞核中的vRNP结合，形成可能的M1-vRNP复合物，然后转运到胞质溶胶中。在本研究的第二部分中，通过反向遗传技术研究了NA蛋白的保守序列（其胞质结构域和跨膜结构域的连续序列）在质膜中的组装过程中的意义。这表明两个连续区域在感染性病毒颗粒的形成中起重要作用。