Oligomerization of Uukuniemi virus nucleocapsid protein.
Oligomerization of Uukuniemi virus nucleocapsid protein.
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DOI：
10.1186/1743-422x-7-187
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发表时间：
2010-08-10
期刊：
影响因子：
4.8
通讯作者：
Plyusnin A
中科院分区：
文献类型：
--
作者：
Katz A;Freiberg AN;Backström V;Schulz AR;Mateos A;Holm L;Pettersson RF;Vaheri A;Flick R;Plyusnin A
Uukuniemi病毒（UUKV）属于布尼亚病毒科（Bunyaviridae）中的静脉病毒属。作为人类的非致病性病毒，UUKV在许多研究中作为安全的布尼亚病毒模型，解决了病毒基因的组织和调控、基因组复制、结构和组装等基本问题。本研究的重点是UUKV核衣壳（N）蛋白的寡聚化，它在病毒复制的几个步骤中起着重要作用。目的是定位参与N蛋白寡聚化的结构域，并详细研究该过程。进行了一系列集中在蛋白质N末端和C末端的实验，首先完全或部分删除推定的N-N相互作用结构域，然后引入氨基酸残基的点突变。突变策略基于N蛋白二级和三级结构的计算机建模。在化学交联，免疫荧光，哺乳动物双杂交，微型基因组，和病毒样颗粒形成试验的N蛋白突变体进行了研究。数据表明，UUKV-N蛋白的寡聚化能力取决于N蛋白分子两端是否存在完整的α螺旋，并且N末端区域的特定结构在N-N相互作用中起着至关重要的作用。该结构由两个α-螺旋形成，富含具有芳香族（W7、F10、W19、F27、F31）或长脂肪族（I14、I24）侧链的氨基酸残基。此外，在哺乳动物双杂交和微型基因组测定中，一些N-末端突变（例如I14 A、I24 A、F31 A）影响N蛋白的功能。UUKV-N蛋白在化学交联和哺乳动物双杂交试验中具有形成寡聚体的能力。在突变分析中，一些引入的单点突变在哺乳动物双杂交和微型基因组测定中都消除了N蛋白的功能，这表明特别是UUKV-N蛋白的N-末端区域对于N-N相互作用是必需的。
Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact α-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two α-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays. UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.