Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: Self-association analysis and nucleic acid binding characterization
Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: Self-association analysis and nucleic acid binding characterization
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DOI：
10.1021/bi0609319
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发表时间：
2006-10-03
期刊：
影响因子：
2.9
通讯作者：
Jiang, Hualiang
中科院分区：
文献类型：
--
作者：
Luo, Haibin;Chen, Jing;Jiang, Hualiang
冠状病毒核衣壳(N)蛋白在病毒粒子组装过程中包裹基因组RNA形成长螺旋核衣壳。由于N蛋白齐聚通常是这一过程中的关键步骤，对这种齐聚反应的表征将有助于理解核衣壳形成的可能机制。严重急性呼吸综合征冠状病毒(SARS-CoV)的N蛋白是最近被发现通过其羧基末端自结合的。在本研究中，为了进一步详细了解该C末端的缔合特征，我们用尺寸排斥层析和化学交联方法系统地研究了它的齐聚作用。我们的结果清楚地表明，SARS-CoV N蛋白的C末端结构域不仅可以形成二聚体，还可以形成三聚体、四聚体和六聚体。对6个缺失突变体的进一步分析表明，残基343-402是该C末端齐聚的必要条件和充分条件。虽然该片段含有许多带电残基，但离子强度的差异对其齐聚没有影响，表明SARS冠状病毒N蛋白C末端的自结合过程中没有静电作用力。凝胶漂移分析结果表明，SARS-CoV N蛋白C末端也能与核酸结合，363-382残基是主要的相互作用伙伴，表明该片段可能与基因组RNA结合位点有关。核酸结合可促进SARS-CoV N蛋白C末端形成高阶低聚物，提示SARS-CoV N蛋白可能与病毒基因组RNA结合，触发长核衣壳的形成。
Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.