Alminaite Agne, Backström Vera, Vaheri Antti, Plyusnin Alexander
Department of Virology, Haartman Institute, PO Box 21, FIN-00014 University of Helsinki, Finland.
Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland.
J Gen Virol. 2008 Sep;89(Pt 9):2167-2174. doi: 10.1099/vir.0.2008/004044-0.
The nucleocapsid (N) protein of hantaviruses (family Bunyaviridae) is the most abundant component of the virion; it encapsidates genomic RNA segments and participates in viral genome transcription and replication, as well as in virus assembly. During RNA encapsidation, the N protein forms intermediate trimers and then oligomers via 'head-to-head, tail-to-tail' interactions. In previous work, using Tula hantavirus (TULV) N protein as a model, it was demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein and that the hydrophobic 'a' residues from the second alpha-helix are especially important. Here, the importance of charged amino acid residues located within the coiled-coil for trimer formation and oligomerization was analysed. To predict the interacting surfaces of the monomers, the previous in silico model of TULV coiled-coils was first upgraded, taking advantage of the recently published crystal structure of the N-terminal coiled-coil of the Sin Nombre virus N protein. The results obtained using a mammalian two-hybrid assay suggested that conserved, charged amino acid residues within the coiled-coil make a substantial contribution to N protein oligomerization. This contribution probably involves (i) the formation of interacting surfaces of the N monomers (residues D35 and D38, located at the tip of the coiled-coil loop, and R63 appear particularly important) and (ii) stabilization of the coiled-coil via intramolecular ionic bridging (with E55 as a key player). It is hypothesized that the tips of the coiled-coils are the first to come into direct contact and thus to initiate tight packing of the three structures.
汉坦病毒（布尼亚病毒科）的核衣壳（N）蛋白是病毒粒子中含量最丰富的成分；它包裹基因组RNA片段，参与病毒基因组的转录和复制，以及病毒组装。在RNA包裹过程中，N蛋白形成中间三聚体，然后通过“头对头、尾对尾”相互作用形成寡聚体。在之前的研究中，以图拉汉坦病毒（TULV）N蛋白为模型，证明N末端完整的卷曲螺旋结构对于N蛋白的寡聚化能力至关重要，并且来自第二个α螺旋的疏水“a”残基尤为重要。在此，分析了位于卷曲螺旋内的带电荷氨基酸残基对三聚体形成和寡聚化的重要性。为了预测单体的相互作用表面，首先利用最近公布的辛诺柏病毒N蛋白N末端卷曲螺旋的晶体结构，对之前的TULV卷曲螺旋计算机模拟模型进行了升级。使用哺乳动物双杂交试验获得的结果表明，卷曲螺旋内保守的带电荷氨基酸残基对N蛋白寡聚化有重大贡献。这种贡献可能涉及（i）N单体相互作用表面的形成（位于卷曲螺旋环末端的残基D35和D38以及R63显得尤为重要）和（ii）通过分子内离子桥接稳定卷曲螺旋（E55起关键作用）。据推测，卷曲螺旋的末端首先直接接触，从而启动三个结构的紧密堆积。
