Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
J Virol. 2012 Oct;86(19):10547-57. doi: 10.1128/JVI.01201-12. Epub 2012 Jul 18.
Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5' RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5' γ-phosphate is a common feature of the negative-strand RNA [(-)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (-)RNAs (of group A rotaviruses) have a 5' GG dinucleotide sequence. As the only rotavirus protein with 5' RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (-)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (-)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5' consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5' GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.
轮状病毒非结构蛋白 NSP2 是一个功能性八聚体，对于形成富含病毒的空泡至关重要，富含病毒的空泡是双链 RNA（dsRNA）轮状病毒基因组复制和包装的特有场所。作为复制中间体的一个组成部分，NSP2 也与各种复制相关的活动有关。除了序列非依赖性的单链 RNA 结合和螺旋解链活性外，NSP2 还表现出单体相关的核苷和 5' RNA 三磷酸酶（NTPase/RTPase）活性，这些活性由狭窄酶切中的保守 H225 残基介导。缺乏 5' γ-磷酸是轮状病毒包装 dsRNA 片段中的负链 RNA [(-)RNA] 的一个共同特征。引人注目的是，所有 (-)RNAs（A 组轮状病毒）都有 5' GG 二核苷酸序列。作为唯一具有 5' RTPase 活性的轮状病毒蛋白，NSP2 参与从轮状病毒 (-)RNA 中去除 γ-磷酸。为了了解 NSP2 如何在不依赖于序列的 RNA 结合特性的情况下识别 (-)RNA 并在催化裂隙内水解 γ-磷酸，我们确定了 NSP2 与负链轮状病毒 RNA 5' 保守序列复合物的晶体结构。我们的研究表明，结合寡核苷酸的 5' GG 与 NSP2 酶切裂隙中高度保守的残基广泛相互作用。尽管这些残基提供了 GG 特异性相互作用，但表面等离子体共振研究表明，C 端螺旋和酶切裂隙外的其他碱性残基解释了 NSP2 对非序列依赖性 RNA 的结合。我们的研究中的一个新发现，可能对富含病毒的空泡形成具有影响，即 NSP2 的 C 端螺旋表现出两种不同的构象，并进行结构域交换相互作用，从而形成 NSP2 八聚体链。
