The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein.
The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein.
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DOI：
10.1371/journal.ppat.1003624
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发表时间：
2013-09
期刊：
影响因子：
6.7
通讯作者：
Tao YJ
中科院分区：
文献类型：
--
作者：
Zheng W;Olson J;Vakharia V;Tao YJ
具有分段基因组的病毒的基因组包装通常是一个复杂的问题。这对于流感病毒和其他正粘病毒尤其如此，其基因组由多个反义RNA组成，这些反义RNA以核糖核蛋白（RNP）复合物的形式被取代。为了更好地了解正粘病毒RNP的结构特征，使它们被包装，我们确定了鱼正粘病毒，传染性鲑鱼贫血病毒（ISAV）（Isavirus属）的核蛋白（NP）的晶体结构。ISAV-NP作为RNP的主要蛋白组分，具有类似于流感病毒NP的双叶结构。由于无RNA和RNA结合的ISAV NP在溶液中形成稳定的二聚体，我们能够使用重组蛋白和合成寡核苷酸测量NP RNA结合亲和力以及化学计量。我们的RNA结合分析显示，每个ISAV-NP结合1012 nt的RNA，比最初基于群体平均值对甲型流感病毒NP估计的24-28 nt短。通过电子显微镜和动态光散射的结果进一步证实了12-nt的化学计量。考虑到ISAV和流感病毒的RNP具有相似的形态和尺寸，我们的研究结果表明，无NP的RNA可能存在于正粘病毒RNP上，并且选择性RNP包装可能通过直接RNA-RNA相互作用来完成。正粘病毒是RNA病毒家族，包括各种类型的流感病毒。正粘病毒的基因组由多个负义单链RNA分子片段组成，每个片段以杆状双螺旋核糖核蛋白（RNP）复合物的形式包装。不同的RNP如何相互作用以确保特定的基因组包装是一个长期存在的问题，对于我们理解正粘病毒复制和流感病毒基因重排至关重要。我们的研究鱼类正粘病毒，传染性鲑鱼贫血病毒（ISAV），表明其核蛋白（NP），它形成的病毒RNP的蛋白骨架骨架，具有双叶结构的流感病毒NP。由于ISAV-NP在溶液中形成稳定的二聚体，我们能够通过生物化学测定、电子显微镜和动态光散射来确定ISAV-NP RNA结合的化学计量。我们的研究结果表明，每个ISAV-NP结合12-nt RNA，比最初基于群体平均值估计的甲型流感病毒的24-28 nt短。我们建议，NP-游离RNA存在于正粘病毒RNP上，并且这样的RNA区域可能在基因组包装期间介导特定的RNP-RNP相互作用。进一步阐明RNA介导的RNP-RNP相互作用将有助于我们确定正粘病毒（包括流感病毒）基因重排的分子基础。
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. Orthomyxoviruses are a family of RNA viruses that include the various types of influenza viruses. The genome of orthomyxoviruses consists of multiple segments of negative-sense, single-stranded RNA molecules, each packaged in the form of rod-shaped, double-helical ribonucleoprotein (RNP) complexes. How different RNPs interact with each other to ensure specific genome packaging is a long-standing question and crucial to our understanding of orthomyxovirus replication and influenza virus gene reassortment. Our study of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV), shows that its nucleoprotein (NP), which forms the protein scaffold backbone of the viral RNP, has a bi-lobular structure like the influenza virus NP. Because ISAV-NP forms stable dimers in solution, we were able to determine ISAV-NP RNA binding stoichiometry by biochemical assays, electron microscopy and dynamic light scattering. Our results indicate that each ISAV-NP binds ∼12-nt RNA, shorter than the 24–28 nts originally estimated for the influenza A virus based on population average. We propose that NP-free RNA exists on orthomyxovirus RNPs, and such RNA regions likely mediate specific RNP-RNP interactions during genome packaging. Further elucidation of the RNA-mediated RNP-RNP interactions will help us determine the molecular basis of gene reassortment by orthomyxoviruses including the influenza viruses.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.4
作者：
COMPANS, RW;CONTENT, J;DUESBERG, PH
通讯作者：
DUESBERG, PH
影响因子：
5.4
作者：
Carter, Stephen D.;Surtees, Rebecca;Barr, John N.
通讯作者：
Barr, John N.
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
6.7
作者：
Chenavas S;Estrozi LF;Slama-Schwok A;Delmas B;Di Primo C;Baudin F;Li X;Crépin T;Ruigrok RW
通讯作者：
Ruigrok RW