FLU VIRUS RNP COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 甲型流感病毒NP的晶体结构最近在我们实验室被确定。NP在晶体中以三聚体的形式折叠成两个结构域，具有与横纹病毒NP完全不同的拓扑结构。由402至428残基组成的短尾环可能在NP寡聚中发挥重要作用，因为该区域的单个残基突变导致寡聚完全丧失。在两个结构域之间的界面上，在NP三聚体的外部发现了一个大的带正电的凹槽。外部RNA结合位点表明，与非片段RNA病毒不同，流感病毒RNPs中的RNA很可能暴露在RNA中。这种结构差异解释了以前的结果，即流感RNPs与VSV和狂犬病病毒等非节段(-)链RNA病毒的RNPs之间存在根本差异。例如，流感病毒RNPs中的病毒RNA被RNase消化，而副流感病毒和弹状病毒RNPs中的病毒RNA完全抵抗RNase消化。此外，聚乙烯硫酸盐(PVS)是一种带负电荷的聚合物，能够完全取代流感病毒RNP的RNA，而对VSV的RNP没有影响。 为了为我们的外部RNA结合模型提供明确的证据，并阐明RNP的结构和组装，我们建议确定NP：RNA复合体的结构。不幸的是，即使经过广泛的试验，我们也未能获得任何NP-RNA复合体的晶体，这使得冷冻-EM重建成为我们的下一个选择。NP三聚体的大小约为220kD，具有三重旋转对称性。通过比较NP和NP：RNA复合体的结构差异，不仅可以确定RNA的结合部位，还可以确定RNA结合引起的构象变化。因此，本研究将为RNP的结构组织及其在病毒感染过程中的功能提供重要信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The crystal structure of influenza A virus NP has recently been determined in our laboratory. Organized as trimers in the crystal, NP folds into a two-domain structure with a topology completely different from that of the rhabdovirus NP. A short tail loop, consisting of residues 402 to 428, is likely to play an important role in NP oligomerization, as single-residue mutation in this region causes a total loss of oligomerization. A large positively charged groove was identified at the exterior of the NP trimer at the interface between the two domains. An external RNA binding site indicates that RNA is likely to be exposed in the influenza virus RNPs, different from the situation in non-segmented RNA viruses. This structural difference explains previous results that showed fundamental differences between influenza RNPs and the RNPs of non-segmented (-)-strand RNA viruses like VSV and rabies virus. For example, the viral RNA in influenza virus RNPs is digested by RNase treatment, whereas the viral RNA in the RNPs of parainfluenza viruses and rhabdoviruses is completely resistant to RNase digestion. In addition, polyvinylsufate (PVS), a negatively charged polymer, is able to completely displace RNA from influenza virus RNP, whereas it has no effect on RNPs from VSV. To provide a definite evidence for our external RNA binding model and to elucidate RNP structure and assembly, we propose to determine the structure of the NP:RNA complex. Unfortunately we have not been able to obtain any crystals of NP-RNA complex even after extensive trials, and that leaves cryo-EM reconstruction as our next choice. The size of the NP trimer is ~220kD with a three-fold rotation symmetry. By comparing structural differences of NP and NP:RNA complexes, we will be able to determine not only the RNA binding site, but also the conformational changes induced by binding of RNA. Therefore, this study will provide critical information for the structural organization of RNP and its function during virus infection.