FLU VIRUS RNP COMPLEX

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们实验室最近测定了甲型流感病毒NP的晶体结构。 在晶体中组织为三聚体，NP折叠成具有与弹状病毒NP完全不同的拓扑结构的双结构域结构。 由残基402至428组成的短尾环可能在NP寡聚化中起重要作用，因为该区域中的单个残基突变导致寡聚化的完全丧失。在两个结构域之间的界面处的NP三聚体的外部鉴定出大的带正电荷的凹槽。外部RNA结合位点表明RNA可能暴露在流感病毒RNP中，这与非节段RNA病毒中的情况不同。这种结构差异解释了先前的结果，这些结果显示流感RNP与非分段（-）链RNA病毒（如VSV和狂犬病病毒）的RNP之间存在根本差异。例如，流感病毒RNP中的病毒RNA被RNA酶处理消化，而副流感病毒和弹状病毒的RNP中的病毒RNA对RNA酶消化完全耐受。 此外，聚硫酸乙烯酯（PVS），一种带负电荷的聚合物，能够完全取代流感病毒RNP的RNA，而它对VSV的RNP没有影响。 为了给我们的外部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复合物的结构。不幸的是，即使经过广泛的试验，我们也无法获得任何NP-RNA复合物的晶体，这使得冷冻EM重建成为我们的下一个选择。NP三聚体的大小为~220kD，具有三重旋转对称性。 通过比较NP和NP：RNA复合物的结构差异，我们不仅能够确定RNA结合位点，而且还能够确定由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.