The structure and function of native bunyavirus ribonucleoproteins

病毒有可能导致全球危机，最近的COVID-19疫情就说明了这一点。考虑到未来的大流行准备，世界卫生组织已经公布了一份仅11种病毒的清单，这些病毒被认为是高度优先研究的，其中包括来自布尼亚病毒目（也称为布尼亚病毒）的3种病毒，突出了它们对全球公共卫生的重要性。布尼亚病毒的遗传物质由RNA组成，RNA被病毒核蛋白覆盖，并与病毒聚合酶（负责产生更多病毒RNA的病毒蛋白）相互作用。RNA、核蛋白（NP）和聚合酶一起形成核糖核蛋白复合物（RNP）。这种复合物的主要功能是允许病毒基因的表达，允许病毒劫持细胞并迫使其产生新的病毒;因此，RNP在感染过程和疾病中起着核心作用。然而，布尼亚病毒RNP结构的许多基本方面仍然未知，例如核蛋白单元如何彼此连接，以及聚合酶如何连接到RNA链。为了理解RNP结构的这些重要方面，该提案将建立在我们最近发表的关于模型本扬病毒Bunyamwera病毒（BUNV）的NP-RNA复合物结构的突破性结果的基础上。在这项工作中，我们从感染性病毒中纯化了RNP，并使用显微镜方法的组合对其进行了表征。这使我们能够生成NP-RNA链的原子模型，这首次最终表明它是一个灵活的螺旋，这种灵活性对许多病毒功能至关重要。该模型显示了NP分子如何连接形成螺旋链，揭示了RNP灵活性的分子基础。我们通过使用一种被称为复制子的非感染性系统来证实我们的模型，该系统允许我们突变参与形成这种柔性螺旋的NP区域，并表明这些变化导致RNP功能的降低。在这项提议中，我们的目标是首次研究来自最致病的布尼亚病毒的RNP的结构。首先，我们将改进我们纯化BUNV RNP的方案，以改进我们发表的结构，以及可视化也含有病毒聚合酶的RNP。然后我们将使用先进的电子显微镜和原子力显微镜的方法（这两者都允许接近原子分辨率的分子），以提高我们对RNP内的NP排列的理解，并阐明病毒聚合酶如何以及在哪里附着在RNA链内。我们将利用我们从BUNV获得的专业知识，从WHO优先名单上的高致病性布尼亚病毒中产生RNP结构。为了做到这一点，我们将利用相同病毒家族中致病性较低的病毒，这是一种有效的方法，因为已知每个家族中物种的RNP结构是相似的。至于BUNV，我们将纯化大量的这些病毒，纯化它们的RNP并使用显微镜方法对其进行成像。这将使我们能够生成不同RNP的高分辨率模型，我们将使用我们基于实验室的复制子系统进行测试。为了将我们的发现转化为最具致病性的布尼亚病毒，我们将在计算上调整我们的模型以适应WHO列出的病毒，我们将使用这些病毒的特异性复制子系统来测试这些模型。最后，对于一种布尼亚病毒，我们将直接成像RNP在RNP背景下正在进行的RNA合成。值得注意的是，这一信息是完全缺乏任何布尼亚病毒。这将使我们能够将最近的病毒聚合酶的原子模型与真实RNP.Overall中的相应构象相关联，我们将提供对存在的一些最危险的病毒的RNP的排列及其RNA合成机制的理解。反过来，这将加强对这些病毒的流行病防备。

Viruses have the potential to cause global crisis, as illustrated by the recent COVID-19 pandemic. In view of future pandemic preparedness, the World Health Organisation has published a list of just 11 viruses that are considered a high priority for research, and this includes 3 viruses from the order Bunyavirales (also known as bunyaviruses) highlighting their importance to worldwide public health. The genetic material of bunyaviruses is made of RNA, which is covered by the viral nucleoprotein and interacts with the viral polymerase (the viral protein responsible for producing more viral RNA). Together, RNA, nucleoprotein (NP) and polymerase form the ribonucleoprotein complex, or RNP. A primary function of this complex is to allow expression of the viral genes, allowing the virus to hijack the cell and force it into making new viruses; thus, the RNP plays a central role in the infection process and disease. However, many fundamental aspects of bunyavirus RNP structure remain unknown, such as how the nucleoprotein units connect with each other, and how the polymerase is attached to the RNA chain. To understand these important aspects of RNP structure, this proposal will build on our recently published ground-breaking results on the structure of the NP-RNA complex from the model bunyavirus, Bunyamwera virus (BUNV). In this work we purified RNPs from infectious viruses and characterised them using a combination of microscopy approaches. This allowed us to generate an atomic model of the NP-RNA chain, which conclusively showed for the first time that it is a flexible helix, and this flexibility is critical for many viral functions. The model showed how the NP molecules link up to form a helical chain, revealing the molecular basis for RNP flexibility. We confirmed our model by using a non-infectious system known as a replicon, which allowed us to mutate regions of the NP involved in forming this flexible helix, and show that these changes caused a reduction in RNP function.In this proposal we aim to study, for the first time, the structure of RNPs from the most pathogenic bunyaviruses.First, we will improve our protocol for purifying BUNV RNPs, to improve on our published structure, as well as visualizing RNPs that also contain the viral polymerase. Then we will use advanced electron microscopy and atomic force microscopy approaches (both of which allow close to atomic resolution of molecules) to improve our understanding of the NP arrangement within the RNP, and to characterise how and where the viral polymerase is attached within the RNA chain.Secondly, we will use the expertise we have gained with BUNV to generate RNP structures from the highly pathogenic bunyaviruses on the WHO priority list. To do this we will take advantage of less pathogenic viruses within the same virus families, a valid approach since the RNP structures of species within each family are known to be similar. As for BUNV, we will purify large amounts of these viruses, purify their RNPs and image them using microscopy approaches. This will allow us to generate high resolution models of the different RNPs, which we will test using our lab-based replicon systems. To translate our findings to the most pathogenic bunyaviruses, we will computationally adapt our models to the viruses listed by the WHO, and we will test these models using replicon systems specific for these viruses.Finally, for one bunyavirus, we will directly image on-going RNA synthesis by the RNP within the context of an RNP. Of note this information is completely lacking for any bunyavirus. This will allow us to correlate recently atomic models of the viral polymerase, with their corresponding conformations within an authentic RNP.Overall, we will provide an understanding of the arrangement of the RNPs of some of the most dangerous viruses in existence and of their mechanism of RNA synthesis. In turn, this will enhance epidemic preparedness against these viruses.