Phase transitions underlying viral replication: roles of biomolecular condensates in virus assembly

迫切需要新的药物来对抗威胁人类、动物和植物健康的病毒。大多数现有的抗病毒药物抑制病毒附着/进入或靶向关键酶，需要新的抗病毒靶点来开发新的治疗方法和对抗抗病毒耐药性。许多病毒生命周期中的一个关键过程是称为病毒工厂的动态细胞器的形成。越来越多的证据表明，一些病毒工厂通过液-液相分离（LLPS）形成，包括SARS-CoV-2，流感和麻疹病毒。这些隔室集中病毒复制酶并从免疫传感器隔离复制中间体。靶向相分离是一种新兴的范式，可能是发现广谱抗病毒药物的基础。我们最近发现，轮状病毒，一个大的进化类RNA病毒，采用类似的机制，通过LLPS 1形成复制工厂。轮状病毒（RV）仍然是儿童急性胃肠炎的主要原因，尽管有疫苗，但每年仍有超过20万儿童死亡，主要发生在低收入国家。轮状病毒复制工厂代表了由至少两种病毒蛋白在翻译后修饰和核酸结合调节的过程中自发相互作用形成的复杂功能胶体。该研究项目将专注于解剖这些病毒浓缩物的理化性质，以了解它们的动态构象和翻译后修饰如何影响病毒工厂的电荷介导组装，并在此过程中确定未来治疗干预的目标。为了定量描述这些缩合物的形成，我们将使用最近开发的高通量微流体平台PhaseScan 1，2检查重组产生的病毒蛋白的二元和三元混合物以及体外病毒RNA的观察到的相变。这些发现将引导我们定义一种新的病毒复制冷凝物形成模型，该模型涉及蛋白质特异性属性（翻译后修饰，构象）及其高度选择性的RNA组成（同源病毒转录物的分配和非病毒RNA的排除）。我们将应用基于质谱（MS）的蛋白质组学工具（正交有机相分离3和基于邻近的生物素化方法）来定义体内病毒复制浓缩物的分子组成。为了进一步验证我们的发现，将使用超分辨率DNA-PAINT显微镜方法4来定义原位病毒复制工厂的完整分子图像。在早期和晚期感染点期间，病毒生物分子凝聚物的已识别蛋白质居民将通过最近开发的最先进的机器学习方法5进行检查，以揭示驱动LLPS的氨基酸特征。这些残基在病毒复制性缩合物形成中的作用将通过诱变进一步测试。此外，我们将确定和检查的病毒生物分子凝聚物的相行为的翻译后修饰的作用。从这些方法中获得的见解将成为寻找可作为RNA病毒前瞻性治疗药物模板的化合物的基础，并提高我们对病毒蛋白协同相互作用的基本理解，这些病毒蛋白自发形成复杂的生物缩合物，这些生物缩合物是病毒复制的基础。将与利兹大学的质谱设备合作筛选小分子化合物分配到这些生物分子冷凝物中，以确定这些分子分配到冷凝物中的程度。然后在体外（PhaseScan）和体内（病毒复制试验）测试成功的命中，以确定其破坏相分离和病毒复制的效力。

There is an urgent need for new drugs to combat viruses that threaten human, animal and plant health. Most existing antivirals inhibit virus attachment/entry or target key enzymes, and new antiviral targets are needed to develop novel treatments and counter antiviral resistance. One key process in the life cycle of many viruses is the formation of dynamic organelles called viral factories. There is increasing evidence that some viral factories form via liquid-liquid phase separation (LLPS), including SARS-CoV-2, influenza and measles virus. These compartments concentrate viral replication enzymes and sequester replication intermediates from the immune sensors. Targeting phase separation is an emerging paradigm that may underlie discovery of broad-spectrum antivirals.We have recently discovered that rotaviruses, a large evolving class of RNA viruses, employ a similar mechanism of formation of replication factories via LLPS1. Rotaviruses (RVs) remain a major cause of acute gastroenteritis in children, and despite vaccines being available are responsible for >200,000 child deaths annually, predominantly in low-income countries. Rotavirus replication factories represent complex functional colloids formed by spontaneous interactions of at least two viral proteins, during the process modulated by posttranslational modifications and nucleic acids binding. This research project will focus on dissecting the physicochemical properties of these viral condensates to understand how their dynamic conformations and posttranslational modifications that affect charge mediate assembly of viral factories, and in doing so, identify targets for future therapeutic intervention. To quantitatively describe the formation of these condensates, we will examine the observed phase transitions of binary and tertiary mixtures of recombinantly produced viral proteins, as well as viral RNAs in vitro using the recently developed high throughput microfluidics platform PhaseScan1,2. These findings will lead us to define a new model of viral replicative condensate formation that addresses protein-specific attributes (posttranslational modifications, conformation), and their highly selective RNA composition (partitioning of cognate viral transcripts and exclusion of non-viral RNAs). We will apply mass spectrometry (MS)-based proteomics tools (Orthogonal Organic Phase Separation3 and proximity-based biotinylation approaches) to define the molecular composition of viral replicative condensates in vivo. To further validate our findings, a super-resolution DNA-PAINT microscopy approach4 will be used to define the complete molecular picture of viral replication factories in situ. Identified protein residents of viral biomolecular condensates during early and late infection points will be examined by the recently developed state-of-the-art machine learning approach5 to reveal the amino acid features that drive the LLPS. The roles of these residues in the formation of viral replicative condensates will be further tested via mutagenesis. In addition, we will identify and examine the roles of posttranslational modifications on the phase behaviour of the viral biomolecular condensates. The insights gained from these approaches will underlie the search for compounds that could serve as drug templates for prospective therapies for RNA viruses, and improve our fundamental understanding of the synergistic interactions of viral proteins that spontaneously form complex biocondensates that underlie viral replication. Partitioning of small molecule compounds into these biomolecular condensates will be screened in collaboration with the mass-spectrometry facility at the University of Leeds, to determine the degree of partitioning of such molecules into the condensates. Successful hits will be then tested in vitro (PhaseScan) and in vivo (virus replication assays) to determine their potency in disrupting phase separation and viral replication.