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

迫切需要新药来对抗威胁人类、动物和植物健康的病毒。现有的抗病毒药物大多抑制病毒附着/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动态细胞器的形成，称为病毒工厂。越来越多的证据表明，一些病毒工厂是通过液-液相分离（LLPS）形成的，包括SARS-CoV-2、流感和麻疹病毒。这些隔室浓缩病毒复制酶，并从免疫传感器中隔离复制中间体。靶向相分离是一种新兴的范例，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（一种不断进化的RNA病毒）通过LLPS1采用类似的机制形成复制工厂。轮状病毒（rv）仍然是儿童急性胃肠炎的一个主要原因，尽管有疫苗可用，但每年仍造成100万至200万儿童死亡，主要发生在低收入国家。轮状病毒复制工厂是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。该研究项目将重点剖析这些病毒凝聚物的物理化学性质，以了解它们的动态构象和影响电荷介导病毒工厂组装的翻译后修饰，并在此过程中确定未来治疗干预的目标。为了定量地描述这些凝聚物的形成，我们将使用最近开发的高通量微流体平台phasescan1,2，检查观察到的重组产生的病毒蛋白的二元和三级混合物的相变，以及体外的病毒rna。这些发现将引导我们定义一种新的病毒复制凝聚形成模型，该模型解决了蛋白质特异性属性（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割和非病毒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和基于接近度的生物素化方法）来确定体内病毒复制凝聚物的分子组成。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来原位定义病毒复制工厂的完整分子图。新近开发的最先进的机器学习方法将对感染早期和晚期病毒生物分子凝聚体中已确定的蛋白质居民进行检查，以揭示驱动LLPS的氨基酸特征。这些残基在病毒复制凝聚体形成中的作用将通过诱变进一步测试。此外，我们将确定和检查翻译后修饰对病毒生物分子凝聚物的相行为的作用。从这些方法中获得的见解将为寻找可作为RNA病毒前瞻性治疗药物模板的化合物奠定基础，并提高我们对病毒蛋白的协同相互作用的基本理解，这些协同相互作用自发形成复杂的生物凝聚体，是病毒复制的基础。将与利兹大学的质谱设施合作筛选小分子化合物进入这些生物分子凝聚体的分配，以确定这些分子进入凝聚体的分配程度。成功的命中将在体外（PhaseScan）和体内（病毒复制试验）进行测试，以确定它们在破坏相分离和病毒复制方面的效力。

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.