Characterisation and therapeutic manipulation of Flaviviridae innate immune evasion

黄病毒科包括丙型肝炎病毒(丙型肝炎病毒)和昆虫传播的登革病毒(DV)和寨卡病毒(ZV)。DV和ZV是新出现的感染，传播到世界上的新地区，部分原因是气候变化扩大了昆虫媒介范围。目前，每年约有6000万人患有有症状的登革热病毒感染，其中1万人死亡。随着新的感染剂的出现和旧病毒的传播，了解如何开发新的抗病毒药物将变得越来越重要。亲环素(Cyclopilins，CyP)是改变靶蛋白形状的宿主酶，但对细胞功能知之甚少。关键是，Cyps被广泛发现是病毒感染的辅助因子，而丙型肝炎病毒、DV和ZV都依赖Cyps进行复制。细胞非常擅长利用称为细胞自主先天免疫的各种机制来保护自己免受感染。这个复杂的系统使用传感器来检测传入和复制的病毒，这些传感器检测病原体特有的分子模式。感应器的触发激活了干扰素的产生，干扰素被分泌出来，并诱导一系列抗病毒蛋白和途径的表达，从而有效地抑制附近细胞的感染。成功的病毒必须避开或对抗这些保护系统。通常情况下，病毒会在我们称之为隐形的过程中躲避传感器。黄毒科通过颠覆细胞内膜系统，将其复制复合体包裹在膜网中来隐藏。这不包括允许不受阻碍的RNA和病毒蛋白生产和病毒组装的关键传感器。我们的初步数据表明，抑制CYPS会揭开丙型肝炎病毒的外衣，将其揭示给包括RNA传感器RIG-I在内的传感器。当病毒对Cyp的利用受到干扰时，干扰素的产生就证明了这一点。CYP抑制是开发抗病毒药物的有效方法，因为抗病毒作用是由宿主的天然免疫系统介导的，而宿主的天然免疫系统是有效的，病毒很难逃脱。针对宿主因素使病毒的行为像病毒一样，不会感染人类，因为它激活并屈服于先天免疫防御。我们的关键发现是发现，如果丙型肝炎病毒的目标细胞有缺陷的先天免疫系统，它就不需要环磷酰胺。这揭示了该病毒利用细胞色素P4逃避先天免疫。在这里，我们的目标是确切地了解黄病毒是如何利用细胞周期蛋白来逃避天然免疫保护系统的。我们将确定所使用的特定细胞周期蛋白，并测试我们的新型细胞周期蛋白抑制剂是否可以抑制复制。我们将确定Cyps针对的病毒蛋白，并研究Cyps在结构上对病毒蛋白的作用，以帮助病毒隐藏。我们的药物系列区分不同的病毒，我们将研究如何区分。我们将测试哪些环磷酰胺是活性药物的靶点，以解释我们所看到的抗病毒特异性。我们将研究膜网络和病毒复制间隔，看看当我们干扰Cyp的活动时，它是如何变化的。当细胞周期蛋白受到干扰时，传感器会进入复制隔间并被激活吗？我们还将研究先天免疫系统，并确定当我们发现病毒时检测到它的活跃传感器。所有这些目标都使用在我们或合作者的实验室中使用的经过验证和测试的技术。我们的工作将产生对宿主因素如何保护病毒免受先天免疫的关键理解，以及这种相互作用如何作为一种新的治疗或预防策略受到干扰。我们的目标是产生新的知识，详细说明黄毒科逃避先天免疫的分子机制以及干扰它的后果。我们认为，在将宿主靶向策略作为有效的治疗方法应用于临床之前，这种新的知识和宿主靶向策略的可操作性的证明是必要的。

Flavirviridae include hepatitis C virus (HCV) and the insect transmitted viruses dengue virus (DV) and Zika virus (ZV). DV and ZV are emerging infections, spreading to new regions of the world, in part due to climate change expanding insect vector ranges. Currently around 60 million people suffer symptomatic DV infection each year with 10,000 deaths. It will be increasingly important to understand how to develop new antivirals as new infectious agents emerge and old agents spread. Cyclophilins (Cyps) are host enzymes that alter the shape of target proteins, but have poorly understood cellular functions. Critically, Cyps are widely found to act as cofactors for viral infection and HCV, DV and ZV all depend on Cyps for replication. Cells are very good at protecting themselves from infection using a variety of mechanisms termed cell autonomous innate immunity. This complex system detects incoming and replicating viruses using sensors that detect molecular patterns that are specific to the pathogen. Sensor triggering activates production interferon, which is secreted and induces expression of a whole host of antiviral proteins and pathways that potently suppress infection in nearby cells. Successful viruses must evade or antagonise these protective systems. Typically viruses hide from the sensors in a process we term cloaking. Flaviviridae hide by subverting the intracellular membrane system, cloaking their replication complexes in a membranous web. This excludes key sensors allowing unhindered RNA and viral protein production and viral assembly. Our preliminary data demonstrate that inhibiting Cyps uncloaks HCV revealing it to sensors including the RNA sensor RIG-I. This is evidenced by production of interferon when Cyp use by the virus is disturbed. Cyp inhibition is a powerful approach to developing antivirals because the antiviral effect is mediated by the host innate immune system, which is potent and difficult for the virus to escape. Targeting host factors makes the virus behave like a virus which doesn't infect humans because it activates and succumbs to innate immune defences.Our key discovery was to find that HCV doesn't require Cyps if its target cells have a defective innate immune system. This revealed that the virus uses Cyps to evade innate immunity. Here we aim to understand exactly how Flaviviridae use Cyps to evade innate immune protective systems. We will identify the specific Cyps used and test whether our novel Cyp inhibitors can suppress replication. We will identify the viral proteins targeted by Cyps and work out what Cyps do structurally to the virus proteins to help the virus hide. Our drug series distinguishes between different viruses and we will work out how. We will test which Cyps are targeted by the active drugs to explain the antiviral specificity that we see. We will study the membranous web and viral replication compartment and see how it changes when we disturb Cyp activity. Do the sensors enter the replication compartment and get activated when Cyps are disturbed? We will also study the innate immune system and identify the active sensors that detect the virus when we reveal it. All of these aims use tried and tested techniques in use in our or collaborators' laboratories. Our work will generate critical understanding of how host factors can protect viruses from innate immunity and how this interaction can be disturbed as a novel therapeutic or prophylactic strategy. Our goal is to generate new knowledge detailing the molecular mechanisms of the process of innate immune evasion by Flaviviridae and the consequences of disturbing it. We propose that this new knowledge and the demonstration of the tractability of host targeting strategies will be necessary before such strategies can be taken to the clinic as effective therapeutics.