Structural characterisation of the calicivirus entry pathway and its role in defining virulence

杯状病毒是一个病毒家族，其引起对人类和兽医学都具有全球重要性的疾病。最值得注意的人类杯状病毒是人类诺如病毒，这种病毒每年在全世界引起约6.85亿例急性胃肠炎;约2亿例病例发生在5岁以下儿童中，导致每年70，000 - 200，000例儿童死亡，主要发生在发展中国家。据估计，每年人类杯状病毒在全世界造成约600亿美元的医疗费用和生产力损失。动物杯状病毒引起严重疾病，通常具有高发病率和死亡率，从兔子的致命出血性疾病，牛的肠炎到猫科动物的急性呼吸道疾病。虽然已经为兔和猫杯状病毒开发了疫苗，但令人惊讶的是，人类杯状病毒疫苗的开发落后于动物杯状病毒。影响杯状病毒毒力的宿主因素仍有待确定，感染的发病机制也知之甚少，尽管很明显，人类和兽医杯状病毒的病毒进化导致新变体的出现。多年来，由于缺乏合适的系统进行病毒的大规模体外繁殖，对人类杯状病毒的超微结构研究一直受到阻碍。尽管最近的突破性研究已经揭示了几种基因型的人诺如病毒可以在来源于干细胞的人肠类中生长，但仍然不可能在体外制备详细结构研究所需的高滴度的人病毒。阐明杯状病毒的结构和生化特性将在基于结构的治疗设计中发挥关键作用，并将为新型杯状病毒疫苗的开发提供信息，以影响健康。因此，我们选择以原子分辨率分析猫杯状病毒（FCV）作为代表性致病性杯状病毒。我们研究了杯状病毒进入和感染的早期阶段，发现当病毒结合其受体时，病毒外壳上形成漏斗状结构，可能允许病毒启动感染。与这一发现同时，我们观察到，新出现的强毒FCV毒株似乎绕过了引起呼吸道疾病的毒株感染所必需的进入步骤;与强毒分离株不同，这些毒株和疫苗毒株对细胞的感染可以被氯喹阻断。该项目汇集了这两条研究链，以检查杯状病毒-宿主在无毒和毒性病毒株中的相互作用，比较两种生物表型，并阐明杯状病毒毒力是否存在共同的结构决定因素。我们将通过检查鼠诺如病毒来确定在FCV中观察到的新结构是否与其他杯状病毒相同。在受体接合后在病毒体上观察到的漏斗状结构的结构和功能将使用脂质膜模拟病毒的脱壳和感染的启动，使我们能够识别对杯状病毒进入至关重要的宿主-病毒相互作用。 我们将比较早期进入要求的一个小组的致病性杯状病毒株分离从爆发的毒性全身性疾病的猫科动物，调查的作用，病毒进入过程中发挥决定病毒的致病性。将产生嵌合病毒以鉴定杯状病毒衣壳中赋予毒力表型的区域。该项目将揭示人类和动物杯状病毒感染早期阶段的基本见解，促进基于结构的方法来设计对人类和动物健康产生重大影响的新型疗法和疫苗。

Caliciviruses are a family of viruses that cause diseases of global importance to human and veterinary medicine alike. Most notable of the human caliciviruses is the human norovirus, a virus that causes ~685 million cases of acute gastroenteritis each year worldwide; ~200 million cases are in children under 5 years, leading to 70,000-200,000 child deaths/year, mostly in developing countries. Every year human caliciviruses are estimated to cost ~$60 billion worldwide related to healthcare costs and lost productivity.Caliciviruses of animals cause severe diseases, often with high morbidity and mortality, from usually fatal haemorrhagic disease in rabbits, enteritis in cattle to an acute respiratory disease in felids. While vaccines have been developed for the rabbit and feline caliciviruses, it is striking that the development of human caliciviral vaccines has lagged behind that of the animal caliciviruses. The host factors that affect calicivirus virulence remain to be identified and the pathogenesis of infection is poorly understood, although it is evident that viral evolution in both human and veterinary caliciviruses leads to the emergence of new variants. Ultrastructural studies of human caliciviruses had been hampered for many years by the lack of suitable systems for the large-scale in vitro propagation of the viruses. Although recent breakthrough studies have revealed that several genotypes of human norovirus can be grown in human intestinal enteroids derived from stem cells, it is still not possible to prepare the high titres of the human viruses in vitro that are required for detailed structural studies. Elucidating the structural and biochemical properties of caliciviruses will play a key role in the structure-based design of therapeutics and will inform the development of novel caliciviral vaccines to impact health. Accordingly, we elected to analyse at atomic resolution the feline calicivirus (FCV) as a representative pathogenic calicivirus. We examined the early stage of calicivirus entry and infection, discovering that when the virus binds its receptor, a funnel-shaped structure forms on viral shell that likely allows the virus to initiate infection. In parallel with this discovery, we observed that emerging virulent FCV strains appear to bypass entry steps that are essential for infection with strains that cause respiratory disease; infection of cells with these and vaccine strains can be blocked by chloroquine, unlike virulent isolates. This project brings together these two strands of research to examine the calicivirus-host interaction in avirulent and virulent strains of virus, comparing the two biological phenotypes and elucidating whether there are common structural determinants of caliciviral virulence. We will determine whether the novel structure observed in FCV is common to other caliciviruses by examining murine norovirus. The structure and function of the funnel-shaped structure observed on virions following receptor engagement will be modelled using lipid membranes to mimic viral uncoating and initiation of infection, enabling us to identify host-virus interactions that are critical for caliciviral entry. We will compare the early entry requirements of a panel of pathogenic strains of calicivirus isolated from an outbreak of virulent systemic disease in felids, investigating the role that the viral entry process plays in determining viral pathogenicity. Chimaeric viruses will be generated to identify regions in the caliciviral capsid that confer the virulent phenotype. This project will reveal fundamental insights into the early stages of infection with caliciviruses of humans and animals, facilitating structure-based approaches to the design of novel therapeutics and vaccines that will have a significant impact on human and animal health.