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

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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.