Norovirus, a member of the Caliciviridae family of small RNA viruses, is the major cause of viral gastroenteritis worldwide, often referred to as ‘winter vomiting disease’. Over a million people were infected with the virus last winter in the UK alone. Frequent outbreaks in hospitals and schools put increased pressure on healthcare services. Once thought of as a self limiting infection, norovirus has more recently been linked with higher mortality rates in older people as well as chronic infection and increased morbidity in immunocompromised patients such as those receiving chemotherapy. Despite being increasingly studied no treatments for control of norovirus infection are available. Noroviruses use a novel yet poorly understood mechanism for viral protein synthesis. This mechanism relies on the interaction of cellular factors with a virus-encoded protein called VPg, which is covalently linked to the viral RNA. Astroviruses, which also cause gastroenteritis, are thought to use a similar mechanism. Because this mode of translation is distinct from normal cellular protein synthesis it will provide targets for the development of new anti-viral therapies. This proposal plans to use a mammalian in vitro translation reconstitution system to delineate the mechanism of VPg-dependent translation initiation used by noroviruses and astroviruses. Highly purified mammalian translation factors and ribosomal subunits will be combined with viral RNA to recapitulate VPg-dependent translation initiation, identifying which factors are essential for this process. Once identified, specific interactions between VPg and cellular factors will be characterised in detail. The potential of developing anti-viral strategies, targeting these interactions, will then be examined. This proposal will reveal novel insights into a key stage in norovirus replication and provide the first detailed analysis of the VPg-dependent mechanism of astrovirus translation.