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Delineating the Novel Mechanism of VPg Dependent Virus Translation Initiation

Final Report Summary - INITIATING NOROVIRUS (Delineating the Novel Mechanism of VPg Dependent Virus Translation Initiation)

Final Report Summary- Initiating Norovirus (Delineating the Novel Mechanism of VPg Dependent Virus Translation Initiation)

Project Background

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’. 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. During viral infection the host cell machinery is coopted to produce viral proteins required for replication and spread. Caliciviruses, including, norovirus, use a novel yet poorly understood mechanism to promote viral protein production. This mechanism relies on the interaction of cellular factors with a viral protein called VPg, which is attached to the viral RNA genome. Because this mode of translation is distinct from normal cellular protein synthesis it may provide targets for the development of new anti-viral therapies. This project will establish in the UK and exploit a powerful technique for studying protein translation mechanisms to investigate the VPg-dependent mode of viral protein production.

Project work and results

The first stage of this project involved the establishment of a mammalian in vitro translation reconstitution system in the Division of Virology, Department of Pathology, University of Cambridge. This system has previously been used to characterise the function of a number of mammalian translation factors and the novel mechanisms of translation used by poliovirus (Sweeney et al., 2014) and Foot-and mouth disease virus (Pilipenko et al., 2000). Mammalian translation factors and ribosomal subunits have successfully been purified from HeLa cell and rabbit reticulocyte lysates through a series of differential solubility, affinity and size dependent strategies (Pisarev et al. 2007). The activity and purity of these factors was confirmed on control RNAs confirming that the assay is functional and can now be used to examine the impact of each of these proteins on VPg-dependent translation. This system can now also be used to examine the role of proteins from different viruses in regulating translation (Strnadova et al., 2015). As well as the in vitro reconstitution system two further experimental techniques have now been established. The first, selective 2’-hydroxyl acylation analyzed by primer extension is a method to detect structure present in RNA. This method was used to determine the structure present at the VPg-proximal region of porcine sapovirus (Hosmillo et al, 2016). The translation initiation factor eIF4A, an RNA helicase, was shown to be required for porcine sapovirus translation, likely due to a requirement to unwind the identified structure. Secondly, directed hydroxyl radical cleavage, a method to examine protein-RNA interaction has also been established. Fe(II) is tethered to specific locations on the surface of RNA binding proteins and generates hydroxyl radicals after treatment with H2O2 and ascorbic acid, cleaving RNA at nearby sites that can be identified.

Impact of the project

Viruses must compete with host cell messenger RNAs for the protein production machinery in cells they infect to establish an infection. The experimental systems now established in the Division of Virology in Cambridge as a direct result of this project will reveal important new insights into how viruses such as norovirus use novel mechanisms to ensure virus proteins are efficiently synthesized. The in vitro reconstituted system will facilitate identification of cellular proteins essential for this function. In concert, RNA structure mapping and protein-RNA interaction mapping will enable us to build a detailed picture of how these viral translation complexes are assembled. A deeper understanding of the molecular mechanisms required for VPg-dependent protein synthesis may identify key conserved differences to canonical cellular translation providing new targets for broad-spectrum anti-virals.

Contact: Dr Trevor Sweeney
Address: Division of Virology,
Department of Pathology,
University of Cambridge,
Level 5 Lab Block,
Addenbrooke’s Hospital,
Hills Road,
CB2 0QQ, UK.