Dissecting the mechanism of translational control during calicivirus infection

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The caliciviruses are a group of important viruses that infect humans and animals; in humans, the noroviruses are a major cause of gastroenteritis outbreaks, often reported in the UK press as they cause significant problems in hospitals and on cruise ships. In animals, these viruses cause a range of diseases that include a 'flu like illness in cats. The human viruses do not grow well in cell culture in the laboratory so we and others use murine norovirus (MNV) as the best model system to study the human viruses. Production of proteins, or protein synthesis, is an essential process in cells. Messenger RNA, or mRNA, contains the information that is decoded into proteins by the host cell protein synthesis machinery called ribosomes and is assisted by a number of protein factors termed initiation factors. One of the key factors involved in this process is named eIF4F and consists of three proteins, eIF4E (that binds to a structure on the mRNAs called a cap to recruit the ribosome), eIF4G (a scaffold protein that bridges the eIF4F complex to the ribosome) and eIF4A (which helps unwind any structure in the mRNA to allow the ribosome to move along it). In order to make new virus particles, viruses must manufacture new virus proteins but they rely on using the host cell's protein synthesis machinery to do this. We have previously demonstrated that caliciviruses use a novel mechanism for synthesising new virus proteins in infected cells. We have made a number of significant advances in the understanding of how the caliciviruses produce viral proteins: i) We have shown that a viral protein called VPg that is attached to the end of the viral mRNA instead of a cap, binds to one of the key proteins in the cell required for protein synthesis, eIF4E. These viruses have therefore evolved a novel 'proteinaceous' cap substitute that mimics the 5' end of a cellular mRNA. ii) We have also shown that other host translation initiation factors (eIF4A) are required for calicivirus protein synthesis and interfering with these proteins inhibits virus replication. iii) Although feline calicivirus and murine norovirus share the common mechanism of VPg-directed protein synthesis, some fundamental differences seem to exist in their requirements for the cellular initiation factor proteins. iv) Our recent work has shown that these viruses can manipulate the eIF4F complex to regulate production of viral proteins. Building on these findings and our expertise, we now wish to carry out a comparative analysis of the process of calicivirus protein synthesis using modern biochemical techniques, with the aim of understanding the cellular factors they require and how they modify these proteins to aid the production of virus proteins. Specifically we will: 1) Use advanced biochemical techniques to identify what cellular initiation factors are found in complex with the calicivirus VPg protein and then investigate what role they play in virus protein production. 2) Analyse the effect of calicivirus infection on the host eIF4F complex to fully understand how the virus can modulate it to its own advantage. This will tell us how these viruses manipulate the host cell to ensure efficient production of viral proteins. If we can fully understand the mechanism of calicivirus protein synthesis, we can identify ways to inhibit virus replication, and so this work will ultimately aid in the development of novel antiviral therapies for this important group of viruses.