Characterisation of norovirus replication complex formation

Members of the calicivirus family are responsible for important diseases of man and animals; noroviruses cause gastroenteritis or projectile vomiting in humans and outbreaks are often seen in hospitals, cruise ships and in military camps. We currently know very little with regards to how these viruses interact with the host cell and how this leads to the production of new virus particles. When noroviruses infect a host cell they have many effects that can alter the normal processes occurring within the host cell. One such effect is to generate replication ?factories? within the host cell where the viral genome is replicated. In order to better understand this important family of viruses, we now aim to characterise how these replication ?factories? are generated to identify methods of controlling outbreaks of these important viruses.

Caliciviruses are important pathogens of both animals and man, however our understanding of the biology of theses viruses is still very limited. This is mainly due to the fact that the viruses which infect humans, namely noroviruses and sapoviruses, do not grow in tissue culture. All positive stranded RNA viruses replicate within the cytoplasm of the infected cell and as such calicivirus infection leads to the formation of cytoplasmic membranous vesicles on which the virus replicates. Such replication complexes occur through a complex network of viral protein-viral protein interactions as well as viral protein-host cell protein interactions. These host cell proteins are likely to represent good targets for anti-viral intervention. During our recent work funded by a Wellcome Trust Career development fellowship, due to finish at the end of April 2007, we identified a number of key interactions between viral proteins within the replication complex. We now wish to extend this work to take advantage of recent advances in the field which now allow the study of norovirus replication in tissue culture. Using a Norwalk virus replicon and the recently identified murine norovirus, combined with the advanced imaging facilities available within the Henry Wellcome Imaging Suite, we will use EM and confocal microscopy to examine the architecture of the norovirus replication complex. This will allow us to determine the origin of the membranes on which noroviruses replicate. We will also identify host cell factors that interact with a key protein involved in replication complex formation, namely the norovirus NS3 NTPase/helicase, as they are likely to represent good targets for anti viral intervention. The overall aim of this proposal is to begin to address the gap in our knowledge of how these important viruses replicate to allow us to identify ways of controlling or preventing outbreaks.