Mouse norovirus replication: role of microtubules and the innate immune system

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Victoria.

Thesis with publications.

Human Norovirus (NV) is now considered the predominant cause of nonbacterial gastroenteritis cases worldwide. Despite the high frequency of infection in the community there still remains no vaccine or anti-viral treatment for NV infection attributed to the lack of suitable culture systems or animal models. The identification of a novel Murine Norovirus (MNV) in immune-deficient laboratory mice have greatly advanced our knowledge of the mechanisms involved norovirus (NoV) replication and pathogenesis. We examined the roles and interactions of several of the non-structural proteins, with focus on the predominantly uncharacterized non-structural protein three (NS3). Through expression studies, we identified that the NS3 protein induced the formation of distinct vesicle-like structures that did not colocalise with specific protein markers for cellular organelles. Instead these vesicular structures colocalised with membranes of high cholesterol content. Using a GFP-tagged MNV-NS3 we observed that these structures were highly motile, and their movement was dependent on intact microtubules. Our other key area of study was the innate immune system, and the methods employed by MNV (as a model for NV) to subvert and manipulate both the recognition and the response elicited during infection. We established that MNV has several avenues to combat the innate immune system. These studies identified that MNV prevents the nuclear translocation of transcription factors that contribute significantly to innate immune activation and function. We further interrogated these observations and found not one, but three candidates NS1-2, NS3 and NS4 when solely expressed were able to inhibit STAT1 nuclear translocation. Although such redundancy is rarely observed in virus replication, we believe this aids in the efficiency of the MNV to down-regulate the innate immune response, which it performs concurrently at various stages of the innate immune pathway to rapidly deplete host defences.