Early Events in Calicivirus Infection.

Caliciviruses are a family of single-stranded positive sense RNA viruses with a nonsegmented genome. Feline Calicivirus (FCV) causes respiratory disease in cats and noroviruses cause gastroenteritis in humans. More recently, murine norovirus (MNV), a murine form of human norovirus, was described. Although human noroviruses (HuNV) cannot be cultured in vitro, MNV and FCV can, making them excellent models for HuNV. Previous studies of calicivirus entry have shown very different pathways for these two viruses. FCV enters cells via a clathrin-dependent or macropinocytic pathway, but nothing is known about what signalling events are involved in this process, where the virus traffics to post-entry or the effects the virus has on cell metabolism. MNV uses a clathrin- and caveolae- independent pathway that is dependent on dynamin, lipid rafts and microtubules. This project aimed at dissecting the early events in FCV and MNV infection; which signalling pathways are involved and what trafficking occurs in the cells. We have used pharmacological inhibitors of signalling pathways (genistein, okadaic acid and wortmannin) and dominant negative inhibitors of proteins involved in endocytic and signalling pathways as well as markers of endosomal compartments for colocalisation studies. We have now shown that FCV entry is dependent on dynamin, traffics from early to recycling endosomes and is dependent on PI3-kinases for entry and replication. MNV-1 traffics to the recycling endosome, possibly from the macropinosome, and is dependent on tyrosine kinases and protein phosphatases. We have also begun to investigate the effects of FCV infection on the metabolism of host cells using BiOLOG technology. These experiments showed that in infected cells there is an overall decrease in metabolism, but an increase in glucose uptake. This study has resulted in further elucidation of the early events in infection of both of these viruses which could aid in the future development of antiviral therapies.