Enteroviruses are common and important human pathogens. While many infections are mild, some strains can cause severe and even fatal disease with symptoms ranging through fever, hand foot and mouth disease, myocarditis, viral meningitis, encephalitis, acute hemorrhagic conjunctivitis, and acute flaccid paralysis. Although poliovirus (the enterovirus that causes poliomyelitis) has been eradicated from much of the globe it remains a threat, and other emerging enteroviruses can also cause severe polio-like symptoms. Currently, vaccines are only routinely available for poliovirus (approved globally) and EV-A71 (approved in China). Enteroviruses are also some of the best studied viruses and serve as a useful model system for understanding the biology of all "positive-sense" RNA viruses which can be found in every virology textbook. The enterovirus genome is ~7400 nucleotides in length and, ever since poliovirus was first sequenced in 1981, was believed to encode only 11 proteins, including enzymatic proteins that replicate the viral genome and structural proteins that package progeny genomes into protein capsids for transmission. These 11 proteins are translated from the genome (which functions directly as a messenger RNA) concatenated end-to-end as a single giant "polyprotein", which is then cut up by one of the viral proteins (a proteolytic enzyme) into the 11 virus proteins. However, we recently showed that this picture was incomplete: in fact one more protein is cryptically encoded within the genome of many enteroviruses. We call the additional protein UP (Upstream Protein) since it is encoded upstream of the polyprotein. Using an organoid system ("mini gut" 3d cell cultures grown in the lab), we went on to show that UP plays an important role specifically in gut epithelial cells where it is involved in releasing newly formed virus particles from membranous compartments. This is exciting because the gut is the site where enteroviruses first replicate on infecting a new host before, potentially, invading other cell types and organs. In contrast, the closely related rhinoviruses replicate in the upper respiratory tract and - perhaps not surprisingly - they ubiquitously lack the UP protein. We published this research in Nov 2018 in Nature Microbiology. However this previous work just scratched the surface of understanding the biological function of the novel UP protein. Many very important questions remained unanswered, such as why do some enteroviruses appear to lack the UP protein, how does UP differ between different enteroviruses, what controls UP expression, with which membranes does UP associate, and how exactly does UP function to facilitate release of virus from membranous compartments. Through five work packages, we will now perform a detailed functional analysis of the UP protein: 1) Bioinformatic analysis - correlation of UP with virus provenance and clinical data 2) Assessing and deciphering the regulation of UP expression 3) Biochemical and functional characterization of the UP protein 4) Biological functionality of UP in human intestinal organoids 5) Physiological role of UP and potential as a vaccine candidate This research is exciting for several reasons. The discovery of a new protein opens up a whole new avenue for enterovirus research that will give new insights into virus biology and pathogenesis. We are ideally poised to exploit this new research direction. Understanding the function of UP may also help explain why some enteroviruses can infect the gut whereas others infect the upper respiratory tract. These findings may aid rapid prediction of the tropism of newly emerging enteroviruses. More importantly, UP knockout viruses could be ideal candidates for attenuated virus vaccines since they grow well in cell culture (to give good vaccine yield) but are attenuated in the gut; further, this strategy could be quickly applied to newly emerging enteroviruses.