A newly discovered protein-coding ORF in enteroviruses: from mechanism to application

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.

Enteroviruses are common and important mammalian pathogens. Pathology in humans ranges from subclinical to acute flaccid paralysis, myocarditis and meningitis. For nearly 50 years, all the viral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF) covering most of the ~7.4 kb genome. However, in Nov 2018 we reported that many enteroviruses actually encode an additional, previously unknown protein, UP (Upstream Protein), in a short ORF upstream of the polyprotein ORF (Lulla et al, Nature Microbiol). We showed that UP is expressed in representative members of the major human species Enterovirus A, B and C and that, at least in Enterovirus B, UP facilitates virus growth specifically in gut epithelial cells where membrane-associated UP facilitates virus release at late stages of infection.

Despite our published findings, many very important questions remain unanswered such as why do some enteroviruses (particularly poliovirus types 2 and 3) appear to lack UP, how exactly does UP function to facilitate release of virus from membranous compartments, and why is this effect specific to gut epithelia? Having established systems for working with gut epithelial organoids and enteroviruses A, B and C, we are perfectly positioned to properly characterize the UP protein biological function. Our project comprises five main work packages:

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

Together these will elucidate the function of the newly discovered UP protein in enterovirus molecular biology and pathogenesis, and lay the groundwork for UP knockout as a possible attenuated virus vaccine strategy.

The research will benefit academics - particularly in the fields of picornavirus molecular virology and pathogenesis, IRES structure and function, viral viroporin/membrane proteins, organoid viral infections, emerging infections, and vaccine development (see Academic Beneficiaries section for details). As this will be the founding research for the newly discovered enterovirus uORF protein, it will lay the groundwork for all future studies of UP.

The research will be relevant to vaccine development for important human enteroviruses and the same strategy could also be used to develop vaccines for veterinary enteroviruses (e.g. E, F, G) particularly in the event of any newly emerging high pathogenicity strains. This will lead to improved quality of life, and wealth generation. Currently, enterovirus vaccines are only routinely available for poliovirus (approved globally) and EV-A71 (approved in China). Generation of UP-deletion viruses may provide a quick strategy for developing vaccine candidates for current and/or emerging enteroviruses. Our collaborative work with Prof. Shih will include a pilot study to test the efficacy of this approach. UP deletion viruses appear to grow as wildtype in standard cell cultures which is ideal for vaccine production yield, yet are attenuated in the gut epithelium - the primary site of virus entry into a host (we have shown this for EV7; part of the proposed work includes testing this for other enteroviruses). By deleting a significant part of the UP region, we will be able to guard against reversion. UP deletion could also be combined with other established vaccine strategies.

Our proposed work will elucidate the differences between different enteroviruses that lack or that contain a functional uORF. A proper understanding of these factors may allow prediction of the entry route (respiratory tract versus gut) and improved prediction of pathogenesis of newly emerging enterovirus strains based on sequence analysis. This could have clinical implications for treatment and patient sampling strategies.

Towards the end of the project, the research has the potential to impact the pharmaceutical industry. The research may define novel pathways and interactions that could be suitable for antiviral drug targeting.

The project also has good training potential for the staff involved. The named PDRA Valeria Lulla has a strong background in molecular virology. While working on this project she will learn some new molecular biology techniques, but also be able to continue to expand her skills in organoid work as that forms a very large component of the proposed experimental work. Dr Lulla will work closely with the lab of Co-I Matthias Zilbauer and in particular senior research assistant Komal Nayak to transfer expertise and knowledge. In return, Komal Nayak will learn a variety of techniques in virology and in particular virus infection of organoids and subsequent assays that she can transfer back to the Zilbauer lab. Dr Lulla has attended University courses in bioinformatics and programming and will be able to put those skills into action in the Firth lab which, being half experimental and half bioinformatic, provides a perfect training environment for this. These skills are very transferable and in high demand. The University of Cambridge also runs a large variety of courses (teaching, public speaking, career planning, leadership, grant/paper reviewing, grant writing, networking, entrepreneurship, conflict resolution, etc) for mentoring and training rising stars.

The research is good value for money because we already have the organoid system and virus expertise in place, allowing us to "hit the ground running". Results are effectively guaranteed (e.g. it is not a "fishing expedition"). Further, the collaborations and systems generated during the project will support other work in enterovirology and in the application of gut organoids to other enteric viruses.