A new player in thousands of years of poliovirus history

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By Valeria Lulla and Andrew Firth

It is likely that polio has plagued humankind for thousands of years. An Egyptian carving (1400 BCE) depicts a young man with a leg deformity similar to those caused by poliovirus. The epidemic and contagious nature of poliomyelitis was discovered in 1907 (by Ivar Wickman), and subsequently polio was shown to be caused by a virus in 1909 (by Karl Landsteiner and Erwin Popper).

Reprinted with permission from Ny Carlsberg Glyptotek, Copenhagen.

Following the development of a reverse genetics system in 1981 by Vincent Racaniello and David Baltimore, the ensuing molecular studies have made this virus one of the best studied so far – a model for replication of all positive-sense RNA viruses which can be found in every virology textbook. The poliovirus genome – functioning as an mRNA – was believed to be translated into a single polyprotein, which could then be processed into 11 individual proteins, including enzymatic proteins that replicate viral genomes and structural proteins that package progeny genomes into protein capsids for transmission.

However, we found that this picture was incomplete: one more protein was cryptically encoded within the genome of poliovirus 1 (the most common serotype) besides many other enteroviruses (the larger group of related viruses to which poliovirus belongs).

The project had its origins when Andrew Firth – originally a mathematician by training – was undertaking his first postdoctoral position back in 2005, in the lab of Dr Chris Brown in the University of Otago. Andrew had been investigating comparative genomic methods for identifying "hidden" genes and, following Chris' advice, was using virus genomes as convenient test cases. Most of the enterovirus species studied appeared to have a short region upstream of the known polyprotein gene that displayed a strong "protein coding signature" indicating that there was an additional gene hiding there. But without the opportunity to do experimental follow up, the project languished. Ten years later, during which time Andrew had moved to Cambridge and started a lab working at the interface of computational and experimental virology, he finally had the opportunity to return to the project.

To experimentally test the computational predictions, Valeria Lulla – who had joined Andrew’s lab in 2016 – created mutant enteroviruses in which expression of the upstream protein was disabled. By comparing wild type with mutant viruses, Valeria was able to confirm expression of the upstream protein during infection of human cell lines with the wild type viruses. This was repeated for echovirus 7 and poliovirus 1, with the poliovirus work being possible thanks to a collaboration with Nicola Stonehouse’s group at the University of Leeds (UK).

The excitement generated by the confirmation of expression of the new enterovirus protein was followed by months of frustration trying to identify its actual function. No phenotypic differences could be found between the wild type and mutant viruses during infection of any cell line tested. Finally, Ian Goodfellow, who’s lab was just next door, asked: “Why don’t you try organoids?” His lab had successfully established a human intestinal organoid system in collaboration with Matthias Zilbauer's group from the Department of Paediatrics in Cambridge. Amazingly, the very first infection experiment revealed a significant difference between growth of wild type and mutant viruses in this system. Further experiments confirmed the involvement of the new protein in virus release from membranous compartments during infection in gut epithelial cells. This was exciting because the gut is the site where enteroviruses first replicate on infecting a new host before, potentially, invading other cell types. In contrast, the closely related rhinoviruses replicate in the upper respiratory tract and – perhaps not surprisingly – they ubiquitously lack the upstream protein.

These findings overturned the long established view that enteroviruses use a single-polyprotein gene expression strategy, and may have important implications for understanding enterovirus pathogenesis. However, many questions remain to be answered, such as why do some enteroviruses (particularly poliovirus types 2 and 3) appear to lack the upstream protein, and how exactly does the upstream protein function to facilitate release of virus from membranous compartments? No doubt further experimental work will begin to shed new light on these questions.

The paper in Nature Microbiology is here: https://rdcu.be/bbZ93

Andrew Firth

Wellcome Trust Senior Research Fellow, University of Cambridge