Dr. Julienne Jagdeo is a recent graduate and current postdoctoral fellow in the laboratory of Dr. Eric Jan at the University of British Columbia. Her research is focused on the identification of novel viral protease cleavage substrates during picornavirus infection. We sat down with Dr. Jagdeo to discuss her project, and its implications for future viral therapies.
Why are you interested in studying picornaviruses?
To answer that question, you have to understand that viruses are very opportunistic. They have minimal genes, so they need a host to make more copies of themselves. When a virus infects a cell, they manipulate a variety of cellular pathways and hijack host proteins to facilitate processes in the viral life cycle. One way in which viruses are able to do this, is through the activity of virally-encoded proteases, which not only processes and modulates its own viral proteins, but also those of its host to either block the host antiviral response, or usurp protein function.
This mechanism of host protein hijacking is very well studied in the picornavirus family. This is a family of viruses that infect vertebrates, and many of its members are clinically and agriculturally relevant. The foot and mouth disease virus, avian encephalomyelitis virus, coxsackievirus, rhinovirus, and poliovirus are all members of the picornavirus family. Poliovirus in particular is a very well studied member of the family that is often used as a model virus to study various aspects of viral infection. It encodes for two proteases, one of which is the 3C protease, and we already know of approximately 45 host proteins that it targets that are involved in a variety of cellular pathways. Considering all the host pathways that these proteases are thought to affect, we hypothesized that there must be even more unknown host proteins targeted for cleavage.
What was your strategy for identifying these proteins?
We used TAILS, which is an acronym for Terminal Amine Isotopic Labeling of Substrates. This is a gel-free mass spectrometry approach developed by Dr. Chris Overall’s lab at UBC, and it’s specifically designed to find protease substrates by enriching for N-terminal peptides. For our studies, we used cell lysates that were treated with either wild-type or catalytically inactive poliovirus 3C protease. Once cleavage of substrates was allowed to occur, we added a dimethylation label to the lysates, which binds free amine groups that are found either at the natural N-termini of proteins or the N-termini of protease-generated cleavage products. To better identify the protease generated cleavage products, we added a heavy dimethylation label to the wild-type sample and a light dimethylation label to the mutant sample. This would allow us to more easily identify peptides enriched for in the wild-type sample compared to the mutant sample. We then add trypsin to process the proteins into smaller unlabeled peptide fragments, which can then be removed using a polyglycerol aldehyde polymer that covalently binds to their free N-termini. You are then left with an enriched sample of both natural and protease-generated N-termini peptides remained, which were then analyzed by mass spectrometry. Following peptide identification and statistical analysis, we generated a list of candidate substrates for the poliovirus 3C protease.
Was TAILS successful in identifying true protease substrates?
Yes! We generated a list of 72 candidate substrates, and we were able to validate at least seven of them as new substrates of the poliovirus 3C protease. When we knockdown several of these candidate proteins, we see changes in the production of new virus particles. We have also shown that expression of a cleavage resistant form of one of the validated substrates decreases viral infection. So TAILS has help us identify host protein substrates that are important for the viral life cycle.
Did the list of cleavage targets provide insight into the host pathways targeted by viral infection?
One of the most surprising things was that the protease doesn’t appear to target one particular cellular process or pathway. This suggests that the virus has to target several different pathways and functions in order to successfully complete the viral life cycle.
Another interesting finding was that when we repeated the experiment using cardiomyocyte extracts and the coxsackievirus 3C protease, we identified three proteins that were also identified within our poliovirus TAILS experiments using HeLa extracts at the exact same cleavage site. This suggests that many host substrates of 3C, and their cleavage sites, are conserved between these two closely related viruses.
Could knowledge of these host cleavage substrates be used to design antiviral therapeutics?
Yes! Because TAILS identifies substrates at their cleavage site, we can determine a consensus cleavage sequence for the viral protease. We can use this knowledge to generate a peptide substrate analogue that is highly specific for the protease. If we fuse this peptide with an inhibitor, we can inhibit the catalytic function of the protease. Without the catalytic function of the viral protease, the virus won’t be able to replicate.
Having said that, the problem with developing new direct antiviral therapies is that viruses often develop resistance since they’re prone to introducing mutations when replicating their viral genomes. But if we know of particular host pathways that are upregulated or modified under virus infection, an alternative to direct antiviral therapies would be to target cellular proteins within these pathways instead of targeting the virus. TAILS can help us identify cellular proteins and pathways that are modulated during infection.
Since we have now validated TAILS as a tool to look at host substrates of viral proteases, we can look at host substrate targets for viruses with less well-characterized proteases, like Zika virus and MERS.
Thank you for taking the time to speak with us, Dr. Jagdeo! We wish you all the best in your future career!