The Viral Genome: Moving Beyond Reading Code – Why Not Rewrite It?

When you think of ‘coding’ and ‘decoding’ the first thing that probably comes to mind is computer programming. But what if we could recode viruses? That is exactly what scientists from the Universities of York and Leeds have done. 

Researchers have discovered how to read, decode, and manipulate previously ‘hidden’ viral code. Contained within a virus’s genome, that codes for viral components, sits another hidden code that provides an instruction manual for the formation of viral particles. Professor Reidun Twarock, Professor Peter Stockley and colleagues have not only deciphered these ‘instructions’ — they have rewritten and repurposed this code allowing them to directly regulate viral assembly efficiency. Put simply, they have been able to produce artificial instructions that surpass those found naturally.

“Discovery of the packaging signal code was a joint effort, we carried out the modelling and experimentation together.  Once we had identified the assembly code in satellite tobacco mosaic virus (STNV), we thought, what if we can actually turn the table on viruses and improve the code to make the assembly of viral particles more efficient?” Prof. Reidun Twarock, University of York, UK.

Viruses, among the simplest biological entities, are surrounded by a ‘coat protein’ (CP) shell which, in the case of the virus studied by the team, contains single-stranded RNA (ssRNA). A range of processes make up viral lifecycles, including genome encapsulation and capsid assembly. Recently it has been discovered that the regulation of capsid assembly is, in many ssRNA viruses, controlled by multiple RNA-CP contacts. Scattered across multiple viral genome RNA sites, these sequence-specific contacts, termed packaging signals (PSs), specifically bind CP via a common ‘recognition motif’. This research was published in Proceedings of the National Academy of Sciences (PNAS).

We spoke to Prof. Reidun Twarock, to find out more about this research. Reidun shares the ways in which they investigated these recognition motifs, establishing their importance in viral assembly. She explains the methods used to discover the recognition motifs and identify their cooperative function in enhancing PC assembly. She also discusses the implications this discovery could have for vaccination and gene therapy.

Laura Mason (LM): Could you tell me a little more about your background and the project?

Reidun Twarock (RT): I’m a professor of Mathematical Biology with a background in Mathematical Physics, with expertise in bioinformatics, mathematics and physics. [In this project] my team does the theoretical aspects of the work and the Lab of Peter Stockley — a biochemist, does the experimental work. We have been working together for over 15 years on these discoveries. We feel that this is a topic, where a highly integrated, iterative interdisciplinary approach is required, and we therefore work hand in hand, with theory and experimental angles complementing each other.   

Discovery of the packaging signal code was a joint effort, we carried out the modelling and experimentation together. Once we had identified the assembly code in satellite tobacco mosaic virus (STNV), we thought, what if we can actually turn the tables on that virus. Can we improve the code to make particle assembly more efficient? A virus has the added constraint of having to release the genome at the required point in its life cycle, so that it has to sit on the ‘knife’s edge’ between instability and stability. But, without that problem, you can create a protein shell that is even more stable. We therefore showed that we can optimise the code, thus improving assembly efficiency, which can be exploited in the production of virus like particles.

In many ways it is difficult to produce these particles at sufficient yield and stability. Our invention provides a way of overcoming this bottleneck, by repurposing and optimising this packaging signal assembly code.

LM: Could you tell me more about the code?

RT: Picture the viral genome. The code consists of signals that are given as secondary structure elements presenting a common sequence motif. Obviously, these folds are part of a dynamic ensemble, so there is varying stability for signals in different positions depending on how likely they are to fold and unfold. This is very important, because it tells you the likelihood of having signals of the code present at certain positions of the genome. Identifying these folding propensities in the genome, and identifying the PSs and ways of ablating them through mutation, was my contribution.

LM: How did you use this information to regulate viral assembly?

RT: We know that these signals are required at specific positions in the genome. In particular, there are five such signals in the 5’ leader of the genome, that Peter calls the cassette. As this part of the genome also contains other structural elements with regulatory function, the virus is constrained in how much it can vary the packaging signals. But, if your goal is to create stable particles for vaccination or gene delivery purposes, you have the freedom to make more changes. So, we stabilised these signals. Peter then did competition experiments demonstrating that mutated viral genomes containing these edited signals are much more efficient at forming viral particles, meaning you can optimise the code in ways the virus couldn’t in evolution, as it has other constraints.

But then the question was raised; is it only signals in the 5’ leader that are key to assembly, or are other signals important as well?

To answer this question, we started exploring how to identify and mutate the other signals in the genome. The goal was to induce mutations that leave the secondary structure broadly unchanged, so that we could exclude any effects arising from an inability to compact the genome appropriately to fit into the capsid. We were able to make such mutations of the recognition motifs. We were able to show that even if you have the cassette at the start of the genome, particles are no longer assembling efficiently when the other signals across the genome are missing.

What we have therefore proven is, that you need the whole assembly code. But, by specifically adapting and optimising the cassette at the 5’ leader, you are able to optimize formation of the particles. This opens up new opportunities in drug delivery and gene therapy.

LM: What potential applications can this new understanding be applied to?

There are two important applications for this technology.

The first is vaccination. In this case, it is the epitopes on the outside of your particle that are important, so you can therefore strip away any feature in the viral genome that makes the particle infectious, as long as the signals are kept at the correct places. Particles assemble with more ease around such modified genomes and are more stable than the real virus, and this approach can therefore overcome bottlenecks in the production of safe vaccines.

The second application is gene therapy. In this case, the container is used as a transport vehicle, and those signals are crafted onto the cargo to be packaged — a drug delivery or gene vector approach.

LM: Are these applications, vaccination and gene therapy, things that your team will be taking forward?

RT: Yes, we are currently applying this technology to specific viruses of medical interest. The whole process, from discovery of the packaging signals, all the way through to the exploitation of these systems has been a highly interdisciplinary adventure between Peter and myself. Our collaboration has been instrumental to the success and advancement of the project.

Prof. Reidun Twarock was speaking to Laura Elizabeth Mason, Science Writer for Technology Networks