Science Scribbler: Virus Factory

What are we researching?

Our research group focuses on two areas: first, using 3D image data to answer interesting biological questions; and second, developing novel software to reduce the difficulty of analysing the 3D image data we're interested in. Most of our team is from Diamond Light Source, the UK's national synchrotron. As our research incorporates different scientific disciplines, the background of our team is split between biology, imaging, and computer science.

We work with many different types of 3D imaging data. Right now, we're working with data from a technique called Cryo Electron Tomography (see below for more information) to answer questions about how cells change during a virus infection.

Aerial view of Diamond Light Source on the Harwell Science and Innovation Campus in South Oxfordshire, UK.

What virus are we studying?

The virus we're working with here is a Reovirus. Reoviruses belong to a large family of viruses that infect a wide range of animals and plants. Some members of the family cause widespread disease, notably Rotaviruses, which are responsible for serious gastroenteritis. In contrast, reoviruses themselves, whilst they pervasively infect humans, do not typically produce symptoms, indeed they are being trialled as possible anti-cancer agents, since they specifically replicate in many cancer cells activated for division. They are therefore a good starting point to try to understand the life-cycle of this family of viruses.

What is a virus factory, and why are we studying them?

By virus standards Reoviruses are quite large and are made up of several layers of different protein subunits that self-assemble (capturing and protecting the genome of the virus in the process) in factory-like structures that appear in an infected cell just a few hours after infection. As time goes on particles are released from the cell in increasing numbers, as triple-layered particles. These particles have been isolated, purified and their detailed structure determined. They are icosahedral, which means that the protein subunits are arranged in a symmetrical way, simplifying the problem of self-assembling hundreds of protein subunits to form the virus’s coat.

Detailed structures of reovirus at two different stages. These structures were determined by averaging together many copies of each stage of the virus to reveal details of the structure.

What we are doing now is to use cryo-electron tomography to visualise virus particles in very thin slices cut from infected cells that have been frozen. Our aim is, ultimately, to understand the full life cycle, how the virus gets into the cell, replicates, assembles more copies of itself and finally leaves the cell. In the 3D pictures here we have taken a snapshot 12 hours after infection and are aiming to visualise intermediate steps in the assembly process which have not been visible before, and to then work out how virus assembly is organised in time and space within the cell. Within this snapshot, there will be multiple stages of the virus.

Here are some example cartoons and reference images of each of the stages:

• The earliest stage is the star-like structure (leftmost in image above). This is the newly formed, empty shell of the virus.
• The next stage is the circular, but still empty structure (2nd from the left in image above). This is the expanded form ready to be filled with the RNA genome of the virus.
• Next is the same circular, but now filled structure (3rd from the left in image above). This is the virus filled with its RNA genome.
• Last, is the circular, filled virus with a membrane around it (rightmost in image above). This is the fully packaged, enveloped virus.

How do we take images of cells in 3D?

Electron Tomography is a technique that allows us to collect many different tilted images of the same sample and then later piece everything back together into a 3D volume. This important technique gives us a more life-like picture of what things looks like, even very small things like viruses inside of a cell. We're using this technique at very cold temperatures with the help of the cryogen liquid nitrogen. For more information about tomography, watch the video below!

Before the data can be collected using cryo electron tomography, we had to prepare these cells by first freezing them and then using another technique called Focused Ion Beam Milling or FIB Milling for short. This technique uses an ion beam to remove parts of the cell that we're not interested in imaging, leaving only a small slab of the cell that we're interested in. This slab has to be really thin, otherwise we couldn't use electron tomography to see through it. All of this is done at very cold temperatures using liquid nitrogen!

Here's a picture describing the whole process of preparing the cells: freezing them (in liquid ethane) and then milling them using the FIB to leave just a thin slab (lamella).