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Dr. Lexi Walls Talks Coronaviruses and Cryo-EM

By March 18, 2020No Comments

Dr. Lexi Walls is a recent PhD graduate in the laboratory of Dr. David Veesler at the University of Washington. Her research focuses on using cryo-electron microscopy to study transmission of coronaviruses. We sat down with Dr. Walls to discuss the current SARS-CoV-2 outbreak, and how insights from structural biology are helping inform vaccine development.

Let’s start with the basics. What are coronaviruses?

Good question. Coronaviruses are a large family of viruses that can infect both animals and humans. Before the early 2000s, everyone thought of them as the common cold. These coronavirus infections were mild except for cases (typically in the elderly, young children, or immunocompromised populations) where hospitalization could be required. That all changed in 2002 when Severe Acute Respiratory Syndrome Coronavirus, or SARS-CoV, jumped from an animal reservoir to humans. This was the first time that we had seen this happen with a coronavirus, and also the first time that we had seen a coronavirus be so deadly in otherwise healthy adults. The outbreak lasted about a year and a half, had a 10% fatality rate, and affected approximately 8,000 people globally. Then it was over.

Unfortunately, it happened again in 2012 with a different coronavirus, Middle East Respiratory Syndrome, or MERS. That infection is still ongoing, and it has a shockingly high fatality rate in the 30-40% range. Fortunately, it doesn’t seem to spread very easily from human to human, so less than 3,000 people have been infected even though the infection has been ongoing for eight years.

Does a current treatment or vaccine against coronaviruses exist?

There is no vaccine against any form of coronavirus in humans, but there are a couple of antivirals that have been developed and are undergoing soft clinical trials in light of the current outbreak. They’ve been shown to have a wide breadth, meaning that they block not only MERS-CoV and SARS-CoV, but also SARS-CoV-2. The problem with an antiviral is that it treats those who are already sick. A vaccine that prevents people getting sick altogether would be much better. Unfortunately, researchers have been trying to create a coronavirus vaccine since before the MERS outbreak. It’s been challenging. 

What do we know about the structure of these viruses? 

Coronaviruses are enveloped RNA viruses, and they have one of the largest viral genomes. A membrane protein stabilizes the envelope within the interior of the virus, and within this membrane is a nucleocapsid protein which binds to the single stranded RNA. I’m specifically interested in one of the defining features of the virus, which is a glycan-decorated protein that protrudes from the envelope, called a spike. Many viruses have spikes, but coronavirus spikes are especially large. This is what gives them their name – the crown (or corona) of spikes encircling them. 

We thought the spike would be a really interesting protein to study because upon infection, this is the protein that your immune system recognizes. This means that these spikes could play an important role in vaccine development. Furthermore, the spike is involved in host entry. Without a spike protein, the virus can’t get into our cells. So from both a public health/vaccine and basic biology standpoint, it seemed like a good place to start. 

What is the spike’s role in transmission of the virus?

There’s still a lot about coronavirus transmission that is unknown. In general, it begins with a host cell receptor binding event, where a domain on the exterior of the spike, known as the receptor binding domain, engages with the host cell. Upon engagement, the spike protein is cleaved by a host protease, leading to a structural rearrangement that causes dissociation between the host receptor and spike, and release of something called the fusion peptide. At this point, the membrane fusion machinery takes over to bring the host and viral membranes together, allowing the viral genetic material to enter the host cell.

What aspect of the coronavirus spike are you interested in studying?

When I started working on this project, there was no structure of the spike protein. As you can imagine, it’s really hard to understand a virus if you don’t know its appearance or moving parts. So my first project was to use cryo-electron microscopy (cryo-EM) to identify the structure of the spike, in the hopes of gaining greater insight into the virus as a whole. I was successful, and our lab has gone on to solve many more structures of the spike. These structures have provided insight into the domain architecture, the mobility of different regions, and the location and mobility of glycans.

Since then, I’ve been focusing on the mechanism by which the virus inserts itself into the host membrane. Although I didn’t mention it previously, the coronavirus spike exists in a pre-fusion and post-fusion state, and I’ve solved both of these structures. I’m currently trying to understand what happens between these two states. Can we capture any sort of intermediate structure during this large conformational change? 

What is the advantage of cryo-EM over more classical structural biology techniques like x-ray crystallography or NMR?

The big advantages over other structural biology techniques are that you don’t have to crystallize your protein, you don’t need very much of your protein, and you can see multiple states of your protein as they truly exist. This means that you don’t have to force your protein into any specific conformation — it’s as it exists in its hydrated state in nature. It’s also not size limited. You can look at a 50 KDa protein or an entire cell. It’s a really beautiful technique.

I collaborate with a couple of wonderful groups who spent years trying to crystallize the coronavirus spike, but were ultimately unsuccessful. Now that we know the structure, we believe that the spike is meta-stable by design. It has to go through this large conformational change, so it isn’t happy being in one conformation. And if a protein isn’t happy, it’s not going to crystallize. The heterogeneity of the glycans and number of disulfide bonds also make crystallization challenging. All these factors led to cryo-EM being more successful at determining structure than other structural biology methods.

If there was one research question that you could focus all of your time on, what would it be? 

If you had asked me this question a few months ago, I would have said that I was most interested in understanding and capturing an image of the transition between the pre-fusion and post-fusion state. From an imaging perspective, to be able to physically observe that movement would be amazing. And the insights gained from it could apply to any fusion protein.

Since the outbreak, my focus has obviously shifted to creating different constructs for vaccine design, and looking for any therapies that we can get into the clinic right away. What does the virus look like when it’s binding the host receptor? What does it look like when it’s binding a neutralizing antibody? Can this inform vaccine development? This is now the focus.

So your research priorities and day-to-day work have changed due to the current SARS-CoV-2 outbreak?

Definitely. The biggest change is that I’ve been working 24/7. A lot of researchers in our lab have switched focus from their normal projects to the coronavirus project because any information about the virus is helpful in trying to develop a vaccine. There have also been a lot of new funding opportunities that have come our way recently because of this work. This is fantastic, because up until now, this has been an underfunded area of research.

Thank you for taking the time to discuss your research, Dr. Walls! We wish you the best of luck as you continue to pursue your research.