The Structural Basis of Ebola Viral Pathogenesis by Dr. Saphire (Transcript)

Event: NIH Wednesday Afternoon Lecture

Subject: The Structural Basis of Ebola Viral Pathogenesis

Speaker: Erica Ollmann Saphire, Ph.D., Professor, Department of Immunology and Microbial Science, The Scripps Research Institute

Date: Wednesday, November 06, 2013, 3:00:00 PM






Dr. Collins: Good afternoon, everyone. This is a special day because we are in the first day of the NIH research festival and a special day because we have a remarkable lecturer as part of our regular Wednesday afternoon series who is here to teach us something pretty interesting about viral hemorrhagic fever, specifically Ebola virus.

Erica Ollmann Saphire has an interesting and very productive career bringing her to where she as a professor in immunology and microbial science at The Scripps Research Institute. We did a little digging and found a profile of her in the San Diego Union Tribune where she was called, the virus hunter and various comments were made about her contributions, which are obviously substantial. I won’t comment upon what they called her namely a steel magnolia. I thought that was odd to be putting in a profile of a scientist but you can decide for yourself later on.

She got her undergraduate degree at Rice with a double major in Biochem and Cell Biology and Ecology and Evolutionary Biology and then Ph.D. at The Scripps in the year 2000 and has been there in this remarkable productive enterprise focused on trying to understand how pathogens evade and usurp the innate and adaptive immune responses. She has quite a diversity of projects going on in the lab including Lassa and Marburg and Ebola fever and she is an expert in incorporating different approaches to understanding this, including immunology virology and X-ray crystallography.

So it is a great pleasure to have her here and I want to just point out that at the end of the lecture, we will have time for some questions and there are microphones in the aisle and again welcome to those of you who are watching on the web. We’ll try to be sure that questions are posed from the microphone so you can hear them and then at 4:00, we’re going to adjourn down the hall for continuation of informal conversations with our speaker but also the actual formal unveiling of the new FAES center, which I think you’ll want to come and have a look at because it is really quite beautiful facility and we’ll have a ribbon cutting, we will have a few hopefully short speeches and that will morph into a poster session where the scientific directors are actually themselves standing by their posters talking about their science giving you a chance to hit them up with really hard questions. So it’s going to be quite an afternoon.

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But, to get us going here, let me ask you please to give a warm welcome to Erica Ollmann Saphire.

Erica Ollmann Saphire – Professor, Department of Immunology and Microbial Science, The Scripps Research Institute

Thank you, Dr. Collins. It’s a real pleasure to be here. My laboratory works on a lot of different viruses. Today I’m going to show you examples from two of them. The first one is Ebola virus, it’s a long filamentous Filovirus and the second one is Lassa virus – this is smaller rounder particle and it belongs to the arenavirus family.

Now what these viruses in common is a similar disease. They both cause hemorrhagic fever and the symptoms look quite similar especially at first. Whereas Ebola is thankfully quite rare, Lassa is unfortunately extremely common. There are hundreds of thousands of cases of Lassa fever every year in Western Africa and it’s the hemorrhagic most frequently transported to the United States and Europe.

Now what else these viruses have in common is a very small genome. Ebola encodes just seven genes; Lassa only 4. So whereas you have 20,000 – 25,000 genes and you can make 20,000, 25,000 proteins. These viruses make only a few.

So, using this very limited protein toolkit, how does this virus achieve all the different functions of the virus life cycle from attachment to a new host cell, fusion, entry, replication and coding, transcriptions, assembly and exit and some of the more sophisticated functions of immune evasion through lots of different pathways – how do they do that with only a very few proteins at their disposal?

This is the genome of Lassa virus. Those are at 7 genes; this is the genome — that was Ebola and this is Lassa, it’s 4 genes in RNA segments.

So how does a handful of proteins conspire to create such extraordinary pathogenesis in hemorrhagic fever? The answer is that each protein at these viruses do encode is essential. These viruses have no junk. Many of these proteins are multi-functional and some are extremely adaptable. By studying the proteins that these viruses make, we see the vulnerabilities of the virus, the Achilles’ Heel, the place where we can target a drug or vaccine or antibody.

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But perhaps more importantly, we can understand something more about proteins themselves. Because evolution has compelled these proteins to be remarkable, to do more with less than other proteins. By studying what these proteins are capable of, we learn something more about the capabilities of proteins and molecular biology in general.

So I’ll show you a few examples. The first one comes from the first step of the virus life cycle. So the first step – the virus has to find and attach to a new host cell. This is achieved by the glycoprotein called GP. Both of these viruses express only one protein on their surface, the glycoprotein called GP and it is solely responsible for attachment and infusion with that cell.

So Ebola virus is filamentous. This is a cartoon of what the virus might look like, it’s got a membrane envelope, that’s green surrounding the nucleocapsid. And studying to the surface of these glycoprotein spikes. Those for Ebola virus form 450 killadalton trimers and they are quite heavily glycosylated.

So the question you might ask is – if this spike is important for attachment and entry, what does it look like and how does it work?

We had to make about 140 versions of this GP to get one that would crystallize well and we had to grow about 50,000 crystals to get one with the [frac bone]. Before we have a structure, we typically think of a protein as a schematic like this with an N terminus and C terminus. This GP is cleaved in the producer cell, with GL2 sub units. A GP1 which mediates receptor binding and GP2 which mediates fusion. So the GP1 has receptor binding domains and the GP2 has heptad repeats that undergo a conformational change and collapse the six-helix bundle driving membrane fusion. Also in GP1 is this unusual mucin-like domain, it’s very heavily glycosylated. Each domain is about 75 killadalton, it’s 3 in the trimer, there is a lot of unstructured protein and carbohydrate.

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So this is the crystal structure of the Ebola virus GP. The first one we solved — you can see the 3GP1 subunits in blue and green, these mediate receptor binding and they are tied together at the bottom by the GP2 fusion subunits.

Now there is something interesting here. When you think about a fusion peptide for Flu or HIV, it’s a hydrophobic peptide that’s tucked up inside the structure. Well here the fusion loop is tacked under the outside like a [flice water]. The one that belongs to this monomer reaches around the outside of the trimer and binds into the next one over. In order to get this molecule of the crystallized, we had to exize that mucin-like domain.

But we want to understand what the real GP looks like on the viral surface. So it’s got these heavily glycosylated domains attached at the top. Well note GP containing that mucin domain crystallizes, so we had to use a different technique which was small angle X-ray scattering, tiny x-rays and protein molecules tumbling around in solution, you get a low resolution view, maybe 10 angstrom resolution. And in this, that it turns out this is the solution scattering envelope of the complete fully glycosylated Ebola virus GP, with all of its sugars and all of its mucin like domains. So the crystal structure I am showing you in the ribbon, in the center for scale, these are the mucin-like domains attached. So they effectively triple the size of the molecule. When Peter Kwong coined the term glycan shield, this is hell of a glycan shield. They reach about 100 angstrom away from the core of GP and they are quite flexible. So I would actually expect the actual width of this domain to be half that, I think we’re visualizing solution, a lot of flexibility in waving our [camera].

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