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

Video link: Youtube





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.

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.

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.

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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.

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].

The salient feature of this is that these mucin-like domains are massive and they dominate the structure of the GP.

So this is what is on the virus surface. How does it work? How does it find and get into a new host cell? Well, this I’m showing again the crystal structure, I am coloring the surface white, patches that are colored pink, are areas that mutagenesis tells us are important for infectivity. They are a little bit sequestered inside the bowl shape the trimer makes. The rest of these are most important for a receptor binding are very sequestered. In fact, inside structure underneath this domain. So that is sort of a representation of where the mucin-like domains are. The parts that are important for the receptor binding are these pink ones and they are underneath these domains called the glycan cap, these have a lot of glycan attached to them.

So, does this make sense and how on earth is this a functional receptor binding site underneath this entire canopy of protein and carbohydrates?

The answer is that it is known from biology that GP needs to be cleaved by host capsaicin enzymes for infection to occur. The filovirus is different the requirement for some cleavage but it’s especially important for Ebola virus.

So, why? Well, in solving the crystal structure, we see that all of this structure, the glycan cap and the whole mucin like domain are attached by a single polypeptide tether that connects residue 189 to 213. And that piece of polypeptide is disordered. So something that is disordered in a crystal structure is flexible and it’s moving around. So this looks like a pretty attractive cleavage site.

If proteases were to cleave on that yellow loop, this would be the effect, which is much better exposure of the receptor binding sites. Now we are not making that up. This is actually the crystal structure now cleaved GP and another lap simultaneously shows that yes, cleavage indeed strips off 85% of the mass of GP1 leaving the receptor binding sites exposed.

So this is what the protein looks like on the viral surface. What do we learn from this? Receptor binding probably doesn’t happen at the viral surface. Just by looking at the structure alone, you can see spots needed to bind that receptor are not accessible. They are not well exposed in this kind of protein. Instead, the virus that bears this surface enters cells by macropinocytosis.

Once in the endosome, this is cleaved to strip off all that surface sugar in these mucin-like domains leaving the receptor binding site well exposed and allowing binding by the receptor Niemann-Pick C1, NPC1. And this binding site is right there where the glycan cap used to be.

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So what we see here is one polypeptide, that results in two different biologically relevant manifestations. This is the molecule subject to antibody surveillance and this is the molecule that’s functional for receptor binding.

So what does that mean to the immune response? Well, nothing good. Many can be clipped right off. In fact, in a lot of vaccination studies, these sites can be immunodominant. You can see that any antibody that binds to these mucin-like domain epitopes is going to be cut right off in the endosome leaving a perfectly functional receptor binding core that is now antibody free. So those kinds of antibodies don’t neutralize.

The essential conserved sites are not well exposed. So for example, all of these filoviruses share the same receptor, so that’s a conserved binding site, it’s an essential site for the molecule. You’d love to target that with an antibody. It’s completely or partially hidden under the glycan cap and the viral surface, so the antibody might not see it unless you found a way to engineer the antibody.

Because of this conundrum, we are left with a bit of a puzzle that neutralization and protection don’t always correlate for Ebola virus. So neutralization is your ability to inactivate the virus in vitro. Protection is your ability to save the animal in vivo. So for example, antibodies like this, this is the human kz52 from the survivor of the Kikwit, Zaire outbreak neutralizes brilliantly and doesn’t protect the primate. Antibodies like these, including two that bind the mucin like domains, and one that binds up with the glycan cap don’t neutralize but they do protect the primate. So this doesn’t make a lot of sense, leaving you wondering what works here.

We had this result years before and it really cooled everybody’s opinion on antibodies against Ebola virus, maybe it wasn’t even going to be possible to protect animals but then it turns out that you can. These are quite protective even if you wait long enough for hemorrhagic fever to develop. The difference might be that these are given in a cocktail as this was given alone. So does that mean we have to have a cocktail? It’s the replicative capacity or the length of the number of spike of Ebola virus such that we need to have multiple antibodies against multiple sites and if so, which ones do we put together? I mean two-thirds of this cocktail is mucin, does that mean that mucin works? Or is this one the champ that binds the top? We don’t know.

Now in the field we have about 200 different monoclonal antibodies identified against the virus. What do you put together in a cocktail. Well now I’m going divert a little bit from my theme about the proteins of the virus and then tell you how we can use the structure to get at that problem.

This is the website that the viral hemorrhagic fever immunotherapeutic consortium – you will be able to find this link through Scripps very soon. What this is that more than 20PIs in 7 different countries have gotten on the same page. We put almost all the antibodies known against these viruses together in one pool. We blinded them and then compare them side-by-side to see what is more effective. We’re going to try to see if we can correlate efficacy in-vitro and in animals and – to understand how to put together the right cocktail. Right now we have three from the army in a cocktail that neutralized and we have three from Canada in a cocktail that neutralized what if the most effective cocktail is one from japan and one from the army and one from Hamilton. We won’t know until we put them all together. And so it’s nice that everyone is on the same page in the same study.

Okay. So until we make that cocktail, let’s assume that viral infection will proceed. So the next step of viral infection — after the viral membrane is fused with the host endosome membrane, genetic material enters and the virus starts to replicate.

Now, something important happens here. Most people die from Ebola virus infection – 50% to 90%. Some people live. What is the difference? The difference seems to be that those people that survived the Ebola virus infection tend to generate an early and strong immune response against the virus and the viral titer starts to drop by around day 4. Those people that ultimately succumb to the Ebola virus infection are more likely to be characterized by a very poor immune response and their viral titers get quite high — 10 to the 9 to 10 to the 10 per mil at the time of death. So for this decision point to occur by around day 4, that means that the innate immune system is quite important in making this decision of survival or not survival.

So what is the viral factor at play in this innate immune decision point? One of them is a protein called VP35 — viral protein 35 Kilodaltons, that’s how it got its name. It’s a component of the nucleocaps in replication complex. That’s his day job. It also has another job – it’s interferon antagonist. And what it does in that function is to bind double-stranded RNA. Now you typically would only have double-stranded RNA in the context of a viral infection. And so it is a pathogenesis through the molecular pattern. Your innate immune system has sensors that go looking for double-stranded RNA and when they sense they mount to antiviral response, not having to know what the virus is, just the presence of a viral infection.

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