The beauty and the beast – cryo-electron tomographic reconstruction of two enveloped viruses

Speaker:      Sai Li

Department: Bionanoscience

Subject:       The beauty and the beast – cryo-electron tomographic reconstruction of two enveloped viruses.

Location:     TU Delft

Date:            03-03-2016

 Author:        Katja Slangewal

The spikes on the surface of a virus, even if you buy the most expensive light microscope, you won’t be able to see them. Sai Li from Oxford University told us what you can do instead. As the title of his seminar already reveals, there are ‘beautiful’ viruses and, as he calls them, ‘beasty’ viruses. Sai Li used the ‘beautiful’ Tula virus to explain how you can reconstruct the spikes and he used the ‘beasty’ Lassa virus to show what these reconstructions can tell you.

The envelopes of viruses can be visualized by using cryo-electron tomography and subtomogram averaging. The main idea of this process is to combine x-ray crystallization and cryo-EM imaging. See table 1 for the advantages and weaknesses of both methods.

Table 1: Advantages and weaknesses of crystallization and Cryo-EM

Crystal structure by X-rays Cryo-EM
+ High resolution + sample preparation, complete, in situ
– sample preparation, partial structure, artifacts – Low resolution

First I will explain the process for the Tula virus (the beauty) and afterwards I will continue with the Lassa or ‘beasty’ virus. The first reason why the Tula virus is a beauty is because it is a non-pathogenic virus. The virus also lacks a matrix, the glycol protein spikes serve as a matrix instead. This makes it easier to retrieve the structure of the spikes. The spikes of the Tula virus exist of a pair of glycoproteins: GN and GC. GN is a tetramer which attaches to the surface of the virus and GC is a class-II fusion protein. GC enables the Tula virus to merge membranes with a host cell.

In order to visualize the spikes of the Tula virus, pictures of the frozen virus are taken using the Electron Microscope. These pictures are sorted in two groups: containing spikes and not containing spikes. An average of the ones with spikes is taken to model the surface of the Tula virus and to form a 3D density chart of the virus. Sai Li found 3 regions on the surface that contain spikes. He called it an elegant spike interconnection, because it was EM friendly.

Than the crystal structure of GN of the Puumala virus (which is 80% identical to the Tula GN) was used to find the best mathematical fitting of the protein in the density chart formed before. At first the third domain of GC did not seem to fit. However this domain is connected by a flexible linker, which is hard to crystalize. By using some clever tricks Sai Li managed to proof that also this third domain does fit into the density chart. In the end a well conserved area of the spike was clearly exposed. This indicates an antibody binding site, which is useful for virus research.

Secondly the Lassa virus was discussed. The Lassa virus is ‘beasty’ because it is dangerous for humans and there is no treatment yet. Also this virus has a defense mechanism which produces fake viruses with almost the same features, which makes it difficult to purify the virus. It took Sai Li and his group a year to purify this virus.

The surface of the Lassa virus was reconstructed in the same way as the Tula surface, although there were more difficulties with this ‘beasty’ virus. In contrary to the EM friendly spike distribution of the Tula virus, the distribution of spikes on the Lassa virus is totally random. This means that information of neighbors can’t be used by reconstructing the shape of the spikes.  Another ‘beasty’ characteristic was the fusing pH of the Lassa virus. Normally viruses fuse at a pH of 4-5, the Lassa virus fuses at a pH of 3-4. Finally the Lassa virus can’t fuse when the protein Lamp1 is absent. The question is when the spikes do enable fusion and how important the pH and the Lamp1 protein are. By using cryo-electron tomography Sai Li found the answers.

Sai Li used virus-like particles without a genome to avoid danger. He looked at the spikes while changing the pH to see what happened with the volume of the spikes. See table 2 for the results of this experiment.

Table 2: volume changing after changing the pH

pH 7 5 3
Volume 100% 88% 46%

Sai Li also made combined images of the spikes in different pH, like he did with the Tula virus. Apparently the spikes open at a pH of 5 and they lose subpart GP1, which explains the difference in volume. There already was a supposed binding site of Lamp1, based on the amino-acid composition of both proteins. During the research it became clear that this binding site is only available after opening of the spike (see figure 1). This information is important in order to find treatment for this ‘beasty’ virus.

Lassa virus spike at different pH

Figure 1: The Lassa spike opening at pH 5, at pH 3 the GP1 subpart is lost. http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005418

I liked the talk of Sai Li, it really showed how physics and biology are combined to Nanobiology. I was able to follow most of what Sai Li told and the ‘red line’ was totally clear. Sometimes I found it hard to understand the technical terms, especially during the part about the method. However, he used really clear images and movies which made it much easier to understand the story. I also really liked that he had a sense of humor. I learned a lot during this seminar. I already knew much about viruses and a bit about EM and crystalizing proteins. This seminar brought these three together in a nice way. I find it interesting to learn more about viruses and how to visualize them, although I am not sure I would like to study this myself. It sounded like a lot of programming and physics and only a biological context. Nevertheless Sai Li gave a really nice talk.

 

 

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