Speaker: Elizabeth R Wright
Department: Department of Pediatrics, Emory University
Subject: The Pathways Traveled, Structural Studies of Mononegales Virus Assembly
Location: Delft University of Technology
Date: 19 May 2017
Author: Romano van Genderen
Professor Wright started by giving us an overview of different size scales and the techniques used when studying life on that scale. One thing she pointed at is that electron microscopy, her field of interest, is improving on two different terrains. It can now both make pictures of smaller and smaller structures, but also on a bigger scale.
Afterwards, she discusssed the advantages of Cryo-EM. She told us that since it does not use any staining methods, it can measure a specimen in its native state, unlike techniques such as crystallography. Also, there are few to no artifacts visible from the preparation of the specimen.
Next, she showed us the recent advances in the field of Cryo-EM. We now can not only prepare better samples due to techniques like active substrates, but also gather better data thanks to phase plates and process the data more efficiently, not only because of more processing power of contemporary computers, but also because of advantages in auto-segmentation algorithms. But according to her, the most important improvement was the invention of direct electron detectors. These DDCs are far more useful than the previously used CCD cameras, which turn electrons into photons that are then counted. This conversion leads to loss of signal. These new cameras therefore give better signals, and can also be used to record videos in real-time with framerates up to fifty frames per second.
Her lab is currently using these new tools to their best abilities, leading to research on correlative microscopy, overlaying EM and fluorescence images to better locate compounds in the cell. They also study enveloped viruses, the topic of the rest of her talk.
She started by explaining the structure of such a virus, a completely new topic to me. To be specific, she studied paramyxo and pneumoviruses. These viruses have glycoproteins on their surface, a lipid envelope, matrix proteins and a nucleocapsid protein that surrounds and protects their RNA genome. These viruses are very common, for example the common measles belongs to this class.
Figure 1: The structure of a myoxivirus
This class has commonly been regarded as hard to purify. Firstly, people are unsure whether or not it still looks like its native configuration after purification. Also, there are a lot of artifacts and damage introduced by current purification methods. And to finish things off, you also only get very small numbers of virus particles back from it.
This is why professor Wright wanted to improve this method. She used a method currently used in protein purification, using nickel-NTA along with histadine tags binding to it. But in her case she incorporated the nickel-NTA into the cryo-EM grid and added HIS tags to the surface glycoproteins. This makes the grid attract the viruses and leads to far better yields and also removes a lot of artifacts from the sample.
Next, she wanted to study the interior of these viruses during virus assembly and release. There were two common hypotheses on the location of the membrane protein during these processes. One says that the matrix protein covers the inside of the capsid. The other says that it forms a protective layer around the nucleocapsid protein for even more protection. Using cryo-EM, she was able to directly see the matrix protein, and that it does not cover the nucleocapsid protein.
Also, she saw a mesh of fusion proteins, proteins that play a role in binding to the host. These proteins form a two-layered lattice, but there is something weird about this lattice. It seems to have a hole in it. This hole does seem to be the same size as the protein on the host’s surface, suggesting that this a binding pocket.
The final part of her talk made the topic a bit more practical. She talked about how her techniques were used to discover more about the virus known as RSV. This virus causes asthma in newborns and there is currently no vaccine known against it. Her research found that this virus is filamentous when secreted. Also, that its structure can be discovered in large detail by using cryo-EM. One peculiar fact she found was that the RSV-F fusion protein forms a hexamer-of-trimers when in its pre-fusion form. This knowledge can be used to develop a vaccine for this virus.
I did really enjoy the first half of the talk, where the techniques and their advantages were discussed. I did notice a lack of disadvantages, a fact that I find very suspicious to say the least…
On the other hand, the second half was not that interesting, because the main points got drowned in all the details about the virus and its shape. Also, the images were not understandable, even when she told what we were supposed to see. But perhaps that was the fault of the lighting or the screen in the room.