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Structural Openings to Understand and Prevent Tick Borne Encephalitis

Periodic Reporting for period 1 - 2STOP_TBE (Structural Openings to Understand and Prevent Tick Borne Encephalitis)

Reporting period: 2018-05-18 to 2020-05-17

Infectious diseases have been accompanying mankind throughout its whole history. We know of many devastating epidemics caused by pathogenic bacteria of viruses. But we but also know of glorious successes in fighting and preventing such epidemics that become possible as we learn more about the microbes that cause diseases. One of the diseases threatening humans is tick borne encephalitis (TBE). As comes from its name, TBE is an inflammatory disease of central nervous system that is spread by ticks. This severe, often debilitating or even fatal disease is caused by tick-borne encephalitis virus (TBEV). Although there is a vaccine to prevent TBE, only a small portion of people is protected, and there is no antiviral treatment to offer to those who will get ill. Europe is at risk, as areas where TBEV is found localise to many European countries. In addition, due to the global warming, ticks transmitting the virus expand their habitats and bring the disease to new areas. During the last three decades, the number of TBE cases has increased over four times and is likely to increase more. There is an unmet need for antivirals against TBEV, but their development was for long time hampered by the our very limited knowledge about this virus.

Developing antivirals is a challenging task. There are several strategies to tackle it, but none of them can be successful without detailed understanding about the virus we are targeting. We need to know the fine details about how the virus is built and how viral proteins function. We need to know how viruses mutate and identify which parts of the virus are likely to change over time, and which are not. We need to know which proteins of our bodies are important for the virus to grow, and which will inhibit it.
The goal of the project 2STOP_TBE was to provide important knowledge about TBEV that can be used for discovery of therapeutic measures against this virus. We sought to provide the details about the architecture of TBEV, about the important changes of viral structure happening the life cycle, describe how viral proteins function and mutate, and identify which cellular proteins are important for the virus. Such knowledge will allow to target the virus, viral proteins or even host proteins supporting development of antivirals.
This project combined molecular virology, which studies viral infections in biological systems, and structural biology, which investigates 3D structure of proteins and their complexes. A central goal was to study the structure of TBEV particles at different stages of life cycle. As viruses are very small and cannot be seen using regular light microscope, we looked at TBEV particles and proteins using cryo-electron microscopy. In this method, biological samples are frozen at -190C preserving their natural state, and then are imaged using electron microscope. Like words are made of letters, proteins are long chains of small molecules called amino acids. The resolution of contemporary electron microscopes is so high, that using electron micrographs we can build 3D models in which individual amino acids of proteins are identified. In this project we built such a model of TBEV virion, which is infectious, and also a model of non-infectious immature TBEV. By comparing these two models we know which exact parts of the virus conformationally change to make it infectious. This knowledge can help to design drugs that will not allow the virus to maturate and thus will inhibit infection. Having built a high-resolution model of TBEV virion, we can make a similar model of the virion in complex with antiviral drugs, explaining their mechanism of action. This will be done in collaboration with another group from Europe that develop antivirals. Another important information that we obtained, is electron microscopy data about how viral genome is encapsulated in the TBEV particle. Such data can provide information about how the viruses packages its genome in the virion. Genome packaging is very important for viruses, as if they fail to do it, they cannot be infectious anymore. These data allow can be used to develop another strategy of TBEV inhibition by preventing correct genome encapsulation.

As viruses are so small, they have to manipulate the infected cells in order to replicate themselves and to escape from immune responses. Understanding virus-host interactions can provide insights into viral inhibition, and we addressed them from two different perspectives. First, we looked at the structure and function of viral proteins that are involved in virus host interactions. We purified TBEV polymerase NS5---an enzyme that copies viral genomes---and developed and activity test that allows us to screen libraries of chemical compounds to find NS5 inhibitors. We also looked at viral protein NS1, which is a major regulator of TBEV-host interactions. We obtained initial data about NS1 and NS5 structure and continue working towards the high-resolution 3D models of these proteins. Viral non-structural proteins such as NS1 and NS5 usually mutate slower that proteins of the virion, and therefore represent promising targets for antivirals.

Second, we investigated which cellular proteins of the cell are needed by the virus. For this, we turned off almost every single gene in the cells one by one using a method called CRISPR/Cas9 genetic screening. Turning off some of the genes rendered cells resistant to TBEV, and we identified 13 cellular proteins that are critical for this virus. Inhibiting these proteins can be another promising strategy for TBEV drug discovery. Cellular proteins are usually less prone for mutation and therefore such host-directed antivirals can be efficient in a long run.

Finally, are viruses always mutate, we addressed which parts of viral proteins are genetically stable, and which are variable. We compared sequences of some viral proteins from different TBEV strains using a method called sequence alignment and mapped the results of structural models of viral proteins. Such an analysis allows to identify druggable regions of viral proteins and provides useful information to predict drug and vaccine efficacy.
The project provided previously unavailable information about TBEV structure and function, which can be used by academic and industrial parties for discovery of antivirals and improvement of vaccines and diagnostics. One scientific article is already published in open access journal and is available for reading to everyone. Other data from the project is being prepared for publication in open access scientific journals and for deposition in publicly accessible databases. In addition to the impact on drug discovery, this project also impacts the connectedness of European science. Parts of the project were carried out in collaboration with different research groups in Finland and Sweden, we are also collaborating with a research group in Belgium on antiviral discovery and will continue to expand our collaboration networks further. Spin-offs from the project will give rise to new projects in which we will continue to tackle pathogenic viruses.

Project results were disseminated beyond academic community, increasing general public awareness about TBEV and other pathogenic viruses, as well as about how viruses are studies and how therapeutics can be developed.
Tackling deadly viruses is challenging but fun