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Viruses caught red-handed

In order to cause an infection, viruses need to bind to body cells and take possession of them. Prof. Thilo Stehle, biochemist at the University of Tübingen, wants to capture and accurately study the moment at which a virus binds to a cell. He is carrying out complex X-ray structure analyses and is hoping that further insights into this process might some time in the future enable the development of drugs that target the first step of a viral infection.

Prof. Thilo Stehle from the Interfaculty Institute of Biochemistry at the University of Tübingen finds viruses fascinating structures. They are unable to grow or reproduce outside a host cell and use their small genome and economic structure to intrude living cells and programme them in such a way as to enable viral proliferation. “This is an ingenious concept,” said Thilo Stehle, also highlighting the dangerous nature of some viruses, for example those causing serious diseases like AIDS and avian influenza.

Antibiotics are ineffective against viruses. In addition, the antibodies of the human immune system do not always respond effectively to these intruders. Thilo Stehle has started researching the first step of a viral infection, namely the binding of a virus to a cell. “This would, at least theoretically, be an interesting target for drugs. However, our present knowledge is far too limited and we are unable to exploit this possibility. Our work is basic research,” said the scientist whose research group is purifying, crystallising and X-raying the proteins involved in the binding between viruses and cells. The result is a detailed three-dimensional structure of the interaction on the atomic level.

More than the “key and lock principle"

Prof. Thilo Stehle (left) discussing a protein structure model with one of his colleagues. (Photo: Antonie Knierim/University of Tübingen)
Prof. Thilo Stehle (left) discussing a protein structure model with one of his colleagues. (Photo: Antonie Knierim/University of Tübingen)
Thilo Stehle’s team is researching adenoviruses, a type of virus that can lead to a broad range of infections, including colds or gastrointestinal diseases. “Adenovirus infections are generally speaking not very dangerous; but some time ago, a number of young people in the USA unexpectedly died from adenoviral infections,” said the scientist. Coronaviruses might also lead to cold symptoms; so far one case is known – the SARS coronavirus – that developed dangerous modifications from one day to the next, turning a previously relatively harmless infection into a fatal one. “The fact that the dangerous viruses were able to be rapidly eradicated was because China reacted by totally isolating people infected by SARS,” said Stehle hypothesising as to how the viruses had been contained. In addition, a tumour virus is known in mice in which the exchange of a single amino acid prevents the development of tumours. The researcher is convinced that some dangerous alterations in viruses are related to altered binding properties, making the viruses able to attack new tissue types. The viruses can only intrude cells if the cells have receptors that enable the binding of the viruses. That is why Stehle wants to focus on virus-cell contacts and hopefully come up with new ideas as to how these contacts can be modified for therapeutic purposes.
The biochemists structurally analysed the virus-cell binding on the atomic level. “The entire analysis process takes two to three years,” said Stehle. The purification of the proteins at the binding site takes several months. “We are cultivating a crystal that contains absolutely pure proteins. However, it is quite often the case that the crystals do not grow well.”

Diffraction patterns enable conclusions to be made about the protein structure

In order to crystallize a protein, the purified protein undergoes slow precipitation from an aqueous solution. The minute droplet is sealed and the solvent withdrawn. Up to 20 milligrammes of protein crystal are required to determine the protein's three-dimensional structure via X-ray diffraction. “We can carry out all the steps in the institute building, including the X-ray examination,” said Stehle who occasionally also needs to use stronger X-rays.
For example, the X-rays used at the European Synchrotron Radiation Facility in Grenoble (France) are up to 1000 times stronger than those in Tübingen. We often take our crystals to Grenoble, to Switzerland or to the DESY in Hamburg.”

Highly focused X-rays are reflected from the protein crystal. This creates a diffraction pattern that is characteristic of each protein complex. “Complex calculations are required in order to be able to infer the protein structure from the pattern,” explained Thilo Stehle. Sometimes, useful patterns can only be obtained by doping the crystals with heavy metals such as mercury or uranium. “Generally, the method is very accurate,” said Stehle adding that, “we are working with other researchers from around the world via the Internet who help us in our calculations.” The X-ray structure analysis method is also used by other researchers. However, the majority of these researchers are focusing on a different topic, thereby they are not in direct competition.
The three-dimensional presentation of the virus-cell contacts reveals the complexity of the binding process. Not all viruses dock to the cell according to the key and lock principle, which fit perfectly to each other. Some viruses use folding mechanisms or a kind of two-point binding, in which the second binding is only possible upon successful first binding. “For example, HI viruses depend on such two-point binding mechanisms,” said Thilo Stehle adding that, “so far nobody has succeeded in blocking the binding of the virus to the cell.” Drugs used for the treatment of viral infections are based on different mechanisms. For example, neuraminidase inhibitors that are used against influenza viruses, prevent viruses produced in the infected cells from being released. Protease inhibitors are used to fight HI viruses as they prevent viral replication by inhibiting viral proteases.
Diffraction pattern gained from the X-ray structure analysis of a protein crystal. (Photo: Antonie Knierim/University of Tübingen)
“Adenoviruses are also used in gene therapy, for example as vehicles to transport specific genes into body cells,” said the scientist explaining that the therapy focuses on repairing defective genes that cause severe diseases.
“At present, adenovirus types that infect many different tissues are used. It would be far more interesting to develop viral vehicles that are specific for blood cells,” said Stehle imagining that the redesigning of the viral surface might enable viruses to bind to specific receptors only. But research into this topic is at a very early stage.”

Thilo Stehle and his group of researchers are also using X-ray structure analysis to gain new insights into the evolution of viruses. “It is assumed that viruses did not evolve before the cells since the viruses depend on the cells for replication,” said Stehle who accidentally discovered a common feature of adenoviruses and reoviruses that cause diarrhoea in children. “Reoviruses carry a fibre-like spherical protein on their surface (Sigma 1), which is very similar to the binding proteins of adenoviruses. The protrusions have a similar structure, and some of them also bind to the same cell receptors.

“The two proteins are related in evolutionary terms,” said Stehle explaining that the two viruses nevertheless belong to two different families with different genomes. Adenoviruses possess a DNA genome, reoviruses an RNA genome. It can potentially be assumed that the two viruses had a common ancestor or that they have exchanged their genes at some stage during evolution. “This can happen if two viruses infect the same tissue,” said Stehle.

Source: University of Tübingen – 5th May 2008
Further information:

Prof. Thilo Stehle
Interfaculty Institute of Biochemistry
Hoppe-Seyler-Straße 4
72076 Tübingen

Tel.: +49 (0) 70 71/2 97 30 43
Fax: +49 (0) 70 71/29 55 65
E-mail: thilo.stehle@uni-tuebingen.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/press-release/viruses-caught-red-handed