Viruses provide Ghent researchers with a key to cellular secrets
Researchers from Ghent University and VIB have developed a new and more effective way to study cellular processes using the HIV virus
Every cell in the human body contains thousands of different proteins, which can only fulfil their functions when they are working together. Scientists are therefore keen to know which proteins associate together, but often the only way to get at these protein machines is to break the cell open.
“The first step in a classical study of protein complexes essentially turns the highly organised cellular structure into a big messy soup,” explains Sven Eyckerman, who has developed Virotrap with colleagues in Ghent. Once mixed up in this way, it is hard to work out which proteins interact together and how the cell mechanism functions.
The search for an alternative was inspired by the way viruses reproduce. Once attached to a host cell, the virus hijacks the cell’s internal machinery and uses it to make copies of itself. A new genetic core is created and then wrapped up in a shell, forming a new virus particle. These particles are then released from the cell and go on to repeat the process.
Proteins from the host cell are often caught up in these particles, so Eyckerman and his colleagues wondered if they could make a virus pick up specific proteins from the interior and bring them out. They tried this with a safe, stripped-down version of HIV, the virus that causes Aids.
“HIV is well-studied, of course, and it is also used as a system to deliver genes to cells,” Eyckerman explains. “We were already using the virus in that way, so we had the tools in the lab. We just had to retune it a little bit in order to make it work.”
Introducing just one HIV protein, called the Gag protein, into a cell is enough to kick-start the production of empty virus particles. To the protein, the researchers attached a “bait” protein, calculated to bind to the protein complex within the cell and draw it into the developing virus particles.
It took us about a year to figure out a system that lets us purify the particles in a single step
Collecting the virus particles once they have been released into the cell culture medium proved a challenging step. “It’s a big volume of medium, with only a few, very small particles,” Eyckerman explains.
The established methods, such as ultracentrifugation, he adds, are not satisfactory. “It took us about a year to figure out a system that lets us purify these particles in a single step.”
A further challenge was to ensure that the purification process did not interfere with analytical methods, such as mass spectrometry, used to identify the proteins collected. “It’s a pretty complex sample,” Eyckerman adds, “so we have also devised our own data-analysis tools.”
Three years into developing the method, they have confirmed that it worked. Pairs of proteins that were already known to interact inside the cell could be extracted using Virotrap, along with new complexes for future study.
Virotrap is now being used to investigate the basic functions of cells, such as the pathways that are set in motion during inflammation, and even the way cells react to viruses. “I think it’s a nice idea to use a virus system to understand anti-viral pathways,” Eyckerman says.
The method has also been adapted so that small molecules, such as potential drugs, can be attached to the Gag protein. This will allow researchers to investigate how such molecules behave inside the cell, and fine-tune their effects. Eyckerman says this is something the pharmaceutical industry is particularly interested in.
Flemish Institute for Biotechnology (VIB)
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