< Back to previous page
Project
Introduction of analytical separation techniques in virology to study interaction at the molecular level of picornaviridae (FWOAL435)
Using a cell-free system for the production of virus material and CE as separation technique, we want to study some unsolved issues regarding the structure and replication of picornaviruses, such as the function of some viral proteins, the mechanism of initiation of protein- and RNA-synthesis, and the different steps in the morphogenesis. It should also enable us, besides the above-mentioned topics : (i) to detect subviral particles formed during the replication cycle of picornaviruses (morphogenesis), (ii) to study the interactions between the viral genome and subviral particles, which leads to the formation of new virions, (iii) to study interactions between viral RNA and cellular proteins, (iv) to consider interactions between viral and cellular proteins, (v) to monitor interactions between viral RNA and viral proteins with particular attention for the determination of the stoichiometry and affinity of, and finally (vi) to find and identify new targets for potential antiviral drugs.
As already mentioned, viral components can only be detected when they are fluorescently-labeled. This can be achieved using either a derivatisation reaction or through intercalation with a fluorophore. A possible problem that can occur is when one has to detect the de novo synthetized viral molecules in a complex mixture of proteins from the cell extract, because the reaction is not specific. In the past, radioactive precursors were added to solve this problem, as the radioactivity was only incorporated in the de novo synthetized viral molecules. To decrease the use of radioactivity and because it is not so evident to measure radioactivity as CE detection, we want to introduce the use of non-natural aminoacids in the molecular virology. Up till now, researchers were limited to the use of 20 naturally occurring aminoacids (occasionally radioactively labeled). However, due to progresses in the chemical biology, we now have access to a wider variety of chemically modified aminoacids.
Depending on their chemical modifications, these aminoacids can be used for different applications. They can be employed in the detection of viral proteins because they already exhibit better fluorescent properties, such as the 5-hydroxy-L-tryptophane [15], or because they posses new reactive groups-such as alkynes-which makes it possible to label the proteins selectively with a fluorescent agent. Another possibility is to incorporate photo-active cross linkers with leucine and methionine to study the protein-protein interactions. The interesting property of these non-natural aminoacids is that the original proteins activity and functionality is preserved when they are incorporated. With both the fluorescent labeling, and the introduction of non-natural aminoacids, the analytical CE parameters have to be optimized. This is also the case for the conditions of the derivatisation reactions. Such optimizations will be tackled in this project, preferably by an experimental design approach.
As already mentioned, viral components can only be detected when they are fluorescently-labeled. This can be achieved using either a derivatisation reaction or through intercalation with a fluorophore. A possible problem that can occur is when one has to detect the de novo synthetized viral molecules in a complex mixture of proteins from the cell extract, because the reaction is not specific. In the past, radioactive precursors were added to solve this problem, as the radioactivity was only incorporated in the de novo synthetized viral molecules. To decrease the use of radioactivity and because it is not so evident to measure radioactivity as CE detection, we want to introduce the use of non-natural aminoacids in the molecular virology. Up till now, researchers were limited to the use of 20 naturally occurring aminoacids (occasionally radioactively labeled). However, due to progresses in the chemical biology, we now have access to a wider variety of chemically modified aminoacids.
Depending on their chemical modifications, these aminoacids can be used for different applications. They can be employed in the detection of viral proteins because they already exhibit better fluorescent properties, such as the 5-hydroxy-L-tryptophane [15], or because they posses new reactive groups-such as alkynes-which makes it possible to label the proteins selectively with a fluorescent agent. Another possibility is to incorporate photo-active cross linkers with leucine and methionine to study the protein-protein interactions. The interesting property of these non-natural aminoacids is that the original proteins activity and functionality is preserved when they are incorporated. With both the fluorescent labeling, and the introduction of non-natural aminoacids, the analytical CE parameters have to be optimized. This is also the case for the conditions of the derivatisation reactions. Such optimizations will be tackled in this project, preferably by an experimental design approach.
Date:1 Jan 2008 → 31 Dec 2009
Keywords:Virology, Separation, Microbiology, Analytical chemistry
Disciplines:Biological sciences, Chemical sciences, Pharmaceutical sciences