Résumés disponibles (92) :

COLIN Beatrice (INSERM U1177 - Cyril COUTURIER)
Session :

 

@mail :  beatrice.colin@univ-lille2.fr      tél. :  +33(0)320877268


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Titre de la communication :
Validation of a new amplified reporter system
Auteurs (et leurs adresses) de la communication :
COLIN Béatrice, COUTURIER Cyril, DEPREZ Benoit INSERM U1177 \"Drugs & Molecules for Living Systems\" Laboratoire de Pharmacochimie moléculaire Institut Pasteur de Lille 1 rue du Professeur Calmette 59019 LILLE CEDEX
Résumé de la communication :
Nowadays, many viruses remain without preventive or curative treatment, leading to less or more serious epidemics with negative consequence for an increasingly large part of the population. Facing this growing threat and these ever-changing viruses, we developed a cellular virus infection tracking test. This test is based on the cleavage of a cell encoded probe by one of the virus protease upon the infection process. The signal detection can be measured by two reading modes suitable with high throughput: the Bioluminescence Resonance Energy Transfer (BRET) and the Fluorescence Confocal Microscopy or High Content Screening (HCS). Using the Hepatitis C virus (HCV) as a model, our assay showed high sensitivity to follow native HCV infection but was limited using plasmid or pseudo-particles expressing the HCV protease. Indeed, unlike HCV replicative viruses allowing the production of a large amount of protease, a weaker expression is not sufficient to induce a drastic change of both readout signals. To circumvent this problem and in order to enhance the sensitivity of the system, we introduced a relay enzyme: the Tobacco Etch Virus protease (TEV). We modified the settings influencing the energy transfer to gain the best basal BRET signal and the higher decrease after cleavage by the TEV protease relay enzyme. For this purpose, we optimized the distance between the donor and acceptor of the energy transfer and their relative orientation. Finally, to improve the signal-to-noise ratio we engineered the probe to promote its nuclear accumulation in the basal state and its release in cytosol after cleavage by TEV protease. In order to do this, nuclear localization sequences were added to the cell encoded probe and led to the monitoring of TEV activity also by HCS, as the fluorescent part of the probe is able to switch from nucleus to whole cell upon TEV action. To conclude, we succeeded in developing a highly sensitive cell encoded probe that responds to an exogenously expressed virus protease monitored with the BRET and HCS method. This research program will now focus on the validation of the assay using pseudo-particules expressing NS3/4 protease form HCV and native HCV virus. In a generic application, in the future, this Test should be used with other viruses protease like Chikunkunya (ChikV) or Zika (ZIKV).

COLIN Beatrice (INSERM U1177 - Cyril COUTURIER)