An in vitro assay for flavivirus NS2B/NS3 serine protease
Abstract number: 1134_03_387
Bessaud M., Peyrefitte C.N., Pastorino B., Grandadam M., Tolou H.J.
The genus Flavivirus comprises more than 70 viruses. Many of them cause severe human diseases. The genome of flavivirus, a single strand RNA molecule with positive polarity, is translated into a large polyprotein that is processed into structural and non-structural proteins by both viral and cellular proteases. The virus-encoded protease is a binary complex constituted by the NS3 protein and its co-factor, NS2B. As the viral protease plays a critical role in the virus replication cycle, it represents one of the main targets for antiviral therapy against members of the Flavivirus genus.The aim of this study was to develop an in vitro assay using the protease of several flaviviruses belonging to different groups in order to characterize their enzymatic properties, such as temperature, pH and salt-sensitivity and substrate specificity.
Sequences encoding the viral proteinase were located on the genome of 8 flaviviruses. NS2B/NS3 proteinase were expressed as hexahistidine-tagged recombinant proteins and then purified by immobilized-metal affinity chromatography. Their enzymatic properties were characterized in vitro using BAPNA, a chromogenic substrate for trypsin-like proteases.
The protease moiety of the 8 flaviviruses were successfully produced and purified. 5 of them exhibited activity towards BAPNA. Effect of temperature, ionic strength and pH on enzymatic activity were determined. Our results suggest that the hydrophilic domain of NS2B is necessary for proteolytic activity.
The system we developed will allow us to establish a screening test so as to identify or to design inhibitors active as antiviral drugs against one or more pathogenic flaviviruses. The lack of activity of 3 of the 8 proteases we assayed could indicate slight differences in flaviviral proteases selectivity and activity.
|Session name:||XXIst ISTH Congress|
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