The non-structural protein 4A (NS4A) is a versatile polypeptide that is imminent for growth, maturity, and replication of the hepatitis C. virus. It sandwiches with A1 and A0 regions forming a 3-way anti-parallel sheet of the NS3 apoenzyme to resulting in conformation organization, active site ordering, and substrate binding. We have been interested in developing strategies for interference with NS4A-activation of NS3 protease. To move forward in this direction, it was fundamental to have sufficient knowledge of NS4A affinity towards NS4A. Old studies by Lin et al (1995) and Tomei et al (1995) confirmed that the central region NS4A21`-34` is sufficient for NS3 activation (See Figure below). For our surprise, we found no data available for quantification of NS4A affinity to NS3. Differential static light scattering technique (DSLS) on NS3 alone and after incubation with NS4A revealed that NS4A moderately stabilize NS3 (ΔTagg = 2.83±0.24). More important, the aggregation temperature shift was sensitive to specific experimental factors such as incubation time, temperature and relative ratio. It was also observed that gentle shaking of the incubation vessel controls the standard error and enhance the reproducibility of the experiment. Affinity determination by fluorescence anisotropy (FA) method via titration of the NS3 with FTIC-Ahx-NS4A was also affected by the experimental conditions. Shaking at room temperature (25 °C) for at least 45 minutes was necessary to obtain reproducible results for the determination of the dissociation constant (Kd). Results of FA test revealed that NS4A binding to NS3 is not tight (1.87 0.1µM at 1.87 µM NS3).

Lessons taken from the study of HCV-NS3 protease affinity to its NS4A cofactor at different conditions

255th National Meeting (2018)

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