RECOMBINANT DENGUE VIRUS TYPE-1 NS3 PROTEIN EXHIBITS SPECIFIC VIRAL-RNA BINDING AND NTPASE ACTIVITY REGULATED BY THE NS5 PROTEIN

The full-length dengue virus NS3 protein has been successfully express ed as a 94-kDa GST fusion protein in Escherichia coil. Treatment of th e purified fusion protein with thrombin released a 68-kDa protein whic h is the expected molecular mass for the DEN1 NS3 protein. The identit y of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncodi ng region of the dengue virus RNA genome and NTPase activity. We demon strated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'- NCR), although sites outside the stem-loop may also participate in bin ding. Using various unlabeled homopolymeric and heteropolymeric RNAs a s competitors for binding, it was further shown that the DEN1 NS3 prot ein exhibits preferential binding to a 94-nt RNA transcript from the 3 '-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay , we found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. A lthough the NS3 protein was able to utilize all four ribonucleoside tr iphosphates as its substrates, the NS3 protein showed a distinct prefe rence for purine triphosphates (i.e., ATP and GTP), The addition of po ly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which con trasts with the reports for other flaviviral NS3 proteins. However, NT Pase activity was specifically stimulated by the viral NS5 protein, wh ich was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120- min reaction. These data suggest that the NTPase activity of the NS3 p rotein may be regulated by the viral NS5 protein during virus replicat ion. (C) 1998 Academic Press.