FIG. 5.

NS4B has GTPase activity, which is mediated by the NBM. Equal amounts of purified GST, GST-NS4B, and the NBM mutant proteins GST-NS4B(GV), GST-NS4B(IN), GST-NS4B(KS), and GST-NS4B(KR) were incubated with [γ-³²P]GTP. Aliquots were collected every 15 min and subjected to TLC to allow separation of hydrolyzed ³²P_i from GTP, followed by autoradiography and phosphorimager analysis. (A) Representative TLC plate. Locations of GTP and ³²P_i standards are indicated on the left. (B) GTPase activity of wild-type NS4B (⧫) and G129V (▴), I131N (▪), K135S (•), and K135R (×) mutant proteins is plotted as a function of time. When present, any detectable hydrolysis of GTP in the GST control was used for background subtraction purposes. Each data point represents the average of at least four independent determinations. The error bars represent standard deviations.