Biochemical and kinetic analysis of RNA capping. (A) Analysis of capped RNA by denaturing PAGE followed by autoradiography. Lane 1, no RNA; lane 2, RNA reacted with purified p65; lane 3, RNA reacted with virion and lane 4, no protein. Arrow indicates the position of the radiolabelled RNA band. (B) Analysis of only GpppG (i) and nuclease P1 resistant labeled capped RNA (ii) by HPLC. The peak indicated the position of GpppG. (C) Transfer of labelled GMP moiety by p65 to a di phosphate ended viral RNA. Lane 1, 5' triphosphate ended RNA initiating with ‘G’ base; lane 2, 5' di-phosphate ended RNA initiating with ‘G’ base; lane 3, di-phosphate ended RNA initiating with ‘ A’ base; lane 4, 5' tri phosphate ended RNA initiating with ‘A’ base; lane 5, BSA as negative control. Arrow indicates the position of the respective band. (D) Analysis of p65-GTP interaction reaction kinetics. Double reciprocal plot showing the activity of p65 as a function of GTP concentration in the presence of 50 nM (), 100 nM (▲) and 150 nM () RNA.