FIG. 4.
Effect of various conditions on the activity of norovirus His-Pro−Pol. (A) Effect of the enzyme concentration on the amount of incorporated UMP. One, 2, and 3 μM concentrations of His-Pro−Pol were assayed in a 15-μl reaction mixture containing 3 mM MgCl2, 100 μM UTP, and 5 μCi of [α-32P]UTP. The mixture was incubated for 20 min at 30°C and stopped with 0.5 M EDTA. Six microliters of the final mixture was spotted onto a DE81 membrane for counting. Final concentrations of the recombinant polymerase are indicated below the graph. The incorporated UMP is normalized against the value of 3 μM His-Pro−Pol (100%). (B) The polymerase activity of His-Pro−Pol (1 μM) was analyzed following incubation for 30 min at various temperatures (Tinc) (indicated on abscissa). The RdRp assay was performed in a 30-μl reaction mixture which contained 10 μM UTP, 10 μCi of [α-32P]UTP, 3.3 mM MgCl2, and the other components described in Materials and Methods. The UMP incorporation rates are given in percentages of the value obtained at 30°C (100%). (C) Effect of NaCl on the His-Pro−Pol activity. The 30-μl reaction mixture was incubated for 20 min at 30°C and contained 1 μM His-Pro−Pol, 10 μM UTP, 10 μCi of [α-32P]UTP, 3.3 mM MgCl2, and increasing concentrations of NaCl (0 to 50 mM), indicated below the gel. The negative control was an RdRp assay of His-Pro−Pol without MgCl2 and NaCl. The UMP incorporation rates (ordinate) are normalized against the amount of incorporated UMP in an RdRp assay containing 3.3 MgCl2 and no NaCl (100%). (D) Effect of divalent (magnesium and calcium) and monovalent (potassium) cations on the polymerase activity. His-Pro−Pol (1 μM) was incubated at 30°C for 20 min with 3.3 mM (CH3COO)2Mg, MgSO4, MgCl2, CaCl2, KCl, or H2O (negative control) in a 15-μl reaction mixture containing 10 μM UTP and 5 μCi of [α-32P]UTP. The amount of incorporated UMP (ordinate) is normalized against values obtained with magnesium acetate (100%).