Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Table 1

Fig. 6. Ribonucleolytic activity of severe acute respiratory syndrome (SARS)-CoV nonstructural protein (nsp)14 [polyprotein (pp)1ab residues 5903-6429]. (A) Coomassie brilliant blue-stained 12.5% SDS-polyacrylamide gel showing the expression and amylose-affinity purification of the maltose-binding protein (MBP)-nsp14 fusion protein. Lanes 1 and 2, insoluble and soluble proteins, respectively, from a lysate of isopropyl b-D-thiogalactoside (IPTG)-induced Escherichia coli [pMal-SARS-CoV-nsp14] cells; lane 3, amylose column flow-through fraction; lanes 4-6, wash fractions; lanes 7 and 8, proteins eluted with maltose-containing buffer. (B) SDS/PAGE of mutant forms of MBP-nsp14, which were purified as described for the wild-type protein. Lane 1, MBP-nsp14_D5992A/E5994A; lane 2, MBP-nsp14_N6140A; lane 3, MBP-nsp14_D6145A; lane 4, MBP-nsp14_H6170A, lane 5, MBP-nsp14_D6175A. (C) [³²P]-labeled ssRNA3 was incubated with equal amounts of wild-type and mutant MBP-nsp14 fusion proteins, respectively, for 30 min at 37°C. Reaction products were analyzed by denaturing PAGE and autoradiography. Lane 1, reaction without protein; lane 2, reaction with MBP-nsp14; lane 3, reaction with MBP-nsp14_D5992A/E5994A; lane 4, reaction with MBP-nsp14_N6140A; lane 5, reaction with MBP-nsp14_D6145A; lane 6, reaction with MBP-nsp14_H6170A; lane 7, reaction with MBP-nsp14_D6175A.

Fig. 7. Divalent ion dependence of the SARS-CoV exoribonuclease (ExoN) activity. (A) SDS-polyacrylamide gel showing the Ni-NTA-purified, C-terminally His-tagged protein nsp14-HC. Molecular weight markers are indicated. (B) 5'-[³²P]-labeled ssRNA3 was incubated with nsp14-HC at 37°C for 30 min in the absence of divalent ions (lane 2) or in the presence of 5 mM Mg²⁺ (lane 3), 5 mM Mn²⁺ (lane 4), 5 mM Ca²⁺ (lane 5), 0.5 mM Zn²⁺ (lane 6), and 5 mM Zn²⁺ (lane 7). Lane 1, incubation of RNA3 without protein. Reaction products were analyzed by denaturing PAGE and autoradiography.

Fig. 8. 5'-[³²P]-labeled (lanes 2 and 3) and 3'-[³²P]-labeled ssRNA3, respectively, were incubated with nsp14-HC (lanes 3 and 5) or with buffer alone (lanes 2 and 4) for 30 min at 37°C. Reaction products were analyzed by denaturing PAGE and autoradiography. Positions of RNA size markers are given.

Table 1. Oligoribonucleotides and oligonucleotides used in this study as size markers and ExoN substrates