FIG 1.

NiRAN-mediated NMPylation of nsp9 by SARS-CoV and TGEV nsp12. (A and C) Nsp12-mediated NMPylation of nsp9 in the presence of different nucleotides. A 0.05 μM concentration of SARS-CoV nsp12-His₆ (A) or TGEV nsp12-His₆ (C) was incubated in a standard NMPylation assay with 4 μM SARS-CoV nsp9-His₇ (A) or TGEV-His₆ (C) in the presence of the indicated [α-³²P]NTP (lanes 3 to 6). Protein UMPylation was not detected in reaction mixtures containing nsp9 or nsp12 alone (lanes 1 and 2). (B and D) Nsp9-specific UMPylation activities of recombinant SARS-CoV (B) and TGEV (D) nsp12 variants containing the indicated amino acid substitution(s) in the active sites of the NiRAN (lane 4) or RdRp (lane 5) domain. The left (A and B) and right (C and D) panels show the data obtained for SARS-CoV and TGEV, respectively. The sizes of marker proteins (in kilodaltons) are given on the left, and the position of nsp9 is indicated. The column diagrams depict relative NMPylation activities compared to (UMPylation) activities obtained in reaction mixtures containing UTP. Data obtained in three independent experiments were included in the analysis (means and standard errors of the means [SEM] are given). *, P ≤ 0.05; **, P ≤ 0.01.