Coronavirus replication–transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit

Figures

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  Fig. 1.. Self-NMPylation activity of HCoV-229E nsp12. (A) HCoV-229E nsp12-His6 (106 kDa) was incubated for 30 min with the indicated [α-32P]NTPs in the presence of 6 mM MnCl2 (for details, see Materials and Methods). Reaction products were separated by SDS-PAGE and stained with Coomassie brilliant blue. (B) Radiolabeled proteins were visualized by phosphorimaging. Positions of nsp12-His6 and protein molecular mass markers (in kilodaltons) are indicated in A and B. (C) Radioactive signal intensities (mean values ± SEM) were determined from three independent experiments. *P ≤ 0.05. Signal intensities (in percentages) are given in relation to UTP.

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  Fig. 2.. HCoV-229E nsp12-mediated UMPylation of nsp9. HCoV-229E nsp12-His6–mediated protein UMPylation activity was assessed using a range of protein substrates, including bovine serum albumin, MBP-lacZα, and a range of C-terminally His6-tagged HCoV-229E nsps encoded by ORF1a. The proteins were incubated for 10 min with [α-32P]UTP in the absence (A) or presence (B) of nsp12, as described in Materials and Methods. In the Top of A and B, the Coomassie brilliant blue-stained SDS-polyacrylamide gels are shown, and, in the Bottom of A and B, the corresponding autoradiograms are shown. Positions of protein molecular mass markers (in kilodaltons) are given to the left. Also indicated is the position of nsp12-His6 (B, Top) and the radioactive signal observed upon incubation of nsp12-His6 with nsp9-His6 (B, lane 7), the latter indicating nsp12-His6–mediated transfer of [α-32P]UMP to nsp9-His6 (12.9 kDa), which was not observed for other proteins tested.

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  Fig. 3.. Biochemical and virological characterization of HCoV-229E NiRAN-mediated nsp9 NMPylation. (A and B) Role of nucleotide cosubstrate used in the reaction. Nsp12-His6 and nsp9-His6 were mixed and incubated in the presence of different [α-32P]NTPs in the standard NMPylation assay. (A, Top) The Coomassie-stained nsp9-His6 separated by SDS-PAGE. (A, Bottom) An autoradiogram of the same area of the gel. (B) Relative activities (mean values ± SEM) in the presence of the indicated nucleotide cofactors were determined from three independent experiments. *P ≤ 0.05. (C) Role of metal ions. Shown is a standard NMPylation assay in the presence of [α-32P]UTP and different metal ions, each at 1 mM. In C, Top, the Coomassie-stained nsp9-His6 is shown, and, in C, Bottom, the corresponding autoradiogram is shown. Sizes of marker proteins (in kilodaltons) are indicated to the left in A and C. (D) Mutant forms of HCoV-229E nsp12-His6 carrying the indicated amino acid substitutions were incubated with nsp9-His6 in the presence of [α-32P]UTP, as described in Materials and Methods. Radiolabeled nsp9-His6 produced in the NMPylation reaction was detected by phosphorimaging (D, Top), and relative…

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  Fig. 4.. Mono-NMPylation of nsp9 in the presence of nsp12 and UTP or GTP. Shown are deconvoluted intact protein mass spectra of HCoV-229E nsp9 (SI Appendix, Table S1) (A–D). (A) nsp9 alone, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 in the presence of UTP, (D) nsp9 + nsp12-His6 in the presence of GTP.

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  Fig. 5.. Roles of proteolytic processing and N-terminal residues of nsp9 in nsp12-mediated UMPylation. (A) Requirement of a free nsp9 N terminus for nsp9 UMPylation. Nsp7-11-His6 was preincubated at 30 °C in the presence or absence of recombinant Mpro (nsp5-His6) in NMPylation assay buffer containing UTP. After 3 h, the NMPylation assay was started by the addition of nsp12-His6, as described in Materials and Methods. Reactions containing nsp5-His6 (lane 1) and nsp9-His6 (lane 2) were used as controls. After 10 min, the reactions were terminated and the reaction mixtures separated by SDS-PAGE. Proteins were stained with Coomassie brilliant blue (A, Top). Nsp7-11-His6 precursor and processing products resulting from nsp5-His6–mediated cleavage are indicated to the right. Note that (due to their small size) nsp7 and nsp11-His6 are not detectable in this gel and reactions supplemented with nsp5-His6 (lanes 1 and 4; position of nsp5-His6 is indicated by a closed circle) or nsp9-His6 (lane 2) contain small amounts of MBP (indicated by open circles) as a residual impurity resulting from their expression as MBP fusion proteins (SI Appendix, Table S1). (B) Nsp9-His6 variants lacking one or two…

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  Fig. 6.. SARS-CoV-2 nsp12-mediated NMPylation of nsp9. (A) Coomassie-stained SDS-polyacrylamide gel showing the recombinant proteins used in the NMPylation assay. As controls, mutant proteins with active-site replacements in the NiRAN domain (K4465A) and the RdRp domain (DD5152/3AA) of SARS-CoV-2 nsp12 were used. Residue numbering is according to the position in pp1ab. (B) Autoradiogram of an UMPylation assay using nsp9-His6 and [α-32P]UTP as substrates for nsp12-His6 (wild type [wt] and mutants). Molecular masses (in kilodaltons) of marker proteins are indicated to the left.