Figure 2.

Nsp12 has guanylyltransferase activity. (A) Schematic of nsp13 5′ triphosphatase activity followed by a canonical GTase reaction mechanism, which consists of nucleotidylation of the enzyme (E) with GTP (Gppp), then transfer of GMP (Gp) to a diphosphorylated RNA substrate (ppN-RNA). The ³²P isotope of α-³²P-GTP is indicated in red. (B) Purified SARS-CoV-2 nsp13 was visualised by SDS PAGE, the arrow indicates the His6-ZBasic-tagged nsp13 band. (C) 5′ triphosphatase activity of purified nsp13 was tested by incubation with γ-³²P-ATP for the indicated amount of time. The γ-³²P-ATP substrate and inorganic phosphate (Pi) product were visualised by denaturing PAGE and autoradiography, arrows indicate the anticipated bands (left). AP was used as a positive control, and as a negative control nsp13 was inactivated by heating to 70°C for 5 min prior to the reaction, then was incubated with the γ-³²P-ATP substrate for 5 mins. Quantification of γ-³²P-ATP substrate and Pi product is from n = 2 independent reactions, data are mean ± s.e.m. (right). Pi present in the untreated γ-³²P-ATP stock was subtracted from all samples during quantification. (D) SARS-CoV-2 nsp7 and nsp8 were expressed and purified from E. coli cells and visualised by SDS PAGE. Arrows indicate bands corresponding to the proteins of interest. (E) SARS-CoV-2 nsp12 was expressed and purified from Sf9 cells and visualised by SDS PAGE. The arrow indicates the purified nsp12 band. (F) The indicated SARS-CoV-2 proteins were incubated with α-³²P-GTP and diphosphorylated RNA, then radiolabelled RNA products were visualised by denaturing PAGE and autoradiography. The arrow indicates the anticipated product, and the asterisk denotes a faster mobility product which could result from contaminating RNA kinase activity in some protein preparations, or decapping of the anticipated product by a phosphatase such as residual nsp13. (G) Nsp12 was incubated with diphosphorylated RNA and α-³²P-UTP or α-³²P-GTP, then radiolabelled RNA products were visualised by denaturing PAGE and autoradiography. The arrow indicates the anticipated product (top). Quantification of the product is from n = 3 independent reactions (bottom). Data are mean ± s.e.m., analysed by one-way ANOVA. ***P<0.01. (H) Nsp12 or vaccinia capping enzyme were incubated with α-³²P-GTP and diphosphorylated RNA for the indicated amount of time. Radiolabelled RNA products were visualised by denaturing PAGE and autoradiography (left), the arrow indicates the anticipated product. Quantification of the product is from n = 3 independent reactions, data are mean ± s.e.m. (right). (I) Schematic of AP and RppH activity on the diphosphorylated RNA substrate (left). Diphosphorylated RNA was treated with AP or RppH, then incubated with nsp12 or vaccinia capping enzyme and α-³²P-GTP (top). The arrow indicates the anticipated product. Quantification of the product is from n = 2 independent reactions (bottom). Data are mean ± s.e.m., analysed by one-way ANOVA. ***P < 0.001. (J) Schematic of AP and RppH activity on the capped RNA product (left). Diphosphorylated RNA was incubated with nsp12 or vaccinia capping enzyme and α-³²P-GTP, then reaction products were treated with AP and RppH (top). The arrow indicates the anticipated product. Quantification of the product is from n = 2 independent reactions (bottom). Data are mean ± s.e.m., analysed by one-way ANOVA. **P < 0.01.