Figure 4.

The nsp12 NiRAN domain alone is a guanylyltransferase enzyme. (A) The NiRAN domain from SARS-CoV-2 nsp12 was expressed and purified from E. coli, then visualised by SDS PAGE. The arrow indicates the purified NiRAN domain band. (B) Full-length nsp12 and the purified NiRAN domain were tested for GTase activity using α-³²P-GTP and a diphosphorylated RNA substrate, 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 two-way ANOVA. ***P < 0.001. (C) Schematic of AP and RppH activity on the diphosphorylated RNA substrate (left). Diphosphorylated RNA was treated with AP or RppH, then incubated with purified NiRAN domain and α-³²P-GTP (right). 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. (D) Schematic of AP and RppH activity on the capped RNA product (left). Diphosphorylated RNA was incubated with purified NiRAN domain and α-³²P-GTP, then reaction products were treated with AP and RppH (right). 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.