SARS-CoV-2 Nsp14 interacts with human SIRT1.

Abstract

A. Co-purification of endogenous sirtuins SIRT1, 2, 3, 6 and 7 after transfection of Nsp14-strep in HEK293T cells, as shown by western blot. Loading and purification controls are the same as in Fig 1B. B. Strep-tag affinity-purification and western blot after transfection of HEK293T cells with Nsp14-strep and SIRT1 WT and H355Y catalytic mutant, showing that the interaction with Nsp14 is lost in H355Y mutant. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 inhibitor Ex-527. High concentrations of Ex-527 prevent the interaction. D-E. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 specific activator SRT1720 (D) or non-specific activator resveratrol (E). Both drugs were cytotoxic at high concentrations and the apparent decrease in SIRT1 binding correlated with a similar decrease in the input lanes. F. In vitro desuccinylation activity of purified SIRT5 incubated with increasing concentrations of Nsp14, showing no effect. G. In vitro methyltransferase activity of purified Nsp14 incubated with increasing concentrations of SIRT5, showing no specific effect. Unmethylated GpppG cap-analog was used as a substrate.</p