SARS-CoV-2 Nsp14 interacts with human SIRT5.

Abstract

A. Cartoon representation of the protein structure of Nsp14/Nsp10 (PDB 7N0B) and SIRT5 (PDB 3YIR) shows the Nsp14 N-terminal ExoN domain and C-terminal MTase domain. B. Affinity-purification of Nsp14-strep and co-purification of endogenous SIRT5 after transfection in HEK293T cells, as shown by western blot. C. Immunofluorescence of transfected Nsp14-Strep and endogenous SIRT5 in A549 cells. D. CETSA in HEK293T cells transfected with Nsp14-step and/or SIRT5, showing an increase in the stability of SIRT5 and Nsp14 by western blot. E. Western blot showing the absence of SIRT5 in SIRT5-KD HEK293T cells. F. Strep-tag affinity-purification or Flag-tag immunoprecipitation, followed by western blot, after transfection with Nsp14-strep, Nsp10-flag and SIRT5 expression constructs. SIRT5 does not interact with Nsp10. 0.5 μg of each construct or of empty control plasmids were transfected in SIRT5-KD HEK293T cells in a six-well plate. G. Strep-tag affinity-purification and western blot after transfection of Nsp14-strep, SIRT5 and increasing concentrations of Nsp10-tag indicate competitive binding of SIRT5 and Nsp10. 0.5 μg of Nsp14-strep and SIRT5 plasmid were used in a 6-well plate, with 0, 0.5, 1 or 2 μg of Nsp10-Flag.</p