Extended Data Fig. 6: NAMPT is a protein phosphoribosylase.
From: Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins
a, Phosphoribosyltransferase activity in NAD+ synthesis of NAMPT wildtype (WT), H247E and D219A that were purified from bacteria. NAMPT proteins purified to high homogeneity were validated by Coomassie staining and shown on the left. b, HSV-1 titer in sgNAMPT HeLa cells reconstituted with NAMPT WT, H247E and D219A mutants at 24 and 48 h.p.i., n = 3. Statistical significance was calculated using two-way ANOVA. Data are presented as mean values\(\pm\)SD. c, Quantification of the total phosphoribosylated peptides normalized to the total peptides (top panel), the total phosphoribosylated viral peptides normalized to the total peptides and total viral peptides (2nd and 3rd panel from top) in lysates of HeLa cells stably expressing wildtype NAMPT or the NAMPT-H247E mutant, and that of the phosphoribosylated viral peptides normalized to the total viral peptides of HSV-1 virions produced from HeLa cells expressing wildtype NAMPT or NAMPT-H247E (bottom panel). d, A list of identified phosphoribosylated and ADP-ribosylated (only in VP22) sites within their corresponding peptides, mapped to HSV-1 proteins. Numbers in parentheses indicate the corresponding phosphoribosylated residues within HSV-1 proteins. Please see Supplementary Fig. 1. e, Two-dimensional gel electrophoresis and immunoblotting analysis of VP22 in sgNAMPT 293 T cells and those reconstituted with NAMPT expression. f, Coomassie staining of purified GST-VP22, GST-NAMPT, GST-NAMPT-H247E (top panel) and GST-TARG1 (bottom panel). g, Detection of phosphoribose in reactions containing buffer, NAMPT, NAMPT-H247E, or TARG1 by LC-MS with GST-VP22 as the substrate. h, Two-dimensional gel electrophoresis and immunoblotting analysis of VP22-E257A in 293 T cells transiently expressing the NAMPT-H247E mutant with antibody against V5 (VP22). i, Phosphoribose in serial dilutions was determined by LC-MS, which serves as a standard for phosphoribose released from the NAMPT phosphoribosylase reaction. j, The phosphoribosylase activity of NAMPT H247E analyzed by released phosphoribose that is quantitatively determined by liquid chromatography-mass spectrometry. Statistical significance was calculated using unpaired two-tailed t-tests.
