Extended Data Fig. 2: NAMPT restricts HSV-1 lytic replication.

a, HSV-1 growth curve in sgNAMPT HeLa cells reconstituted with NAMPT expression, n = 3, P = 0.0014. b, NAMPT enzymatic activity in NAD⁺ synthesis was determined with or without FK866 (10 nM), n = 3. P = 0.0002. c, HSV-1 titer in HepG2 cells treated with vehicle (DMSO) or FK866 (10 nM), with or without nicotinamide riboside (NR, 0.1 mM), n = 3, P = 0.0003 for FK866 versus DMSO. FK866 was added at 96 hours before HSV-1 infection and treatment was extended during infection. NR was added immediately before HSV-1 infection. d, HSV-1 titer in the medium of shCTL and shNAMPT HepG2 cells at MOI = 0.1 determined by plaque assay, n = 5, P = 0.0297. e, HSV-1 titer in the medium of 293 T cells that transiently express FLAG-NAMPT in increasing dose as indicated by plasmid amount at 24 h.p.i (MOI = 0.1), n = 3. f-g, Growth curve of HSV-1 in the medium of shCTL and shNAMPT mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFF) at MOI = 0.1, with NAMPT knockdown validated by immunoblotting with indicated antibodies using whole cell lysates. h-i, Diagram (h) and summary (i) of PRPP levels and its effect on HSV-1 replication in HeLa cells by NAM, NAPRT expression or UPRT (UMP synthetase) depletion. j, HeLa cells, grown with exogenous NAM (0.1 mM), were mock- or HSV-1-infected as indicated in (h). PRPP were determined by LC-MS at 12 h.p.i. and viral titer in the medium was determined by plaque assay using Vero monolayer, n = 3 for viral titration, n = 4 for NAM and PRPP analysis. Diagram was created with Biorender.com. Statistical significance was calculated using two-way ANOVA for a, b, c, d, f, and g, unpaired two-tailed t-tests for j. Data are presented as mean values\(\pm\)SD. Graphics created with BioRender.com.