Extended Data Fig. 5: NAMPT is incorporated into HSV-1 virions.
From: Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins
a, Representative immunofluorescence images of HSV-1-infected sgNAMPT HeLa cells reconstituted with FLAG-NAMPT using antibodies to FLAG (NAMPT), UL19, and gB. Scale bars, 5 μm. b-c, Cryo-electron microscopy analysis of NAMPT in HSV-1-infected sgNAMPT HeLa cell reconstituted with HA-NAMPT. Scale bars, 500 nm. d, Purified HSV-1 virions, along with whole cell lysates and exosomes, were analyzed by immunoblotting with antibodies against known exosome markers and HSV-1 structural proteins. e, Transmission electron microscopy analysis of purified HSV-1 virions after permeabilization with 1% Triton X-100 and immunogold-staining with antibodies to UL19 (10 nm gold) and HA (NAMPT, 25 nm gold). f, Top panels: Immunoblotting analysis of purified HSV-1 virions treated with protease K, in the absence or presence of Triton X-100 (1%), with antibodies to NAMPT and viral proteins. Bottom panel: Diagram of sensitivity to proteinase K of HSV-1 envelope glycoproteins, tegument protein UL37, and capsid protein UL19 with or without Triton X-100. Proteins were indicated by symbols with distinct shapes and colors. g, Immunoblotting analysis of whole cell lysates (WCL) of HeLa cells stably expressing APEX and NAMPT-APEX infected with HSV-1 for biotinylation assay, with H2O2 serves as a positive control. h, HSV-1 proteins, including UL21, gD, UL37, VP16, VP22, UL32, UL17, UL47, and gB precipitated with endogenous NAMPT in transfected 293 T cells. i-j, NAMPT interactions with VP22 (I), UL37, UL18, and UL38 (J) in transfected 293 T cells were analyzed by co-immunoprecipitation and immunoblotting. k, Immunoblotting analysis of HSV-1 structural proteins with indicated antibodies using virions produced from shCTL and shNAMPT HepG2 cells that were normalized against the UL19 capsid protein.
