Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins

Abstract

As obligate intracellular pathogens, viruses activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of salvage nicotinamide adenine dinucleotide (NAD⁺) synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses through unknown mechanisms. Here, we show that NAMPT restricts herpes simplex virus type 1 (HSV-1) replication by impeding the virion incorporation of viral proteins owing to its phosphoribosyl-hydrolase (phosphoribosylase) activity, which is independent of the role of NAMPT in NAD⁺ synthesis. Proteomics analysis of HSV-1-infected cells identifies phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins, which are de-phosphoribosylated by NAMPT in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Loss of NAMPT renders mice highly susceptible to HSV-1 infection. Our work describes an additional enzymatic activity of a metabolic enzyme in viral infection and host defence, offering a system to interrogate the roles of protein phosphoribosylation in metazoans.

Extended Data Fig. 1 ∣. Metabolic alterations by HSV-1 during lytic replication.

a, Heatmap of metabolites profiled in mock- and HSV-1-infected HepG2 cells at 12 hours post-infection (h.p.i) (MOI = 5). b, Cell viability of 293 T cells depleted with NAMPT, NMNAT1, NRK, or NADSYN, n = 3. c, Quantification of NAD⁺ and NMN in shNAMPT HeLa cells infected with HSV-1, supplemented with nicotinic acid (NA, 0.1 mM) or nicotinamide riboside (NR, 0.1 mM). d-f, Infection diagram (d) and HSV-1 titers (e, n = 3) in shCTL and shNAMPT HeLa cells with or without supplementation of NR, P = 0.0217 (no NR), P = 0.0028 (0.05 mM NR), P < 0.0001 (0.1 mM NR), P = 0.0005 (0.2 mM NR). Exogenous NR increases the abundance of the NAD⁺-related metabolites in sgNAMPT HeLa cells in a dose-dependent manner, n = 5 (f). Diagram was created with Biorender.com. g, HSV-1 titer in shCTL and shNAMPT HeLa cells without or with supplementation of nicotinamide riboside (NR, 0.1 mM) at 12 h.p.i. (MOI = 0.1), n = 3. h, Growth curve of HSV-1 is determined by intracellular virus in sgCTL and sgNAMPT HeLa cells, n = 4, P < 0.0001. Statistical significance was calculated using unpaired two-tailed t-tests, for d and f; two-way ANOVA for h. Data in b, d, f, h, and g are presented as mean values±SD. Graphics created with BioRender.com.

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±SD. Graphics created with BioRender.com.

Extended Data Fig. 3 ∣. NAMPT restricts HSV-1 replication in mice.

a, Lysates of indicated tissues of wildtype and NAMPT-KO mice were analyzed by immunoblotting with indicated antibodies. b, Body weight of wildtype and NAMPT-KO mice was determined immediately before HSV-1 infection, n = 11. c, Hematolysin & Eosin (H&E) staining and UL37 immunohistochemistry staining of the liver of HSV-1-infected Nampt^fl/fl and Nampt^−/− mice, with boxed region shown below. Scale bars, 50 μm. d, Body weight of wildtype (n = 13), NAMPT-KO (n = 10) and NAMPT-KO mice with nicotinamide riboside (NR) supplementation via intraperitoneal injection (n = 10) was determined immediately before HSV-1 infection. Statistical significance was calculated using unpaired two-tailed t-tests for b and d. Data are presented as mean values±SD.

Extended Data Fig. 4 ∣. Loss of NAMPT does not compromise innate immune response against HSV-1.

a, NAMPT-KO in HepG2 cells was validated by immunoblotting with indicated antibodies using whole cell lysates. b-c, Heatmap of cytokine gene expression in sgCTL and sgNAMPT HepG2, with Sendai virus (SeV, 30 HA unit/ml) (b) and HeLa cells infected with HSV-1 (MOI = 2) (c). n = 3 for sgCTL HepG2 infected with Sendai virus for ISG15 and IRF7 analysis, n = 3 for sgCTL HepG2 mock group for IRF7 analysis, n = 4 for all the rest analyses. d, The mRNA of indicated inflammatory genes was determined by reverse transcription and real-time PCR using the liver of wildtype (n = 4 for mock, n = 6 for HSV-1 infection), NAMPT-KO (n = 4 for mock, n = 5 for HSV-1 infection) and NAMPT-KO mice with nicotinamide riboside (dose: 400 mg/kg/day, n = 4 for mock, n = 5 for HSV-1 infection) at 3 days post-infection of HSV-1 (2 × 10⁷ PFU, intravenous). ND, not detected. Data in c and d are presented as mean values±SD.

Extended Data Fig. 5 ∣. NAMPT is incorporated into HSV-1 virions.

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.

Extended Data Fig. 6 ∣. NAMPT is a protein phosphoribosylase.

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±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 (2^nd and 3^rd 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.

Extended Data Fig. 7 ∣. Phosphoribose of structural proteins promotes their virion incorporation.

a-k, Whole cell lysates of 293 T cells transiently expressing the NAMPT-H247E mutant and HSV-1 proteins, including gD (a), gB (b), gH (c), UL21(d), UL18 (e), UL38 (f), VP16 (g), UL17 (h), UL19 (i), UL37 (j), and UL47 (k), were analyzed by two-dimensional gel electrophoresis and immunoblotting with antibodies to the V5 epitope (HSV-1 proteins) and FLAG (NAMPT). l-m, Two-dimensional gel electrophoresis and immunoblotting analysis of purified intracellular and extracellular HSV-1 virions (produced from sgNAMPT 293 T cells) with antibodies to gD (l) and quantification by densitometry of gD species (m). Numbers at the bottom (a and l) and on x-axis (m) indicate the gD species with distinct charge status.

Extended Data Fig. 8 ∣. Virion incorporation of phosphoribosylation-resistant mutants of HSV-1 proteins.

a-e, Wild-type and phosphoribosylation-resistant mutants of HSV-1 proteins, including UL37 (a), VP16 (b), UL47 (c), UL18 (d), and UL32 (e), incorporated in extracellular HSV-1 virions and expressed in HSV-1-infected cells were analyzed by immunoblotting with indicated antibodies. All HSV-1 proteins were tagged with either the V5 epitope (VP16, UL18 AND UL32) or FLAG epitope (UL37 and UL47).

Extended Data Fig. 9 ∣. Functional characterization of phosphoribosylation in HSV-1 replication using recombinant HSV-1 carrying phosphoribosylation-resistant mutations.

a, Schematic illustration of engineering recombinant HSV-1 using the bacteria artificial chromosome (BAC) system. b, Mutations engineered in the HSV-1 genome were validated by sequencing (right panels). c, Mutation of gB-D66A and -A66D (revertant) in the HSV-1 genome was confirmed by sequencing. d-e, Growth curve of HSV-1 containing phosphoribosylation-resistant mutants of UL46 (D439, 440 A), UL47 (E636A) (d), VP16 (D6A, E7A, D314A) and UL17 (D339A) (e) in HepG2 cells (MOI = 0.1) was determined by plaque assays using Vero cells. f, Growth curve of parental wildtype (WT) HSV-1 and recombinant HSV-1 carrying gB-A66D (revertant) in HepG2 cells (MOI = 0.1) was determined by plaque assay using Vero cells. Statistical significance was calculated using two-way ANOVA.

Extended Data Fig. 10 ∣. Phosphoribose of structural proteins promotes HSV-1 entry and replication.

a, Entry analysis of HSV-1 virions treated with NAMPT or NAMPT-H247E, with or without ATP (2 mM), n = 3. Statistical significance was calculated using unpaired two-tailed t-tests, P < 0.0001 (+ATP, UL37), P = 0.0031 (+ATP, UL48), P = 0.1455 (−ATP, UL37), P = 0.5709 (−ATP, UL48). b, Two-dimensional gel electrophoresis and immunoblotting analysis of HSV-1 virions treated with wildtype NAMPT (WT), the NAMPT-H247E mutant or TARG1 with antibody against gB. Red arrows indicate the new species produced by NAMPT or TARG1 treatment. c, Quantification of NAM, NMN and phosphoribose in the supernatant of the in vitro phosphoribosylase reactions by mass spectrometry, n = 3. Statistical significance was calculated using unpaired two-tailed t-tests, P < 0.0001 for NAMPT + HSV-1 group versus NAMPT + PRPP group in NAM and phosphoribose analysis. ND, not detected. d, Summary of biochemical and functional characterization of the site-specific phosphoribosylation of HSV-1 proteins. Red arrows indicate the residues whose phosphoribosylation-resistant mutations displayed significant effect on HSV-1 infection. Intensity of colour indicates the degree of effect and open circles indicate no phenotype. Statistical significance was calculated using unpaired two-tailed Student’s t-tests and two-way ANOVA analysis. Data in a and c are presented as mean values±SD.

Fig. 1 ∣. NAMPT restricts HSV-1 lytic replication.

a, Heatmap of the NAD⁺-related metabolites in HSV-1-infected HepG2 cells (multiplicity of infection (MOI) = 2, 12 h post infection (h.p.i.)). b, Schematic illustration of the NAD⁺ synthesis pathways. c, Knockdown of NAMPT (n = 3, P = 0.0003), NMNAT1 (n = 4, P < 0.0001), NRK (n = 3, P = 0.0086) or NADSYN (n = 3, P < 0.0001) in 293T cells validated by RT–qPCR using total RNA. d, HSV-1 titre in the medium of 293T cells with depletion of NAMPT (n = 3, P < 0.0001), NMNAT1 (n = 3), NRK (n = 3) or NADSYN (n = 3). e, HSV-1 titres in control (shCTL) and NAMPT-knockdown (shNAMPT) HeLa cells supplemented with NR (0.1 mM), n = 3, P = 0.0034 for shNAMPT1 versus shCTL, P = 0.0037 for shNAMPT2 versus shCTL. f,g, HSV-1 growth curve in control (sgCTL) and NAMPT-KO (sgNAMPT) HeLa cells supplemented with NR (0.1 mM), n = 3, P < 0.0001. Intracellular PRPP was determined by LC–MS at 12 h.p.i., n = 4 for shCTL, n = 3 for sgNAMPT and sgNAMPT + NR (f). Viral titre in the medium at 48 h.p.i was determined by plaque assay using a Vero monolayer, n = 3, P = 0.0038 for sgNAMPT versus shCTL and P < 0.0001 for sgNAMPT + NR versus shCTL (g). h,i, HeLa cells were infected with lentivirus expressing NAPRT (h) or shUPRT (i). HeLa cells were mock-infected or HSV-1-infected as indicated in (a). Intracellular PRPP was determined by LC–MS at 12 h.p.i (n = 4), and viral titre in the medium was determined by plaque assay using a Vero monolayer (n = 3, P = 0.022 for shNAPRT versus shCTL; P = 0.3486 for shUPRT versus shCTL). Statistical significance was calculated using unpaired two-tailed t-tests for c, d, e, g, h and i; two-way ANOVA for f. Data are presented as mean values ± s.d. in d, e, g, i and j.

Fig. 2 ∣. NAMPT restricts HSV-1 lytic replication in mice.

a, Immunoblotting analysis of the liver of Nampt^fl/fl and Nampt^−/− mice infected with HSV-1, with indicated antibodies. b–e, Age- (12–14-week-old) and gender-matched Nampt^fl/fl and Nampt^−/− mice were infected with HSV-1 (5 × 10⁷ PFU, intravenous injection) and mouse survival was recorded (b), immunohistology was performed with antibodies against NAMPT and HSV-1 antigens (UL37 and UL47) (c), HSV-1 load was determined by plaque assays using liver homogenates, n = 5, P < 0.0001 (d) and viral genome copy number was determined by real-time PCR using extracted total DNA, n = 5, P < 0.0001 (e). f–j, Diagram of the experimental design involving NAMPT deletion via tamoxifen injection, HSV-1 infection, mouse survival, HSV-1 replication and host immune response (f). Age-matched (12–14-week-old) and gender-matched Nampt^fl/fl and Nampt^−/− mice were used in these experiments. NR was injected (400 mg kg⁻¹ d⁻¹) intraperitoneally at the time of tamoxifen induction. The NAD⁺-related metabolites (g) in the liver of wild-type (n = 7), NAMPT-KO (n = 7) and NAMPT-KO with NR supplementation (n = 7) were determined immediately before HSV-1 infection. Mouse survival was recorded after HSV-1 infection (2 × 10⁷ PFU, intravenous), P = 0.0003 for Nampt^−/− versus Nampt^fl/fl, P < 0.0001 for Nampt^−/− + NR versus Nampt^fl/fl (h). HSV-1 replication was determined by plaque assay, n = 6 for Nampt^fl/fl, n = 5 for Nampt^−/− and Nampt^−/− + NR; P = 0.0004 for Nampt^−/− versus Nampt^fl/fl, P < 0.0001 for Nampt^−/− + NR versus Nampt^fl/fl, P = 0.0138 for Nampt^−/− + NR versus Nampt^−/− (i). Viral gene expression by reverse transcription followed by real-time PCR, n = 6 for Nampt^fl/fl, n = 5 for Nampt^−/− and Nampt^−/− + NR; P < 0.0001 for Nampt^−/− versus Nampt^fl/fl, P < 0.0001 for Nampt^−/− + NR versus Nampt^fl/fl, P = 0.0441 for Nampt^−/− + NR versus Nampt^−/− (j). Scale bar, 10 μm. Statistical significance was calculated using the log-rank test for mouse survival data (in b and h), unpaired two-tailed t-tests for d, e, g, I and j. Data are presented as mean values; error bars, s.d. in d, e, g, i and j. Graphics created with BioRender.com.

Fig. 3 ∣. NAMPT is packaged in HSV-1 virions.

a–c, sgNAMPT HeLa cells reconstituted with HA–NAMPT were infected with HSV-1 (MOI = 2) for 12 h. Immunofluorescence image of HSV-1-infected HeLa cells with antibodies to HA (NAMPT), UL19 and TGN46, with boxed region amplified and shown on the right (a). Immunogold staining followed by electron microscopy analysis with antibody against HA (NAMPT, 25 nm gold) and TGN46 (10 nm gold) (b) or HA (NAMPT, 25 nm gold) and UL19 (10 nm gold) (c). d, Immunoblotting analysis of fractions of sucrose gradient ultracentrifugation with indicated antibodies. WCL, whole cell lysate. e, Transmission electron microscopy analysis of purified HSV-1 virions after immunogold staining with antibodies to UL19 (10 nm gold) and HA (NAMPT, 25 nm gold). f, Interaction between NAMPT and de-enveloped HSV-1 virions analysed by co-sedimentation in sucrose gradient ultracentrifugation and immunoblotting with indicated antibodies using pelleted virions. g, Scatter plots of viral proteins identified by mass spectrometry after NAMPT–APEX2-mediated proximity ligation and normalization to the APEX2 control with a cutoff of >1.5-fold in the PSM numbers of viral proteins. h, HSV-1 proteins interact with NAMPT by co-immunoprecipitation in HSV-1-infected 293T cells that stably express FLAG–NAMPT. i, Immunoblotting analysis of purified HSV-1 virions derived from sgCTL and sgNAMPT HepG2 cells infected with HSV-1 (MOI = 0.1, 48 h.p.i.). Scale bar, 100 nm. Imaging data represents three (in a) or two (in b, c and e) independent experiments. PSM, peptide spectrum match.

Fig. 4 ∣. NAMPT is a protein phosphoribosylase.

a, HSV-1 titre in the medium of sgNAMPT HeLa cells reconstituted with wild-type (WT), H247E and D219A mutants of NAMPT at 24 h.p.i. (MOI = 0.1, n = 3) and whole cell lysates were analysed for the expression of NAMPT proteins (bottom panels) by immunoblotting with indicated antibodies. b, Schematic illustration of the procedure to identify phosphoribosylated peptides. iMAC, immobilized metal affinity chromatography. c, Schematic illustration of phosphoribose linked to glutamate, aspartate and arginine residues of peptides. d, Tandem mass spectrum of the VP22 peptide containing phosphoribosylated E74 and D75 (highlighted in red). e, Two-dimensional gel electrophoresis and immunoblotting analysis of 293T cells expressing V5–VP22 with WT or the H247E mutant of NAMPT. f,g, Elution profile by liquid chromatography (f) of phosphoribose cleaved by NAMPT (n = 3), and two-dimensional gel electrophoresis and immunoblotting analysis of GST–VP22 (g) of the in vitro phosphoribosylase reaction. h, The phosphoribosylase activity of NAMPT and NAMPT-H247E was determined by an in vitro reaction containing purified HSV-1 virions and NAMPT proteins, in which the released phosphoribose was quantitatively determined by LC–MS, n = 3 for WT NAMPT reaction with 0.00092 μg and 0.029482 μg virion; n = 4 for WT NAMPT reaction with 0.0018 μg and 0.059 μg virion; n = 5 for WT NAMPT reaction with 0.118 μg virion; n = 7 for the rest of the WT groups; n = 5 for all H247E groups. i, The V_max, K_cat and K_m of NAMPT phosphoribosylase activity determined in this study compared to its phosphoribosyltransferase activity (in NMN synthesis) defined by previous studies. Statistical significance was calculated using unpaired two-tailed t-tests. Data in a, f and h are presented as mean values ± s.d. Graphics created with BioRender.com.

Fig. 5 ∣. Phosphoribose of HSV-1 structural proteins promotes their virion incorporation.

a,b, Two-dimensional gel electrophoresis and immunoblotting analysis of purified HSV-1 virions with antibody to gD (a), and quantification by densitometry of the gD species with distinct charge as indicated by arrows in (b). c, Schematic illustration of the procedure to evaluate the virion incorporation of HSV-1 mutant proteins: 293T cells transiently expressing HSV-1 proteins (WT and phosphoribosylation-resistant mutants) were infected with HSV-1 (MOI = 0.2) and extracellular virions were collected 36 h post infection for immunoblotting analysis to examine HSV-1 structural proteins in reference to their expression in 293T cells. d–g, WT and phosphoribosylation-resistant mutants of HSV-1 proteins, including VP22 (d), gD (e), gB (f) and gH (g), in HSV-1 virions and expressed in HSV-1-infected cells were analysed by immunoblotting with indicated antibodies. All HSV-1 proteins were tagged with the V5 or FLAG epitope. Graphics created with BioRender.com.

Fig. 6 ∣. Phosphoribose of structural proteins is important for HSV-1 replication.

a, Schematic illustration of engineering and characterizing recombinant HSV-1 using BAC. b, Titre of recombinant HSV-1 carrying indicated mutations in the medium of HepG2 cells at 48 h.p.i. (MOI = 0.1, n = 3). Note, recombinant HSV-1 carrying gB-D66A failed to amplify. ND, not detected. VP22-E77A versus WT, P = 0.0002; VP22-E257A versus WT, P = 0.0003; gD-297A versus WT, P = 0.0003. Data are presented as mean values; error bars, s.d. c–f, Growth curve of WT HSV-1 and that containing VP22-E77A or VP22-E257A (phosphoribosylation-resistant) (c), gD-D297A (d), gH-D794A (e) and that containing VP22-R86A, VP22-E149A, VP22-E234A (ADP-ribosylation-resistant) (f) in the medium of HepG2 cells at a MOI = 0.1 were determined by plaque assay using Vero cells. g–h, Immunoblotting analysis of purified virions of recombinant HSV-1 containing VP22-E77A or VP22-E257A (g), gD-D297A or gH-D794A (h) compared to WT HSV-1, with HSV-1 virion normalized to UL19 and whole cell lysates of HSV-1-infected HeLa cells as controls with indicated antibodies. Statistical significance was calculated using unpaired two-tailed t-tests for b; two-way ANOVA for c, d, e and f. Graphics created with BioRender.com.

Fig. 7 ∣. Phosphoribose of structural proteins promotes HSV-1 entry.

a, Schematic illustration of entry analysis using WT and chimeric HSV-1 virions containing gD-D297A, gB-D66A or gH-D794A. b, Entry analysis of HSV-1 using HeLa cells as target cells, with HSV-1 produced from 293T cells transiently expressing WT gD (n = 4, P < 0.0001), gB (n = 4, P < 0.0001) and gH or their corresponding phosphoribosylation-resistant mutants as indicated. c–e, Entry analysis of WT recombinant HSV-1 and that containing VP22-E77A or VP22-E257A mutations; n = 3, P = 0.0004 for VP22-E77A versus WT, P = 0.0013 for VP22-E257A versus WT (c), gD-D297A, n = 3, P < 0.0001 (d) or gH-D794A, n = 3, P = 0.9520 (e). f, Entry analysis of HSV-1 produced from sgCTL, sgNAMPT HeLa cells, or sgNAMPT HeLa cells reconstituted with NAMPT; sgNAMPT versus sgCTL, n = 3, P = 0.0006; sgNAMPT + WT versus sgCTL, P = 0.0006. g–j, Schematic illustration of the in vitro phosphoribosylase reaction and subsequent functional or biochemical analysis using purified HSV-1 virions (g). Entry analysis (WT versus buffer, n = 3, P = 0.003; H247E versus WT, P = 0.1087; TARG1 versus buffer, P = 0.0246) (h), quantification of phosphoribose released in the phosphoribosylase reaction by mass spectrometry (WT versus buffer, n = 3, P < 0.0001) (i) and two-dimensional gel electrophoresis and immunoblotting analysis with anti-gD antibody of HSV-1 virions treated with NAMPT, NAMPT-H247E or TARG1 (j). Statistical significance was calculated using unpaired two-tailed t-tests, for b, f, h and i, two-way ANOVA for c, d and e. Data in b, f, h and i are presented as mean values; error bars, s.d.