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

Nature Metabolism volume 6, pages 2300–2318 (2024)Cite this article

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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.

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Fig. 1: NAMPT restricts HSV-1 lytic replication.
Fig. 2: NAMPT restricts HSV-1 lytic replication in mice.
Fig. 3: NAMPT is packaged in HSV-1 virions.
Fig. 4: NAMPT is a protein phosphoribosylase.
Fig. 5: Phosphoribose of HSV-1 structural proteins promotes their virion incorporation.
Fig. 6: Phosphoribose of structural proteins is important for HSV-1 replication.
Fig. 7: Phosphoribose of structural proteins promotes HSV-1 entry.

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Data availability

All raw datasets used for phosphoribosylated peptide analysis can be found in the PRIDE public repository under project accession number PXD050684. All source data supporting the main figures and extended data figures are published within the paper. Source data are provided with this paper.

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Acknowledgements

We thank S.-I. Imai (Washington University) for providing the Namptfl/fl mice, S. Petteri (Stanford University) and N. Graham (University of Southern California) for LC–MS techniques, J. Hao (Poochon Scientific) for APEX-related protein identification, W. Beatty (Washington University) for electron microscopy analysis, W. Yuan (University of Southern California), D. Knipe (Harvard Medical School) and R. Longnecker (Northwestern University) for providing HSV-1 plasmids, J. Zhao (Cleveland Clinic Foundation) for guidance on generating HSV-1 mutant viruses, G. Cohen (University of Pennsylvania) for anti-gH antibody, C. Zhen (University of Calgary) for anti-VP22 antibody, B. Lomenick (CalTech) for processing MS samples, and N. Graham (University of Southern California) and S. Pitteri (Stanford University) for assistance on mass spectrometry analysis. We are grateful to Y. Zhou, S. Rice, J. Carriere and J. Xiao for their assistance. This work was partly supported by a startup fund from the Herman Ostrow School of Dentistry of the University of Southern California and grants from the National Institutes of Health (AG070904, CA285192 and AI180537) and Infectious Disease Society of America Foundation (Microbial Pathogenesis in AD) (P.F.) and National Natural Science Foundation of China (81821002) (C.H.).

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Author notes

  1. Shu Feng

    Present address: Department of Diabetes and Cancer Metabolism, Beckman Research Institute, City of Hope, Duarte, CA, USA

  2. These authors contributed equally: Shu Feng, Na Xie.

Authors and Affiliations

  1. Section of Infection and Immunity, Herman Ostrow School of Dentistry, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA

    Shu Feng, Na Xie, Yongzhen Liu, Chao Qin, Ali Can Savas, Ting-Yu Wang, Shutong Li, Youliang Rao, Alexandra Shambayate & Pinghui Feng

  2. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and West China, School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, PR China

    Na Xie & Canhua Huang

  3. Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA

    Ting-Yu Wang & Tsui-Fen Chou

  4. Department of Diabetes and Cancer Metabolism, Beckman Research Institute, City of Hope, Duarte, CA, USA

    Charles Brenner

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Contributions

P.F. and S.F. conceived the project. S.F., N.X., Y.L., C.Q., T.W., P.F. and T.C. determined the methodology. S.F., N.X., Y.L., C.Q., A.C.S., T.W., Y.R., S.L. and A.S. performed the investigation. P.F., C.B. and C.H. acquired funding. P.F. and C.H. were responsible for project administration. P.F., C.B., C.H. and T.C. supervised the project. P.F. and S.F. wrote the original draft of the manuscript; P.F., S.F., C.B. and C.Q. reviewed and edited the final version.

Corresponding author

Correspondence to Pinghui Feng.

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Competing interests

C.B. is a chief scientific advisor of ChromaDex and co-founder of Alphina Therapeutics. P.F. is a consultant for Marc J Bern & Partners. All other authors declare no competing interests.

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Nature Metabolism thanks Joseph Baur, Claire Eyers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt, in collaboration with the Nature Metabolism team.

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Extended data

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\(\pm\)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\(\pm\)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 Namptfl/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\(\pm\)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\(\pm\)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\(\pm\)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.

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\(\pm\)SD.

Supplementary information

Supplementary Information

Supplementary Figures

Reporting Summary

Supplementary Table 1

Supplementary Table 1. An Excel file summarizing NAMPT-binding proteins identified by APEX2-mediated biotinylation and mass spectrometry, related to Fig. 3g (source file). Supplementary Table 2. An Excel file summarizing the NAMPT-binding HSV-1 proteins identified by APEX2-mediated biotinylation and mass spectrometry, related to Fig. 3g. Supplementary Table 3. An Excel file summarizing the phosphoribosylated peptides identified from a regular (2 h) run of HSV-1-infected cell lysates of HeLa expressing wildtype NAMPT or NAMPT-H247E mutant, related to Extended Data Fig. 6c and 6d. Supplementary Table 4. An Excel file showing the percentage of phosphoribosylated peptides normalized to the total peptides analyzed in Supplementary Table 3, related to Extended Data Fig. 6c and 6d. Supplementary Table 5. An Excel file providing the raw data for total peptides and proteins identified from an extended (4 h) run of HSV-1-infected cell lysates of HeLa expressing wildtype NAMPT or NAMPT-H247E mutant, related to Extended Data Fig. 6c and 6d. Supplementary Table 6. An Excel file providing the raw data for viral peptides and proteins identified from an extended (4 h) run of HSV-1-infected cell lysates of HeLa expressing wildtype NAMPT or NAMPT-H247E mutant, related to Extended Data Fig. 6c and 6d. Supplementary Table 7. An Excel file showing the percentage of phosphoribosylated viral peptides summarized in Supplementary Tables 5 and 6, related to Extended Data Fig. 6c and 6d. Supplementary Table 8. An Excel file summarizing the quantification of viral and cellular phosphoribosylated peptides summarized in Supplementary Tables 5 and 6, related to Extended Data Fig. 6c and 6d. Supplementary Table 9. An Excel file showing the percentage of phosphoribosylated total (viral and cellular) peptides summarized in Supplementary Tables 5 and 6, related to Extended Data Fig. 6c and 6d. Supplementary Table 10. A list of primers used in this study.

Source data

Source Data Fig. 1

Unprocessed western blots and/or gels, transmission electron microscopy images and immunofluorescence images.

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Statistical Source Data.

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Statistical Source Data.

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Feng, S., Xie, N., Liu, Y. et al. Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins. Nat Metab 6, 2300–2318 (2024). https://doi.org/10.1038/s42255-024-01162-0

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