Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation

Autoři: Wenwu Lin aff001;  Yuyan Liu aff002;  Melissa Molho aff002;  Shengjie Zhang aff001;  Longshen Wang aff001;  Lianhui Xie aff001;  Peter D. Nagy aff002
Působiště autorů: State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China aff001;  Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, United States of America aff002
Vyšlo v časopise: Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation. PLoS Pathog 15(10): e1008092. doi:10.1371/journal.ppat.1008092
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008092


The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

Klíčová slova:

Fermentation – Fluorescence resonance energy transfer – Glycolysis – Leaves – Protein interactions – Reverse transcriptase-polymerase chain reaction – Viral replication – Yeast


1. de Castro IF, Volonte L, Risco C (2013) Virus factories: biogenesis and structural design. Cell Microbiol 15: 24–34. doi: 10.1111/cmi.12029 22978691

2. Belov GA, van Kuppeveld FJ (2012) (+)RNA viruses rewire cellular pathways to build replication organelles. Curr Opin Virol 2: 740–747. doi: 10.1016/j.coviro.2012.09.006 23036609

3. den Boon JA, Ahlquist P (2010) Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 64: 241–256. doi: 10.1146/annurev.micro.112408.134012 20825348

4. Nagy PD, Pogany J (2012) The dependence of viral RNA replication on co-opted host factors. Nature Reviews Microbiology 10: 137–149.

5. Wang A (2015) Dissecting the molecular network of virus-plant interactions: the complex roles of host factors. Annu Rev Phytopathol 53: 45–66. doi: 10.1146/annurev-phyto-080614-120001 25938276

6. Altan-Bonnet N (2017) Lipid Tales of Viral Replication and Transmission. Trends Cell Biol 27: 201–213. doi: 10.1016/j.tcb.2016.09.011 27838086

7. van der Schaar HM, Dorobantu CM, Albulescu L, Strating JR, van Kuppeveld FJ (2016) Fat(al) attraction: Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles. Trends Microbiol 24: 535–546. doi: 10.1016/j.tim.2016.02.017 27020598

8. Shulla A, Randall G (2016) (+) RNA virus replication compartments: a safe home for (most) viral replication. Curr Opin Microbiol 32: 82–88. doi: 10.1016/j.mib.2016.05.003 27253151

9. Kovalev N, Inaba JI, Li Z, Nagy PD (2017) The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast. PLoS Pathog 13: e1006520. doi: 10.1371/journal.ppat.1006520 28759634

10. Carbonell A, Carrington JC (2015) Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27: 111–117. doi: 10.1016/j.pbi.2015.06.013 26190744

11. Andino R (2003) RNAi puts a lid on virus replication. Nat Biotechnol 21: 629–630. doi: 10.1038/nbt0603-629 12776148

12. Hsu NY, Ilnytska O, Belov G, Santiana M, Chen YH, et al. (2010) Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell 141: 799–811. doi: 10.1016/j.cell.2010.03.050 20510927

13. Diamond DL, Syder AJ, Jacobs JM, Sorensen CM, Walters KA, et al. (2010) Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLoS pathogens 6: e1000719. doi: 10.1371/journal.ppat.1000719 20062526

14. Schoggins JW, Randall G (2013) Lipids in innate antiviral defense. Cell host & microbe 14: 379–385.

15. Perera R, Riley C, Isaac G, Hopf-Jannasch AS, Moore RJ, et al. (2012) Dengue virus infection perturbs lipid homeostasis in infected mosquito cells. PLoS pathogens 8: e1002584. doi: 10.1371/journal.ppat.1002584 22457619

16. Syed GH, Amako Y, Siddiqui A (2010) Hepatitis C virus hijacks host lipid metabolism. Trends Endocrinol Metab 21: 33–40. doi: 10.1016/j.tem.2009.07.005 19854061

17. Heaton NS, Randall G (2010) Dengue virus-induced autophagy regulates lipid metabolism. Cell host & microbe 8: 422–432.

18. Heaton NS, Perera R, Berger KL, Khadka S, Lacount DJ, et al. (2010) Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis. Proceedings of the National Academy of Sciences of the United States of America 107: 17345–17350. doi: 10.1073/pnas.1010811107 20855599

19. Wang X, Diaz A, Hao L, Gancarz B, den Boon JA, et al. (2011) Intersection of the multivesicular body pathway and lipid homeostasis in RNA replication by a positive-strand RNA virus. Journal of virology 85: 5494–5503. doi: 10.1128/JVI.02031-10 21430061

20. Nagy PD, Pogany J, Lin JY (2014) How yeast can be used as a genetic platform to explore virus-host interactions: from 'omics' to functional studies. Trends Microbiol 22: 309–316. doi: 10.1016/j.tim.2014.02.003 24647076

21. Nagy PD (2017) Exploitation of a surrogate host, Saccharomyces cerevisiae, to identify cellular targets and develop novel antiviral approaches. Curr Opin Virol 26: 132–140. doi: 10.1016/j.coviro.2017.07.031 28843111

22. Nagy PD (2016) Tombusvirus-Host Interactions: Co-Opted Evolutionarily Conserved Host Factors Take Center Court. Annu Rev Virol 3: 491–515. doi: 10.1146/annurev-virology-110615-042312 27578441

23. Xu K, Nagy PD (2014) Expanding use of multi-origin subcellular membranes by positive-strand RNA viruses during replication. Curr Opin Virol 9C: 119–126.

24. Xu K, Nagy PD (2017) Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants. J Virol 91.

25. Xu K, Nagy PD (2016) Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus. PLoS Biol 14: e2000128. doi: 10.1371/journal.pbio.2000128 27760128

26. Xu K, Nagy PD (2015) RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes. Proc Natl Acad Sci U S A 112: E1782–E1791. doi: 10.1073/pnas.1418971112 25810252

27. Pogany J, Nagy PD (2015) Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro. J Virol 89: 5714–5723. doi: 10.1128/JVI.03711-14 25762742

28. Pogany J, Nagy PD (2012) p33-Independent Activation of a Truncated p92 RNA-Dependent RNA Polymerase of Tomato Bushy Stunt Virus in Yeast Cell-Free Extract. J Virol 86: 12025–12038. doi: 10.1128/JVI.01303-12 22933278

29. Pogany J, White KA, Nagy PD (2005) Specific binding of tombusvirus replication protein p33 to an internal replication element in the viral RNA is essential for replication. J Virol 79: 4859–4869. doi: 10.1128/JVI.79.8.4859-4869.2005 15795271

30. Nagy PD, Strating JR, van Kuppeveld FJ (2016) Building Viral Replication Organelles: Close Encounters of the Membrane Types. PLoS Pathog 12: e1005912. doi: 10.1371/journal.ppat.1005912 27788266

31. Fernandez de Castro I, Fernandez JJ, Barajas D, Nagy PD, Risco C (2017) Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex. J Cell Sci 130: 260–268. doi: 10.1242/jcs.181586 27026525

32. Prasanth KR, Chuang C, Nagy PD (2017) Co-opting ATP-generating glycolytic enzyme PGK1 phosphoglycerate kinase facilitates the assembly of viral replicase complexes. PLoS Pathog 13: e1006689. doi: 10.1371/journal.ppat.1006689 29059239

33. Chuang C, Prasanth KR, Nagy PD (2017) The Glycolytic Pyruvate Kinase Is Recruited Directly into the Viral Replicase Complex to Generate ATP for RNA Synthesis. Cell Host Microbe 22: 639–652 e637. doi: 10.1016/j.chom.2017.10.004 29107644

34. Huang TS, Nagy PD (2011) Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants. J Virol 85: 9090–9102. doi: 10.1128/JVI.00666-11 21697488

35. Wang RY, Nagy PD (2008) Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis. Cell Host Microbe 3: 178–187. doi: 10.1016/j.chom.2008.02.005 18329617

36. Lunt SY, Vander Heiden MG (2011) Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol 27: 441–464. doi: 10.1146/annurev-cellbio-092910-154237 21985671

37. Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324: 1029–1033. doi: 10.1126/science.1160809 19460998

38. Olson KA, Schell JC, Rutter J (2016) Pyruvate and Metabolic Flexibility: Illuminating a Path Toward Selective Cancer Therapies. Trends Biochem Sci 41: 219–230. doi: 10.1016/j.tibs.2016.01.002 26873641

39. Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P (2007) Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol 5: e220. doi: 10.1371/journal.pbio.0050220 17696647

40. Eberhardt I, Cederberg H, Li H, Konig S, Jordan F, et al. (1999) Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity. Eur J Biochem 262: 191–201. doi: 10.1046/j.1432-1327.1999.00370.x 10231381

41. Hohmann S (1993) Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevisiae. Mol Gen Genet 241: 657–666. doi: 10.1007/bf00279908 8264540

42. Snider J, Kittanakom S, Curak J, Stagljar I (2010) Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: a powerful tool for identifying protein-protein interactions. J Vis Exp.

43. Barajas D, Martin IF, Pogany J, Risco C, Nagy PD (2014) Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase. PLoS Pathog 10: e1004087. doi: 10.1371/journal.ppat.1004087 24763736

44. Kovalev N, Pogany J, Nagy PD (2014) Template role of double-stranded RNA in tombusvirus replication. J Virol 88: 5638–5651. doi: 10.1128/JVI.03842-13 24600009

45. Mithran M, Paparelli E, Novi G, Perata P, Loreti E (2014) Analysis of the role of the pyruvate decarboxylase gene family in Arabidopsis thaliana under low-oxygen conditions. Plant Biol (Stuttg) 16: 28–34.

46. Ismond KP, Dolferus R, de Pauw M, Dennis ES, Good AG (2003) Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway. Plant Physiol 132: 1292–1302. doi: 10.1104/pp.103.022244 12857811

47. Panavas T, Hawkins CM, Panaviene Z, Nagy PD (2005) The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus. Virology 338: 81–95. doi: 10.1016/j.virol.2005.04.025 15936051

48. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, et al. (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2: 437–445. doi: 10.1016/s1097-2765(00)80143-4 9809065

49. Chuang C, Prasanth KR, Nagy PD (2015) Coordinated Function of Cellular DEAD-Box Helicases in Suppression of Viral RNA Recombination and Maintenance of Viral Genome Integrity. PLoS Pathog 11: e1004680. doi: 10.1371/journal.ppat.1004680 25693185

50. Kovalev N, Pogany J, Nagy PD (2012) A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis. PLoS Pathog 8: e1002537. doi: 10.1371/journal.ppat.1002537 22359508

51. Wang RY, Stork J, Pogany J, Nagy PD (2009) A temperature sensitive mutant of heat shock protein 70 reveals an essential role during the early steps of tombusvirus replication. Virology 394: 28–38. doi: 10.1016/j.virol.2009.08.003 19748649

52. Wang RY, Stork J, Nagy PD (2009) A key role for heat shock protein 70 in the localization and insertion of tombusvirus replication proteins to intracellular membranes. J Virol 83: 3276–3287. doi: 10.1128/JVI.02313-08 19153242

53. Pogany J, Stork J, Li Z, Nagy PD (2008) In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70. Proc Natl Acad Sci U S A 105: 19956–19961. doi: 10.1073/pnas.0810851105 19060219

54. Imamura H, Nhat KP, Togawa H, Saito K, Iino R, et al. (2009) Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci U S A 106: 15651–15656. doi: 10.1073/pnas.0904764106 19720993

55. Nagy PD (2015) Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection. J Virol 89: 5196–5199. doi: 10.1128/JVI.02973-14 25741009

56. Inaba JI, Nagy PD (2018) Tombusvirus RNA replication depends on the TOR pathway in yeast and plants. Virology 519: 207–222. doi: 10.1016/j.virol.2018.04.010 29734044

57. Barajas D, Xu K, Sharma M, Wu CY, Nagy PD (2014) Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast. Virology 471-473C: 72–80.

58. Palm W, Thompson CB (2017) Nutrient acquisition strategies of mammalian cells. Nature 546: 234–242. doi: 10.1038/nature22379 28593971

59. Yu L, Chen X, Wang L, Chen S (2018) Oncogenic virus-induced aerobic glycolysis and tumorigenesis. J Cancer 9: 3699–3706. doi: 10.7150/jca.27279 30405839

60. Vaishnavi SN, Vlassenko AG, Rundle MM, Snyder AZ, Mintun MA, et al. (2010) Regional aerobic glycolysis in the human brain. Proc Natl Acad Sci U S A 107: 17757–17762. doi: 10.1073/pnas.1010459107 20837536

61. Vlassenko AG, Vaishnavi SN, Couture L, Sacco D, Shannon BJ, et al. (2010) Spatial correlation between brain aerobic glycolysis and amyloid-beta (Abeta) deposition. Proc Natl Acad Sci U S A 107: 17763–17767. doi: 10.1073/pnas.1010461107 20837517

62. Jones W, Bianchi K (2015) Aerobic glycolysis: beyond proliferation. Front Immunol 6: 227. doi: 10.3389/fimmu.2015.00227 26029212

63. Shannon BJ, Vaishnavi SN, Vlassenko AG, Shimony JS, Rutlin J, et al. (2016) Brain aerobic glycolysis and motor adaptation learning. Proc Natl Acad Sci U S A 113: E3782–3791. doi: 10.1073/pnas.1604977113 27217563

64. Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, et al. (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21: 947–962. doi: 10.1002/yea.1142 15334558

65. Jaag HM, Nagy PD (2009) Silencing of Nicotiana benthamiana Xrn4p exoribonuclease promotes tombusvirus RNA accumulation and recombination. Virology 386: 344–352. doi: 10.1016/j.virol.2009.01.015 19232421

66. Li Z, Barajas D, Panavas T, Herbst DA, Nagy PD (2008) Cdc34p ubiquitin-conjugating enzyme is a component of the tombusvirus replicase complex and ubiquitinates p33 replication protein. J Virol 82: 6911–6926. doi: 10.1128/JVI.00702-08 18463149

67. Bachan S, Dinesh-Kumar SP (2012) Tobacco rattle virus (TRV)-based virus-induced gene silencing. Methods Mol Biol 894: 83–92. doi: 10.1007/978-1-61779-882-5_6 22678574

68. Lin W, Wang L, Yan W, Chen L, Chen H, et al. (2017) Identification and characterization of Bamboo mosaic virus isolates from a naturally occurring coinfection in Bambusa xiashanensis. Arch Virol 162: 1335–1339. doi: 10.1007/s00705-016-3191-2 28050737

69. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2: 1565–1572. doi: 10.1038/nprot.2007.199 17585298

70. Pogany J, Nagy PD (2008) Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast. J Virol 82: 5967–5980. doi: 10.1128/JVI.02737-07 18417594

71. Rajendran KS, Nagy PD (2006) Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: requirement of p33:p92 interaction for replicase assembly. Virology 345: 270–279. doi: 10.1016/j.virol.2005.09.038 16242746

72. Rajendran KS, Nagy PD (2003) Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. J Virol 77: 9244–9258. doi: 10.1128/JVI.77.17.9244-9258.2003 12915540

Hygiena a epidemiologie Infekční lékařství Laboratoř

Článek vyšel v časopise

PLOS Pathogens

2019 Číslo 10

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Ulcerative colitis_muž_břicho_střeva
Ulcerózní kolitida
nový kurz

Blokátory angiotenzinových receptorů (sartany)
Autoři: MUDr. Jiří Krupička, Ph.D.

Antiseptika a prevence ve stomatologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Citikolin v neuroprotekci a neuroregeneraci: od výzkumu do klinické praxe nejen očních lékařů
Autoři: MUDr. Petr Výborný, CSc., FEBO

Zánětlivá bolest zad a axiální spondylartritida – Diagnostika a referenční strategie
Autoři: MUDr. Monika Gregová, Ph.D., MUDr. Kristýna Bubová

Všechny kurzy
Kurzy Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se