Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins


Dengue virus (DENV) is a major arthropod-borne human pathogen, infecting more than 400 million individuals annually worldwide; however, neither a therapeutic drug nor a prophylactic vaccine is currently available. Amongst the DENV proteins, non-structural protein 1 (NS1) is one of the most enigmatic, being required for RNA replication, but also secreted from infected cells to counteract antiviral immune response, thus contributing to pathogenesis. Despite its essential role at early stages of the viral replication cycle, the molecular determinants governing NS1 functions are unknown. Here, we used a combination of genetic, high-resolution imaging and biochemical approaches and found that NS1 additionally plays an important role for the production of infectious virus particles. By using a novel trans-complementation system with fully functional epitope-tagged NS1, we show that NS1 interacts with the structural proteins residing in the envelope of the virus particle. An NS1 variant retained in the endoplasmic reticulum still supported efficient DENV particle production, demonstrating that secretion of NS1 is dispensable for virion production. This study expands the list of functions exerted by NS1 for the DENV replication cycle. Given this multi-functional nature, NS1 appears to be an attractive target for antiviral therapy.


Vyšlo v časopise: Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins. PLoS Pathog 11(11): e32767. doi:10.1371/journal.ppat.1005277
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.ppat.1005277

Souhrn

Dengue virus (DENV) is a major arthropod-borne human pathogen, infecting more than 400 million individuals annually worldwide; however, neither a therapeutic drug nor a prophylactic vaccine is currently available. Amongst the DENV proteins, non-structural protein 1 (NS1) is one of the most enigmatic, being required for RNA replication, but also secreted from infected cells to counteract antiviral immune response, thus contributing to pathogenesis. Despite its essential role at early stages of the viral replication cycle, the molecular determinants governing NS1 functions are unknown. Here, we used a combination of genetic, high-resolution imaging and biochemical approaches and found that NS1 additionally plays an important role for the production of infectious virus particles. By using a novel trans-complementation system with fully functional epitope-tagged NS1, we show that NS1 interacts with the structural proteins residing in the envelope of the virus particle. An NS1 variant retained in the endoplasmic reticulum still supported efficient DENV particle production, demonstrating that secretion of NS1 is dispensable for virion production. This study expands the list of functions exerted by NS1 for the DENV replication cycle. Given this multi-functional nature, NS1 appears to be an attractive target for antiviral therapy.


Zdroje

1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI (2013) The global distribution and burden of dengue. Nature 496: 504–507. nature12060 [pii]; doi: 10.1038/nature12060 23563266

2. Nowak T, Farber PM, Wengler G, Wengler G (1989) Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis. Virology 169: 365–376. 2705302

3. Paul D, Bartenschlager R (2013) Architecture and biogenesis of plus-strand RNA virus replication factories. World J Virol 2: 32–48. doi: 10.5501/wjv.v2.i2.32 24175228

4. Belov GA, van Kuppeveld FJ (2012) (+)RNA viruses rewire cellular pathways to build replication organelles. Curr Opin Virol 2: 740–747. S1879-6257(12)00141-1 [pii]; doi: 10.1016/j.coviro.2012.09.006 23036609

5. Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J, Bartenschlager R (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5: 365–375. S1931-3128(09)00098-5 [pii]; doi: 10.1016/j.chom.2009.03.007 19380115

6. Junjhon J, Pennington JG, Edwards TJ, Perera R, Lanman J, Kuhn RJ (2014) Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells. J Virol 88: 4687–4697. JVI.00118-14 [pii]; doi: 10.1128/JVI.00118-14 24522909

7. Winkler G, Randolph VB, Cleaves GR, Ryan TE, Stollar V (1988) Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162: 187–196. 2827377

8. Gutsche I, Coulibaly F, Voss JE, Salmon J, d'Alayer J, Ermonval M, Larquet E, Charneau P, Krey T, Megret F, Guittet E, Rey FA, Flamand M (2011) Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci U S A 108: 8003–8008. 1017338108 [pii]; doi: 10.1073/pnas.1017338108 21518917

9. Akey DL, Brown WC, Dutta S, Konwerski J, Jose J, Jurkiw TJ, DelProposto J, Ogata CM, Skiniotis G, Kuhn RJ, Smith JL (2014) Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science 343: 881–885. science.1247749 [pii]; doi: 10.1126/science.1247749 24505133

10. Youn S, Li T, McCune BT, Edeling MA, Fremont DH, Cristea IM, Diamond MS (2012) Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus. J Virol 86: 7360–7371. JVI.00157-12 [pii]; doi: 10.1128/JVI.00157-12 22553322

11. Avirutnan P, Hauhart RE, Somnuke P, Blom AM, Diamond MS, Atkinson JP (2011) Binding of flavivirus nonstructural protein NS1 to C4b binding protein modulates complement activation. J Immunol 187: 424–433. jimmunol.1100750 [pii]; doi: 10.4049/jimmunol.1100750 21642539

12. Avirutnan P, Fuchs A, Hauhart RE, Somnuke P, Youn S, Diamond MS, Atkinson JP (2010) Antagonism of the complement component C4 by flavivirus nonstructural protein NS1. J Exp Med 207: 793–806. jem.20092545 [pii]; doi: 10.1084/jem.20092545 20308361

13. Chung KM, Liszewski MK, Nybakken G, Davis AE, Townsend RR, Fremont DH, Atkinson JP, Diamond MS (2006) West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H. Proc Natl Acad Sci U S A 103: 19111–19116. 0605668103 [pii]; doi: 10.1073/pnas.0605668103 17132743

14. Wilson JR, de Sessions PF, Leon MA, Scholle F (2008) West Nile virus nonstructural protein 1 inhibits TLR3 signal transduction. J Virol 82: 8262–8271. JVI.00226-08 [pii]; doi: 10.1128/JVI.00226-08 18562533

15. Chatel-Chaix L, Fischl W, Scaturro P, Cortese M, Kallis S, Bartenschlager M, Fischer B, Bartenschlager R (2015) A combined genetic-proteomic approach identifies residues within Dengue virus NS4B critical for interaction with NS3 and viral replication. J Virol. JVI.00867-15 [pii]; doi: 10.1128/JVI.00867-15

16. Mackenzie JM, Jones MK, Young PR (1996) Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220: 232–240. S0042-6822(96)90307-4 [pii]; doi: 10.1006/viro.1996.0307 8659120

17. Youn S, Ambrose RL, Mackenzie JM, Diamond MS (2013) Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis. Virol J 10: 339. 1743-422X-10-339 [pii]; doi: 10.1186/1743-422X-10-339 24245822

18. Lindenbach BD, Rice CM (1997) trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71: 9608–9617. 9371625

19. Khromykh AA, Kenney MT, Westaway EG (1998) trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. J Virol 72: 7270–7279. 9696822

20. Khromykh AA, Sedlak PL, Westaway EG (2000) cis- and trans-acting elements in flavivirus RNA replication. J Virol 74: 3253–3263. 10708442

21. Khromykh AA, Sedlak PL, Guyatt KJ, Hall RA, Westaway EG (1999) Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. J Virol 73: 10272–10280. 10559344

22. Edeling MA, Diamond MS, Fremont DH (2014) Structural basis of Flavivirus NS1 assembly and antibody recognition. Proc Natl Acad Sci U S A 111: 4285–4290. 1322036111 [pii]; doi: 10.1073/pnas.1322036111 24594604

23. Flamand M, Megret F, Mathieu M, Lepault J, Rey FA, Deubel V (1999) Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol 73: 6104–6110. 10364366

24. Crooks AJ, Lee JM, Easterbrook LM, Timofeev AV, Stephenson JR (1994) The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell. J Gen Virol 75 (Pt 12): 3453–3460. 7527836

25. Alcon-LePoder S, Drouet MT, Roux P, Frenkiel MP, Arborio M, Durand-Schneider AM, Maurice M, Le B I, Gruenberg J, Flamand M (2005) The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes: implications for viral infectivity. J Virol 79: 11403–11411. 79/17/11403 [pii]; doi: 10.1128/JVI.79.17.11403-11411.2005 16103191

26. Blitvich BJ, Mackenzie JS, Coelen RJ, Howard MJ, Hall RA (1995) A novel complex formed between the flavivirus E and NS1 proteins: analysis of its structure and function. Arch Virol 140: 145–156. 7646339

27. Young LB, Melian EB, Khromykh AA (2013) NS1' colocalizes with NS1 and can substitute for NS1 in West Nile virus replication. J Virol 87: 9384–9390. JVI.01101-13 [pii]; doi: 10.1128/JVI.01101-13 23760245

28. Kurosu T, Chaichana P, Yamate M, Anantapreecha S, Ikuta K (2007) Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochem Biophys Res Commun 362: 1051–1056. S0006-291X(07)01834-7 [pii]; doi: 10.1016/j.bbrc.2007.08.137 17825259

29. Cheng HJ, Lin CF, Lei HY, Liu HS, Yeh TM, Luo YH, Lin YS (2009) Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies. Exp Biol Med (Maywood) 234: 63–73. 0805-RM-147 [pii]; doi: 10.3181/0805-RM-147

30. Falconar AK (2008) Monoclonal antibodies that bind to common epitopes on the dengue virus type 2 nonstructural-1 and envelope glycoproteins display weak neutralizing activity and differentiated responses to virulent strains: implications for pathogenesis and vaccines. Clin Vaccine Immunol 15: 549–561. CVI.00351-07 [pii]; doi: 10.1128/CVI.00351-07 18160621

31. Falconar AK (1997) The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis. Arch Virol 142: 897–916. 9191856

32. Henchal EA, Henchal LS, Schlesinger JJ (1988) Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. J Gen Virol 69 (Pt 8): 2101–2107. 3404125

33. Lee JM, Crooks AJ, Stephenson JR (1989) The synthesis and maturation of a non-structural extracellular antigen from tick-borne encephalitis virus and its relationship to the intracellular NS1 protein. J Gen Virol 70 (Pt 2): 335–343. 2471787

34. Mason PW (1989) Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169: 354–364. 2523178

35. Muylaert IR, Chambers TJ, Galler R, Rice CM (1996) Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence. Virology 222: 159–168. S0042-6822(96)90406-7 [pii]; doi: 10.1006/viro.1996.0406 8806496

36. Lindenbach BD, Rice CM (1999) Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol 73: 4611–4621. 10233920

37. Suzuki R, de BL, Duarte dos Santos CN, Mason PW (2007) Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1. Virology 362: 374–383. S0042-6822(06)00875-0 [pii]; doi: 10.1016/j.virol.2006.11.026 17289102

38. Somnuke P, Hauhart RE, Atkinson JP, Diamond MS, Avirutnan P (2011) N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement. Virology 413: 253–264. S0042-6822(11)00094-8 [pii]; doi: 10.1016/j.virol.2011.02.022 21429549

39. Wallis TP, Huang CY, Nimkar SB, Young PR, Gorman JJ (2004) Determination of the disulfide bond arrangement of dengue virus NS1 protein. J Biol Chem 279: 20729–20741. doi: 10.1074/jbc.M312907200 ;M312907200 [pii]. 14981082

40. Youn S, Cho H, Fremont DH, Diamond MS (2010) A short N-terminal peptide motif on flavivirus nonstructural protein NS1 modulates cellular targeting and immune recognition. J Virol 84: 9516–9532. JVI.00775-10 [pii]; doi: 10.1128/JVI.00775-10 20592095

41. Kudelko M, Brault JB, Kwok K, Li MY, Pardigon N, Peiris JS, Bruzzone R, Despres P, Nal B, Wang PG (2012) Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses. J Biol Chem 287: 767–777. M111.270579 [pii]; doi: 10.1074/jbc.M111.270579 22105072

42. Li MY, Grandadam M, Kwok K, Lagache T, Siu YL, Zhang JS, Sayteng K, Kudelko M, Qin CF, Olivo-Marin JC, Bruzzone R, Wang PG (2015) KDEL Receptors Assist Dengue Virus Exit from the Endoplasmic Reticulum. Cell Rep. S2211-1247(15)00167-9 [pii]; doi: 10.1016/j.celrep.2015.02.021

43. Kummerer BM, Rice CM (2002) Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 76: 4773–4784. 11967294

44. Vossmann S, Wieseler J, Kerber R, Kummerer BM (2015) A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production. J Virol 89: 4951–4965. JVI.03351-14 [pii]; doi: 10.1128/JVI.03351-14 25694595

45. Apte-Sengupta S, Sirohi D, Kuhn RJ (2014) Coupling of replication and assembly in flaviviruses. Curr Opin Virol 9: 134–142. S1879-6257(14)00202-8 [pii]; doi: 10.1016/j.coviro.2014.09.020 25462445

46. Mateo R, Nagamine CM, Spagnolo J, Mendez E, Rahe M, Gale M Jr., Yuan J, Kirkegaard K (2013) Inhibition of cellular autophagy deranges dengue virion maturation. J Virol 87: 1312–1321. JVI.02177-12 [pii]; doi: 10.1128/JVI.02177-12 23175363

47. Heinz FX, Stiasny K, Puschner-Auer G, Holzmann H, Allison SL, Mandl CW, Kunz C (1994) Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198: 109–117. S0042-6822(84)71013-0 [pii]; doi: 10.1006/viro.1994.1013 8259646

48. Hsieh SC, Wu YC, Zou G, Nerurkar VR, Shi PY, Wang WK (2014) Highly conserved residues in the helical domain of dengue virus type 1 precursor membrane protein are involved in assembly, precursor membrane (prM) protein cleavage, and entry. J Biol Chem 289: 33149–33160. M114.610428 [pii]; doi: 10.1074/jbc.M114.610428 25326389

49. Pong WL, Huang ZS, Teoh PG, Wang CC, Wu HN (2011) RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNA-interacting proteins. FEBS Lett 585: 2575–2581. S0014-5793(11)00514-X [pii]; doi: 10.1016/j.febslet.2011.06.038 21771593

50. Teoh PG, Huang ZS, Pong WL, Chen PC, Wu HN (2014) Maintenance of dimer conformation by the dengue virus core protein alpha4-alpha4' helix pair is critical for nucleocapsid formation and virus production. J Virol 88: 7998–8015. JVI.00940-14 [pii]; doi: 10.1128/JVI.00940-14 24807709

51. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J (1982) Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42: 3858–3863. 6286115

52. Steigemann P, Wurzenberger C, Schmitz MH, Held M, Guizetti J, Maar S, Gerlich DW (2009) Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136: 473–484. S0092-8674(08)01601-2 [pii]; doi: 10.1016/j.cell.2008.12.020 19203582

53. Steinmann E, Brohm C, Kallis S, Bartenschlager R, Pietschmann T (2008) Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J Virol 82: 7034–7046. JVI.00118-08 [pii]; doi: 10.1128/JVI.00118-08 18480457

54. Scaturro P, Trist IM, Paul D, Kumar A, Acosta EG, Byrd CM, Jordan R, Brancale A, Bartenschlager R (2014) Characterization of the mode of action of a potent dengue virus capsid inhibitor. J Virol 88: 11540–11555. JVI.01745-14 [pii]; doi: 10.1128/JVI.01745-14 25056895

55. Gastaminza P, Kapadia SB, Chisari FV (2006) Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J Virol 80: 11074–11081. JVI.01150-06 [pii]; doi: 10.1128/JVI.01150-06 16956946

56. Fischl W, Bartenschlager R (2013) High-throughput screening using dengue virus reporter genomes. Methods Mol Biol 1030: 205–219. doi: 10.1007/978-1-62703-484-5_17 23821271

57. Kumar A, Buhler S, Selisko B, Davidson A, Mulder K, Canard B, Miller S, Bartenschlager R (2013) Nuclear localization of dengue virus nonstructural protein 5 does not strictly correlate with efficient viral RNA replication and inhibition of type I interferon signaling. J Virol 87: 4545–4557. JVI.03083-12 [pii]; doi: 10.1128/JVI.03083-12 23408610

58. Gouttenoire J, Roingeard P, Penin F, Moradpour D (2010) Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B. J Virol 84: 12529–12537. JVI.01798-10 [pii]; doi: 10.1128/JVI.01798-10 20926561

59. Schaller T, Appel N, Koutsoudakis G, Kallis S, Lohmann V, Pietschmann T, Bartenschlager R (2007) Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol 81: 4591–4603. JVI.02144-06 [pii]; doi: 10.1128/JVI.02144-06 17301154

60. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612. doi: 10.1002/jcc.20084 15264254

61. Paul D, Romero-Brey I, Gouttenoire J, Stoitsova S, Krijnse-Locker J, Moradpour D, Bartenschlager R (2011) NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes. J Virol 85: 6963–6976. JVI.00502-11 [pii]; doi: 10.1128/JVI.00502-11 21543474

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