Monomeric Nucleoprotein of Influenza A Virus

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Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.

Vyšlo v časopise: Monomeric Nucleoprotein of Influenza A Virus. PLoS Pathog 9(3): e32767. doi:10.1371/journal.ppat.1003275
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003275

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Zdroje

1. BaudinF, BachC, CusackS, RuigrokRW (1994) Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. Embo J 13: 3158–3165.

2. IseniF, BaudinF, BlondelD, RuigrokRW (2000) Structure of the RNA inside the vesicular stomatitis virus nucleocapsid. Rna 6: 270–281.

3. ArnheiterH, DavisNL, WertzG, SchubertM, LazzariniRA (1985) Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Cell 41: 259–267.

4. HondaA, UedaK, NagataK, IshihamaA (1988) RNA polymerase of influenza virus: role of NP in RNA chain elongation. J Biochem 104: 1021–1026.

5. RuigrokRW, CrepinT, KolakofskyD (2011) Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Microbiol 14: 504–510.

6. MastersPS, BanerjeeAK (1988) Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. J Virol 62: 2658–2664.

7. CurranJ, MarqJB, KolakofskyD (1995) An N-terminal domain of the Sendai paramyxovirus P protein acts as a chaperone for the NP protein during the nascent chain assembly step of genome replication. J Virol 69: 849–855.

8. MavrakisM, MehouasS, RealE, IseniF, BlondelD, et al. (2006) Rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA. Virology 349: 422–429.

9. LeyratC, YabukarskiF, TarbouriechN, RibeiroEAJr, JensenMR, et al. (2011) Structure of the vesicular stomatitis virus N(0)-P complex. PLoS Pathog 7: e1002248.

10. RaymondDD, PiperME, GerrardSR, SmithJL (2010) Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proc Natl Acad Sci U S A 107: 11769–11774.

11. FerronF, LiZ, DanekEI, LuoD, WongY, et al. (2011) The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathog 7: e1002030.

12. RaymondDD, PiperME, GerrardSR, SkiniotisG, SmithJL (2012) Phleboviruses encapsidate their genomes by sequestering RNA bases. Proc Natl Acad Sci U S A 109: 19208–19213.

13. QiX, LanS, WangW, ScheldeLM, DongH, et al. (2010) Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468: 779–783.

14. HastieKM, KimberlinCR, ZandonattiMA, MacRaeIJ, SaphireEO (2011) Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3′ to 5′ exonuclease activity essential for immune suppression. Proc Natl Acad Sci U S A 108: 2396–2401.

15. HastieKM, LiuT, LiS, KingLB, NgoN, et al. (2011) Crystal structure of the Lassa virus nucleoprotein-RNA complex reveals a gating mechanism for RNA binding. Proc Natl Acad Sci U S A 108: 19365–19370.

16. YeQ, KrugRM, TaoYJ (2006) The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444: 1078–1082.

17. NgAK, ZhangH, TanK, LiZ, LiuJH, et al. (2008) Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. Faseb J 22: 3638–3647.

18. NgAK, LamMK, ZhangH, LiuJ, AuSW, et al. (2012) Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein. J Virol 86: 6758–6767.

19. RuigrokRW, BaudinF (1995) Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles. J Gen Virol 76(Pt 4): 1009–1014.

20. TarusB, BakowiezO, ChenavasS, DucheminL, EstroziLF, et al. (2012) Oligomerization paths of the nucleoprotein of influenza A virus. Biochimie 94: 776–785.

21. YeQ, GuuTS, MataDA, KuoRL, SmithB, et al. (2013) Biochemical and structural evidence in support of a coherent model for the formation of the double-helical influenza a virus ribonucleoprotein. MBio 4: e00467–00412.

22. EltonD, MedcalfE, BishopK, DigardP (1999) Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements. Virology 260: 190–200.

23. ChanWH, NgAK, RobbNC, LamMK, ChanPK, et al. (2010) Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities. J Virol 84: 7337–7345.

24. BouloS, AkarsuH, LotteauV, MullerCW, RuigrokRW, et al. (2011) Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein. Virology 409: 84–90.

25. ColomaR, ValpuestaJM, ArranzR, CarrascosaJL, OrtinJ, et al. (2009) The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5: e1000491.

26. ShenYF, ChenYH, ChuSY, LinMI, HsuHT, et al. (2011) E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc Natl Acad Sci U S A 108: 16515–16520.

27. PetriT, DimmockNJ (1981) Phosphorylation of influenza virus nucleoprotein in vivo. J Gen Virol 57: 185–190.

28. KistnerO, MullerK, ScholtissekC (1989) Differential phosphorylation of the nucleoprotein of influenza A viruses. J Gen Virol 70(Pt 9): 2421–2431.

29. AlmondJW, FelsenreichV (1982) Phosphorylation of the nucleoprotein of an avian influenza virus. J Gen Virol 60: 295–305.

30. CompansRW, ContentJ, DuesbergPH (1972) Structure of the ribonucleoprotein of influenza virus. J Virol 10: 795–800.

31. OrtegaJ, Martin-BenitoJ, ZurcherT, ValpuestaJM, CarrascosaJL, et al. (2000) Ultrastructural and functional analyses of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification. J Virol 74: 156–163.

32. HutchinsonEC, DenhamEM, ThomasB, TrudgianDC, HesterSS, et al. (2012) Mapping the phosphoproteome of influenza a and B viruses by mass spectrometry. PLoS Pathog 8: e1002993.

33. ArranzR, ColomaR, ChichonFJ, ConesaJJ, CarrascosaJL, et al. (2012) The Structure of Native Influenza Virion Ribonucleoproteins. Science 338: 1634–1637.

34. MoellerA, KirchdoerferRN, PotterCS, CarragherB, WilsonIA (2012) Organization of the Influenza Virus Replication Machinery. Science 338: 1631–1634.

35. FodorE, PritloveDC, BrownleeGG (1994) The influenza virus panhandle is involved in the initiation of transcription. J Virol 68: 4092–4096.

36. TileyLS, HagenM, MatthewsJT, KrystalM (1994) Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5′ ends of the viral RNAs. J Virol 68: 5108–5116.

37. HagenM, ChungTD, ButcherJA, KrystalM (1994) Recombinant influenza virus polymerase: requirement of both 5′ and 3′ viral ends for endonuclease activity. J Virol 68: 1509–1515.

38. CianciC, TileyL, KrystalM (1995) Differential activation of the influenza virus polymerase via template RNA binding. J Virol 69: 3995–3999.

39. KlumppK, RuigrokRW, BaudinF (1997) Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. Embo J 16: 1248–1257.

40. JorbaN, ColomaR, OrtinJ (2009) Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog 5: e1000462.

41. Resa-InfanteP, JorbaN, ColomaR, OrtinJ (2011) The influenza virus RNA synthesis machine: advances in its structure and function. RNA Biol 8: 207–215.

42. MarklundJK, YeQ, DongJ, TaoYJ, KrugRM (2012) Sequence in the influenza A virus nucleoprotein required for viral polymerase binding and RNA synthesis. J Virol 86: 7292–7297.

43. TarusB, ChevalierC, RichardCA, DelmasB, Di PrimoC, et al. (2012) Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding. PLoS One 7: e30038.

44. KaoRY, YangD, LauLS, TsuiWH, HuL, et al. (2010) Identification of influenza A nucleoprotein as an antiviral target. Nat Biotechnol 28: 600–605.

45. SuCY, ChengTJ, LinMI, WangSY, HuangWI, et al. (2010) High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity. Proc Natl Acad Sci U S A 107: 19151–19156.

46. GerritzSW, CianciC, KimS, PearceBC, DeminieC, et al. (2011) Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers. Proc Natl Acad Sci U S A 108: 15366–15371.

47. KabschW (1993) Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants. Journal of Applied Crystallography 26: 795–800.

48. KabschW (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr 66: 133–144.

49. Collaborative Computational Project n (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763.

50. DeLano WL (2002) The PyMOL Molecular Graphics System. San Carlos, CA USA: DeLano Scientific.

51. MendelCM, MendelDB (1985) ‘Non-specific’ binding. The problem, and a solution. Biochem J 228: 269–272.

52. RocchiaW, SridharanS, NichollsA, AlexovE, ChiabreraA, et al. (2002) Rapid grid-based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: applications to the molecular systems and geometric objects. J Comput Chem 23: 128–137.