1887

Journal of General Virology header logo

Volume 100, Issue 10

RNA processing bodies are disassembled during Old World alphavirus infection Free

Abstract

RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3–4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): alphavirus , nuclear transport , P-bodies , stress granules and transcription
© 2019 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001310
2019-08-16
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/100/10/1375.html?itemId=/content/journal/jgv/10.1099/jgv.0.001310&mimeType=html&fmt=ahah

References

  1. Kedersha N, Anderson P. Mammalian stress granules and processing bodies. Methods Enzymol 2007; 431:61–81 [View Article]
     [Google Scholar]
  2. Parker R, Sheth U. P bodies and the control of mRNA translation and degradation. Mol Cell 2007; 25:635–646 [View Article]
     [Google Scholar]
  3. Hubstenberger A, Courel M, Bénard M, Souquere S, Ernoult-Lange M et al. P-Body purification reveals the condensation of repressed mRNA regulons. Mol Cell 2017; 68:144–157 [View Article]
     [Google Scholar]
  4. Luo Y, Na Z, Slavoff SA. P-Bodies: composition, properties, and functions. Biochemistry 2018; 57:2424–2431 [View Article]
     [Google Scholar]
  5. Zheng D, Ezzeddine N, Chen C-YA, Zhu W, He X et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J Cell Biol 2008; 182:89–101 [View Article]
     [Google Scholar]
  6. Sheth U, Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 2003; 300:805–808 [View Article]
     [Google Scholar]
  7. Coller J, Parker R. General translational repression by activators of mRNA decapping. Cell 2005; 122:875–886 [View Article]
     [Google Scholar]
  8. Brengues M, Teixeira D, Parker R. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 2005; 310:486–489 [View Article]
     [Google Scholar]
  9. Aizer A, Kalo A, Kafri P, Shraga A, Ben-Yishay R et al. Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage. J Cell Sci 2014; 127:4443–4456 [View Article]
     [Google Scholar]
  10. Kamenska A, Simpson C, Vindry C, Broomhead H, Bénard M et al. The DDX6-4E-T interaction mediates translational repression and P-body assembly. Nucleic Acids Res 2016; 44:6318–6334 [View Article]
     [Google Scholar]
  11. Ayache J, Bénard M, Ernoult-Lange M, Minshall N, Standart N et al. P-Body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes. Mol Biol Cell 2015; 26:2579–2595 [View Article]
     [Google Scholar]
  12. Kedersha N, Stoecklin G, Ayodele M, Yacono P, Lykke-Andersen J et al. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 2005; 169:871–884 [View Article]
     [Google Scholar]
  13. Kedersha NL, Gupta M, Li W, Miller I, Anderson P. Rna-Binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol 1999; 147:1431–1442 [View Article]
     [Google Scholar]
  14. Chen R, Mukhopadhyay S, Merits A, Bolling B, Nasar F et al. ICTV virus taxonomy profile: Togaviridae . J Gen Virol 2018; 99:761–762 [View Article]
     [Google Scholar]
  15. Lwande OW, Obanda V, Bucht G, Mosomtai G, Otieno V et al. Global emergence of alphaviruses that cause arthritis in humans. Infect Ecol Epidemiol 2015; 5:29853 [View Article]
     [Google Scholar]
  16. Morrison TE. Reemergence of Chikungunya virus. J Virol 2014; 88:11644–11647 [View Article]
     [Google Scholar]
  17. Rupp JC, Sokoloski KJ, Gebhart NN, Hardy RW. Alphavirus RNA synthesis and non-structural protein functions. J Gen Virol 2015; 96:2483–2500 [View Article]
     [Google Scholar]
  18. McInerney GM, Kedersha NL, Kaufman RJ, Anderson P, Liljeström P. Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation. Mol Biol Cell 2005; 16:3753–3763 [View Article]
     [Google Scholar]
  19. Panas MD, Varjak M, Lulla A, Eng KE, Merits A et al. Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection. Mol Biol Cell 2012; 23:4701–4712 [View Article]
     [Google Scholar]
  20. Panas MD, Ahola T, McInerney GM. The C-terminal repeat domains of NSP3 from the old world alphaviruses bind directly to G3BP. J Virol 2014; 88:5888–5893 [View Article]
     [Google Scholar]
  21. Panas MD, Schulte T, Thaa B, Sandalova T, Kedersha N et al. Viral and cellular proteins containing FGDF motifs bind G3BP to block stress granule formation. PLoS Pathog 2015; 11:e1004659 [View Article]
     [Google Scholar]
  22. Kim DY, Reynaud JM, Rasalouskaya A, Akhrymuk I, Mobley JA et al. New world and old world alphaviruses have evolved to exploit different components of stress granules, FXR and G3BP proteins, for assembly of viral replication complexes. PLoS Pathog 2016; 12:e1005810 [View Article]
     [Google Scholar]
  23. Schulte T, Liu L, Panas MD, Thaa B, Dickson N et al. Combined structural, biochemical and cellular evidence demonstrates that both FGDF motifs in alphavirus NSP3 are required for efficient replication. Open Biol 2016; 6:160078 [View Article]
     [Google Scholar]
  24. Götte B, Panas MD, Hellström K, Liu L, Samreen B et al. Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery. PLoS Pathog 2019; 15:e1007842 [View Article]
     [Google Scholar]
  25. Poblete-Durán N, Prades-Pérez Y, Vera-Otarola J, Soto-Rifo R, Valiente-Echeverría F. Who regulates whom? an overview of RNA granules and viral infections. Viruses 2016; 8:180 [View Article]
     [Google Scholar]
  26. Reineke LC, Lloyd RE. Diversion of stress granules and P-bodies during viral infection. Virology 2013; 436:255–267 [View Article]
     [Google Scholar]
  27. Chahar HS, Chen S, Manjunath N. P-Body components LSM1, GW182, DDX3, DDX6 and xrn1 are recruited to WNV replication sites and positively regulate viral replication. Virology 2013; 436:1–7 [View Article]
     [Google Scholar]
  28. Emara MM, Brinton MA. Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly. Proc Natl Acad Sci U S A 2007; 104:9041–9046 [View Article]
     [Google Scholar]
  29. Ariumi Y, Kuroki M, Kushima Y, Osugi K, Hijikata M et al. Hepatitis C virus hijacks P-body and stress granule components around lipid droplets. J Virol 2011; 85:6882–6892 [View Article]
     [Google Scholar]
  30. Pager CT, Schütz S, Abraham TM, Luo G, Sarnow P. Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules. Virology 2013; 435:472–484 [View Article]
     [Google Scholar]
  31. Pérez-Vilaró G, Scheller N, Saludes V, Díez J. Hepatitis C virus infection alters P-body composition but is independent of P-body granules. J Virol 2012; 86:8740–8749 [View Article]
     [Google Scholar]
  32. Ariumi Y, Kuroki M, Abe K-i, Dansako H, Ikeda M et al. Ddx3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication. J Virol 2007; 81:13922–13926 [View Article]
     [Google Scholar]
  33. Jangra RK, Yi M, Lemon SM. Ddx6 (rck/p54) is required for efficient hepatitis C virus replication but not for internal ribosome entry site-directed translation. J Virol 2010; 84:6810–6824 [View Article]
     [Google Scholar]
  34. Scheller N, Mina LB, Galão RP, Chari A, Giménez-Barcons M et al. Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates. Proc Natl Acad Sci U S A 2009; 106:13517–13522 [View Article]
     [Google Scholar]
  35. Dougherty JD, White JP, Lloyd RE. Poliovirus-mediated disruption of cytoplasmic processing bodies. J Virol 2011; 85:64–75 [View Article]
     [Google Scholar]
  36. Dougherty JD, Tsai W-C, Lloyd RE. Multiple poliovirus proteins repress cytoplasmic RNA granules. Viruses 2015; 7:6127–6140 [View Article]
     [Google Scholar]
  37. Greer AE, Hearing P, Ketner G. The adenovirus E4 11 K protein binds and relocalizes the cytoplasmic P-body component DDX6 to aggresomes. Virology 2011; 417:161–168 [View Article]
     [Google Scholar]
  38. Seto E, Inoue T, Nakatani Y, Yamada M, Isomura H. Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection. Virology 2014; 458-459:151–161 [View Article]
     [Google Scholar]
  39. Abrahamyan LG, Chatel-Chaix L, Ajamian L, Milev MP, Monette A et al. Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of HIV-1 genomic RNA. J Cell Sci 2010; 123:369–383 [View Article]
     [Google Scholar]
  40. Liljeström P, Lusa S, Huylebroeck D, Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 1991; 65:4107–4113
     [Google Scholar]
  41. Pohjala L, Utt A, Varjak M, Lulla A, Merits A et al. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS One 2011; 6:e28923 [View Article]
     [Google Scholar]
  42. Sjöberg EM, Suomalainen M, Garoff H. A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology 1994; 12:1127–1131 [View Article]
     [Google Scholar]
  43. Smerdou C, Liljeström P. Two-helper RNA system for production of recombinant Semliki Forest virus particles. J Virol 1999; 73:1092–1098
     [Google Scholar]
  44. Tamberg N, Lulla V, Fragkoudis R, Lulla A, Fazakerley JK et al. Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus. J Gen Virol 2007; 88:1225–1230 [View Article]
     [Google Scholar]
  45. Scholte FEM, Tas A, Albulescu IC, Žusinaite E, Merits A et al. Stress granule components G3BP1 and G3BP2 play a proviral role early in Chikungunya virus replication. J Virol 2015; 89:4457–4469 [View Article]
     [Google Scholar]
  46. David A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G et al. Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 2012; 197:45–57 [View Article]
     [Google Scholar]
  47. Panas MD, Kedersha N, McInerney GM. Methods for the characterization of stress granules in virus infected cells. Methods 2015; 90:57–64 [View Article]
     [Google Scholar]
  48. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 2006; 7:R100 [View Article]
     [Google Scholar]
  49. Wilczynska A, Aigueperse C, Kress M, Dautry F, Weil D. The translational regulator CPEB1 provides a link between DCP1 bodies and stress granules. J Cell Sci 2005; 118:981–992 [View Article]
     [Google Scholar]
  50. Scheuner D, Song B, McEwen E, Liu C, Laybutt R et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell 2001; 7:1165–1176 [View Article]
     [Google Scholar]
  51. Bensaude O. Inhibiting eukaryotic transcription: which compound to choose? how to evaluate its activity?. Transcription 2011; 2:103–108 [View Article]
     [Google Scholar]
  52. Cougot N, Babajko S, Séraphin B. Cytoplasmic foci are sites of mRNA decay in human cells. J Cell Biol 2004; 165:31–40 [View Article]
     [Google Scholar]
  53. Ross J. mRNA stability in mammalian cells. Microbiol Rev 1995; 59:423–450
     [Google Scholar]
  54. Kedersha N, Cho MR, Li W, Yacono PW, Chen S et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol 2000; 151:1257–1268 [View Article]
     [Google Scholar]
  55. Kudo N, Matsumori N, Taoka H, Fujiwara D, Schreiner EP et al. Leptomycin B inactivates Crm1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc Natl Acad Sci U S A 1999; 96:9112–9117 [View Article]
     [Google Scholar]
  56. Nishi K, Yoshida M, Fujiwara D, Nishikawa M, Horinouchi S et al. Leptomycin B targets a regulatory cascade of CRM1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J Biol Chem 1994; 269:6320–6324
     [Google Scholar]
  57. Soderholm JF, Bird SL, Kalab P, Sampathkumar Y, Hasegawa K et al. Importazole, a small molecule inhibitor of the transport receptor importin-β. ACS Chem Biol 2011; 6:700–708 [View Article]
     [Google Scholar]
  58. Ferraiuolo MA, Basak S, Dostie J, Murray EL, Schoenberg DR et al. A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J Cell Biol 2005; 170:913–924 [View Article]
     [Google Scholar]
  59. Pattnaik AK, Dinh PX. Manipulation of cellular processing bodies and their constituents by viruses. DNA Cell Biol 2013; 32:286–291 [View Article]
     [Google Scholar]
  60. Aizer A, Kafri P, Kalo A, Shav-Tal Y. The P body protein Dcp1a is hyper-phosphorylated during mitosis. PLoS One 2013; 8:e49783 [View Article]
     [Google Scholar]
  61. Rzeczkowski K, Beuerlein K, Müller H, Dittrich-Breiholz O, Schneider H et al. C-Jun N-terminal kinase phosphorylates Dcp1a to control formation of P bodies. J Cell Biol 2011; 194:581–596 [View Article]
     [Google Scholar]
  62. Andrei MA, Ingelfinger D, Heintzmann R, Achsel T, Rivera-Pomar R et al. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 2005; 11:717–727 [View Article]
     [Google Scholar]
  63. Igreja C, Peter D, Weiler C, Izaurralde E. 4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to repress translation. Nat Commun 2014; 5:4790 [View Article]
     [Google Scholar]
  64. Kubacka D, Kamenska A, Broomhead H, Minshall N, Darzynkiewicz E et al. Investigating the consequences of eIF4E2 (4EHP) interaction with 4E-transporter on its cellular distribution in HeLa cells. PLoS One 2013; 8:e72761 [View Article]
     [Google Scholar]
  65. Carrasco L, Sanz MA. The regulation of translation in Alphavirus-Infected cells. Viruses 2018; 10:70 [View Article]
     [Google Scholar]
  66. Sen GL, Blau HM. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol 2005; 7:633–636 [View Article]
     [Google Scholar]
  67. Breakwell L, Dosenovic P, Karlsson Hedestam GB, D'Amato M, Liljeström P et al. Semliki Forest virus nonstructural protein 2 is involved in suppression of the type I interferon response. J Virol 2007; 81:8677–8684 [View Article]
     [Google Scholar]
  68. Garmashova N, Gorchakov R, Frolova E, Frolov I. Sindbis virus nonstructural protein NSP2 is cytotoxic and inhibits cellular transcription. J Virol 2006; 80:5686–5696 [View Article]
     [Google Scholar]
  69. Garmashova N, Gorchakov R, Volkova E, Paessler S, Frolova E et al. The old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff. J Virol 2007; 81:2472–2484 [View Article]
     [Google Scholar]
  70. Eulalio A, Behm-Ansmant I, Schweizer D, Izaurralde E. P-Body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol 2007; 27:3970–3981 [View Article]
     [Google Scholar]
  71. Guzikowski AR, Chen YS, Zid BM. Stress‐induced mRNP granules: form and function of processing bodies and stress granules. Wiley Interdiscip Rev RNA 2019; 10:e1524 [View Article]
     [Google Scholar]
  72. Froshauer S, Kartenbeck J, Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol 1988; 107:2075–2086 [View Article]
     [Google Scholar]
  73. Garcia-Moreno M, Sanz MA, Pelletier J, Carrasco L. Requirements for eIF4A and eIF2 during translation of Sindbis virus subgenomic mRNA in vertebrate and invertebrate host cells. Cell Microbiol 2013; 15:823–840 [View Article]
     [Google Scholar]
  74. Sanz MA, García-Moreno M, Carrasco L. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm. Cell Microbiol 2015; 17:520–541 [View Article]
     [Google Scholar]
  75. Dickson AM, Anderson JR, Barnhart MD, Sokoloski KJ, Oko L et al. Dephosphorylation of HuR protein during alphavirus infection is associated with HuR relocalization to the cytoplasm. J Biol Chem 2012; 287:36229–36238 [View Article]
     [Google Scholar]
  76. Sokoloski KJ, Dickson AM, Chaskey EL, Garneau NL, Wilusz CJ et al. Sindbis virus usurps the cellular HuR protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells. Cell Host Microbe 2010; 8:196–207 [View Article]
     [Google Scholar]
  77. Burnham AJ, Gong L, Hardy RW. Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA. Virology 2007; 367:212–221 [View Article]
     [Google Scholar]
  78. Lin J-Y, Shih S-R, Pan M, Li C, Lue C-F et al. hnRNP A1 interacts with the 5' untranslated regions of enterovirus 71 and Sindbis virus RNA and is required for viral replication. J Virol 2009; 83:6106–6114 [View Article]
     [Google Scholar]
  79. Atasheva S, Garmashova N, Frolov I, Frolova E. Venezuelan equine encephalitis virus capsid protein inhibits nuclear import in mammalian but not in mosquito cells. J Virol 2008; 82:4028–4041 [View Article]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001310
Loading
RNA processing bodies are disassembled during Old World alphavirus infection
J. Gen. Virol. 100, 1375 (2019); https://doi.org/10.1099/jgv.0.001310
/content/journal/jgv/10.1099/jgv.0.001310
/content/journal/jgv/10.1099/jgv.0.001310
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error