title: Chikungunya virus capsid protein contains nuclear import and export signals.
abstract: BACKGROUND
  Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system.
  
  
  METHODS
  EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy.
  
  
  RESULTS
  Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex.
  
  
  CONCLUSIONS
  We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.
authors.affiliation: Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Schillingallee 69, 18057, Rostock, Germany. herchen@med.uni-rostock.de.
authors.forename: Saijo
authors.initials: S
authors.lastname: Thomas
authors.forename: Jagdish
authors.initials: J
authors.lastname: Rai
authors.forename: Lijo
authors.initials: L
authors.lastname: John
authors.forename: Stephan
authors.initials: S
authors.lastname: Schaefer
authors.forename: Brigitte M
authors.initials: BM
authors.lastname: Pützer
authors.forename: Ottmar
authors.initials: O
authors.lastname: Herchenröder
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry; D002646:Chikungunya virus / Q000235:genetics* / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins