title: Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells.
abstract: BACKGROUND
  Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.
  
  
  RESULTS
  Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.
  
  
  CONCLUSIONS
  Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.
authors.affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Metz
authors.forename: Corinne
authors.initials: C
authors.lastname: Geertsema
authors.forename: Byron E
authors.initials: BE
authors.lastname: Martina
authors.forename: Paulina
authors.initials: P
authors.lastname: Andrade
authors.forename: Jacco G
authors.initials: JG
authors.lastname: Heldens
authors.forename: Monique M
authors.initials: MM
authors.lastname: van Oers
authors.forename: Rob W
authors.initials: RW
authors.lastname: Goldbach
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.forename: Gorben P
authors.initials: GP
authors.lastname: Pijlman
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002459:Cell Fusion; D002460:Cell Line; D002646:Chikungunya virus / Q000235:genetics*; D002846:Chromatography, Affinity; D045683:Furin / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D045683:Furin