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.

Figure 1

CHIKV E1 and E2 expression using recombinant baculoviruses. A) Schematic representation of four CHIKV glycoprotein constructs expressed from recombinant baculoviruses. The shaded areas represent transmembrane domains or signal peptides (ss), asterisks indicate predicted N-glycosylation sites, S and F indicate signalase and furin cleavage sites, respectively. The predicted molecular mass of the glycoproteins is indicated. All constructs were equipped with a C-terminal 6 × His-tail. B) Gene expression in the cell and in medium fractions of infected Sf21 cells was analyzed by Coomassie brilliant blue-staining and western blotting using α-His mabs and rabbit α-E1 and rabbit α-E2 polyclonal antisera.

Figure 2

N-linked glycosylation of CHIKV E1 and E2 synthesized with baculovirus vectors. A) Infected Sf21 cells were treated with tunicamycin and cell fractions were analyzed by western blotting using α-E1 and α-E2 polyclonal antisera. B) Glycosylation of recombinant CHIKV E1 and E2 was confirmed by PAS-staining. C) Detection of baculovirus glycoprotein GP64 verified tunicamycin activity.

Figure 3

Furin inhibiton assay on Ac-E3E2 and Ac-E3E2ΔTM infected Sf21 cells. Sf21 cells were infected with Ac-E3E2 and Ac-E3E2ΔTM in the absence (-) or presence (+) of furin inhibitor. The cell fraction (A) and the concentrated medium fraction (B) were analyzed by western blotting using α-E2 polyclonal antiserum. Glycosylated proteins are indicated with an asterisk.

Figure 4

CHIKV E1ΔTM and E2 protein secretion. Both CHIKV E1ΔTM and E2ΔTM were expressed with E3, HBM or 6K^-40 as signal sequence. The cell fraction A) and the medium fraction B) were analyzed by western blot using α-E1 and α-E2 polyclonal antibodies. CHIKV infected Ap61 mosquito cell-lysate was used as a control.

Figure 5

Purification of secreted, recombinant CHIKV E1ΔTM and E2ΔTM proteins. Sf21 cells were infected with Ac-6KE1ΔTM and Ac-E3E2ΔTM and the respective serum-free medium fractions were loaded onto Talon^® spin columns. Bound proteins were eluted 3 times with 150 mM imidazol. The elution fractions were analyzed with CBB (A) and western blotting, using α-E1 and α-E2 polyclonal antisera (B). NP represents non-purified fractions and El.1-3 indicate the subsequent elution fractions. Total protein concentration of the elution fractions (1 ml each) are noted above in μg/ml.

Figure 6

Surface expression of CHIKV E1 and E2 on the plasma membrane. Sf21 cells were infected with recombinant baculoviruses and living cells were immunostained against E1 and E2. Confocal microscopy reveals the presence of CHIKV-E1 and-E2 at the surface of infected Sf21 cells.

Figure 7

Syncytia formation after surface expression of CHIKV-E1 in insect cells. Sf9-ET cells in cholesterol supplemented Sf900 II medium (pH = 6.4) were infected with Ac-6KE1, Ac-E3E2 and Ac-GFP. Cells were incubated 72 h post infection with medium of pH = 5.8, pH = 5.5 and pH = 5.0 for 2 min. Pictures were taken 4 h post induction. Syncytia are indicated with arrows.