cell fusion activity was enhanced in proportion to the concentration of cholesterol
added (Fig. 5A). To confirm the role of cholesterol in cell fusion, the cells were grown
under cholesterol-depleted conditions by addition of MβCD as described, after which
cholesterol was added to specific concentration. Although the cell fusion was observed
under condition of without further treatment (Fig. 5B, left panels), cell fusion was
blocked completely when the cells were depleted of cholesterol (Fig. 5B, middle
panels). After the repletion of 0.15 mg/ml cholesterol, the strong fusion activities were
restored (Fig. 5B, right panels). Taken together, these observations indicate that
CHIKV induced cell fusion is pH- and cholesterol-dependent. In the presence of 0.2
mg/ml of cholesterol at pH 5.8, cell fusion of infected cells proceeded rapidly as
demonstrated by time-lapse photography. The onset of cell fusion occurred after just
30 minutes of incubation and the fusion was complete by 180 minutes (Fig. 6).
3.4. Expression of 6K-E1 can induce cell syncytium formation
The E1 and E2 proteins of several alphaviruses form heterodimers on the viral
envelope, with the E1 protein playing a fundamental role in viral membrane fusion. To
determine whether the CHIKV E1 protein is critical for the induction of cell fusion,
two recombinant baculoviruses (Figs. 1 C and D) carrying partial deletions of the
CHIKV structural genes were generated. The first deletion resulted in a vector that
expressed 6K sequence and E1 protein proteins (Fig. 1C). The 6K sequence is known
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to serve as a signal sequence for SFV E1 protein (Liljestrom and Garoff, 1991). The
second recombinant virus, vAc-CHIKV-26S-ΔE1-Rhir-E encodes C and E2, but
contains an E1 deletion (Fig. 1D). The protein expression profiles and cell fusion
abilities of Sf21 cells infected with these three baculoviruses were verified by Western
blot analyses and fluorescence microscopy. Figure 7A of Western blot shows that the
E1 protein was expressed correctly in both vAc-CHIKV-26S-Rhir-E (lane 1) and
6K-E1-Rhir-E-infected Sf21 cells (lane 3) but not in
vAc-CHIKV--26S-ΔE1-Rhir-E (lane 2) or vAc-Rhir-E-infected cells (lane 4). Cells infected with
vAc-CHIKV-26S-ΔE1-Rhir-E did not fuse, whereas cells infected with
vAc-CHIKV-6K-E1-Rhir-E which expressed E1 protein only showed a clear evidence
of fusion (Fig. 7B). The other protein bands appeared in the Western blot (Fig. 7A)
may represent the non-specific binding of the anti-E1 polyclonal antibodies to the cell
extracts of recombinant baculoviruses infected Sf21 cells. Thus, the E1 protein of
CHIKV is necessary and sufficient to induce fusion at infected Sf21 cells.
4. Discussion
In this study, a baculovirus bi-cistronic expression system was used to demonstrate
the co-expression of EGFP and CHIKV structural proteins in Sf21 cells and induction
of Sf21 cell fusion. Advantages of co-expression of EGFP are to facilitate the
identification of recombinant baculoviruses and the determination of viral titer (Chen
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et al., 2005). More importantly, it made it possible to analyze cell fusion events and to
demonstrate CHIKV membrane fusion requires low pH and cholesterol.
One might argue that the observed Sf21 cell fusion is not mediated by CHIKV
proteins but by baculovirus gp64 protein since gp64 protein can induce insect cell
fusion (Blissard and Wenz, 1992). Two lines of evidence support that Sf21 cell fusion
is induced by CHIKV proteins but not baculovirus gp64. First, induction of cell fusion
by baculovirus gp64 protein occurs only at pH of 5.5 or lower while Sf21 cells
expressing CHIKV proteins fuse with other Sf21 cells, no matter expression of CHIKV
proteins or not, can occur at pH of 6.4 (Fig. 4). Second, Sf21 cells infected by the
control recombinant baculovirus, vAc-Rhir-E, which expressed EGFP but no CHIKV
proteins did not undergo cell fusion at the same culture media condition (Fig. 3A, right
panel). Furthermore, Sf21 cell fusion was blocked proportionally by adding various
dilutions of anti-CHIKV sera (Fig. 3B), indicating that the cell fusion event was
mediated specifically by the CHIKV proteins but not the baculovirus. However,
weaker fusion ability at pH of 5.6 was observed at vAc-Rhir-E-infected Sf21 cells
when comparing with vAc-CHIKV-S26-Rhir-E-infected Sf21 cells (data not shown).
The successful induction of fusion between infected Sf21 cells is attributed to the
proper cleavage of the CHIKV polyprotein into individual structural proteins (Fig. 2A)
and the proper targeting of the E1 and E2 proteins to the cell surface (Fig. 2B).
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Syncytium formation in Sf21 cells infected with the recombinant baculovirus
expressing only the 6K-E1 protein further indicated that the 6K-E1 sequence alone is
sufficient for cell fusion (Fig. 7). The analysis of viral membrane fusion has been done
by virus-liposome interaction assay (Kielian et al., 1996), in which purified and
labeled viruses are incubated with liposomes followed by either ultracentrifugation or
immunoprecipitation and protein quantitation. These methods require virion
purification and are more time-consuming and labor-intensive than the cell-based
analysis developed in this study. Because the Sf21 cell is a cholesterol auxotroph and
grows in a mildly acidic medium covering the pH threshold of CHIKV membrane
fusion, the method described here provides a way to manipulate and study the activity
of class II viral fusion without using viral particles; furthermore it can be performed
under facilities of low-level bio-safety. In addition, it may be valuable for screening
agents for their ability to block viral membrane fusion by CHIKV infection. Others
have been shown or discussed that inhibition of viral membrane fusion using peptides,
small molecules or neutralizing antibodies are effective prevent viral infections
(Gollins and Porterfield, 1986; Kilby and Eron, 2003; Nybakken et al., 2005; Skehel
and Wiley, 2000; Zwick, 2005; Sanchez-San Martin et al., 2009).
In summary, the results presented here show that the expression of CHIKV
structural proteins by recombinant baculoviruses can induce fusion of insect cell.
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Furthermore, it is shows that the CHIKV E1 protein is necessary and sufficient for
syncytium formation. This fusion event is low pH- and cholesterol-dependent that has
been observed with other alphaviruses. In addition, this insect cell-based system may
serve as a tool for studying class II viral membrane fusion, for insights into the process
of CHIKV infection.
Acknowledgements
This work was supported by grants NSC-96-2317-B-033-001 from the National
Science Council of Taiwan and The Center of Excellence Program on Membrane
Technology, the Ministry of Education of Taiwan to T.Y. Wu and a BMRP grant from
the Chang Gung Memorial Hospital to S.J. Lo. The authors thank Dr. Victor Stollar
(UMDNJ-Robert Wood Johnson Medical School, New Jersey, USA) and Dr. Simon
Silver, a visiting professor of CGU for their help in revising the manuscript.
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Fig. 1. Schematic presentation of the recombinant baculovirus transfer vectors. (A)
The bi-cistronic baculovirus transfer vector pBac-Rhir-E, in which the RhPV 5’-UTR
IRES is located between the six MCS cloning sites (NheI, BglII, PstI, KpnI, XbaI, and
EcoRI ) and the EGFP genes. (B) pBac-CHIKV-26S-Rhir-E, in which the CHIKV 26S
subgenomic cDNA is cloned into the BglII and XbaI sites of pBac-Rhir-E. (C)
pBac-CHIKV-6K-E1-Rhir-E, in which a 1.8-kb NheI-NotI fragment containing the
6K-E1 gene is cloned into the pBac-CHIKV-26SRhir-E to replace the 26S subgenomic
cDNA. (D) pBac-CHIKV-26S-ΔE1-Rhir-E, in which the E1 gene is deleted.
Abbreviations: PPH, polyhedrin promoter; EGFP, enhanced green fluorescent protein
gene; Rhir, RhPV 5’-UTR IRES; STOP, translational stop codon.
Fig. 2. Analyses of CHIKV structural protein expression in Sf21 cells. (A) Western
blot analysis. CHIKV structural proteins were detected by rabbit anti-CHIKV E1
(anti-E1), anti-CHIKV E2 (anti-E2) and anti-whole CHIKV (anti-capsid) antibodies,
respectively. Lane 1, purified CHIKV from ultra-centrifugation as a positive control;
lane 2, Sf21 cells infected by vAc-Rhir-E as a negative control; lane 3, Sf21 cells
infected by vAc-CHIKV-26S-Rhir-E. The E1, P62, E2 and capsid proteins of CHIKV
are indicated by arrows to the right of the gels. The respective molecular weights of
proteins are indicated. (B) Immunofluorescence observation of E1 and E2 on the cell
surface of recombinant baculovirus infected Sf21 cells. Sf21 cells infected with either
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vAc-CHIKV-26S-Rhir-E (left panels) or vAc-Rhir-E (right panels) were directly
observed under a fluorescence microscope with a FITC filter for detection of EGFP.
The infected cells were fixed and stained with either anti-CHIKV E1 (E1) or
anti-CHIKV E2 (E2) antibodies and examined with a rhodamine filter for detection of
E1 or E2. Bar represents 25 μm.
Fig. 3. Sf21 cells infected by vAc-CHIKV-26S-Rhir-E induce syncytium formation. (A)
Sf21 cells infected by vAc-CHIKV-26S-Rhir-E without further treatment (left panels),
or with treatment of anti-CHIKV serum (1:200) (middle panels), and infected by
vAc-Rhir-E (right panels) were examined under a fluorescence microscope with a
FITC channel (upper panels) or a bright field (lower panels). The polykaryotic cells
were indicated by arrows. Pictures from upper and lower panels were taken in the same
field. Bar represents 25 μm. (B) The specific inhibition of cell fusion by antiserum in a
dose-dependant manner. Sf21 cells were infected with recombinant baculovirus of
vAc-CHIKV-26S-Rhir-E, at multiplicity of infection M.O.I. of 10 in Sf-900 II
containing 8% FCS. After 1 dpi, cells were incubated with different dilutions (1:200,
1:400, 1:800,1:1600 and 1:3,200) of rabbit anti-whole CHIKV serum in growth
medium for 1 hour at 27, and then replace medium with Sf-900 II (pH 5.8)
containing 2% FCS , 0.1mg /ml cholesterol and a corresponding diluted serum. The
samples were then examined and photographed under an inverted fluorescence
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microscope (IX71; Olympus) at 2 dpi. The “wo” indicates the infected cells without
further treatment. Bar represents 25 μ m. (C) Cell fusion between
vAc-CHIKV-26S-Rhir-E-infected and uninfected Sf21 cells. Uninfected cells (target
cells) were pre-stained with CellTracker RED CMPTX and then co-cultured with
infected Sf21 cells (expression cells) in SF-900 II (pH 5.8) containing 0.2 mg/ml
cholesterol. After 2-hour incubation at 27°C, the cell-cell fusion was observed under a
confocal microscope. Expression cells emitted green florescence, target cells emitted
red florescence and overlay images of both cells appeared in yellow. All pictures were
taken in the same field. (D) Localizations of EGFP-expressing cells, CHIKV envelope
expressing cells and polynuclear fused cells. Sf21 cells were infected with
recombinant baculovirus of vAc-CHIKV-26S-Rhir-E. After 1 dpi, cells were treated
with Sf-900 II (pH 5.8) containing 2% FCS and 0.1mg /ml cholesterol for 2 hours, and
then stained with rabbit anti-whole CHIKV serum at a dilution of 1:800 in Sf-900 and
following incubation within 10 μM Hoechst 33342 for nucleus staining and the
secondary antibody, Alexa Fluor 546-labeled goat anti-rabbit IgG. The sample was
then examined and photographed under an inverted fluorescence microscope (IX71;
Olympus). Infected cells emitted green florescence (upper left panel), CHIKV
envelope protein-expressing cells emitted red florescence (upper right panel), nucleus
emitted blue florescence (lower left panel) and overlay image of above three images
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(lower right panel). And the yellow circle indicated the non-infected cells that did not
express the E1 or E2 proteins as well as the green fluorescence protein. Bar represents
25 μm.
Fig. 4. The effect of pH on cell fusion. Sf21 cells were infected with
vAc-CHIKV26S-Rhir-E in Sf-900 II containing 2% FCS. After 1 dpi, the culture
medium was replaced with Sf-900 II containing 2% FCS and 0.2 mg/ml cholesterol at
pH 5.8, 6.0, 6.2, 6.4 and 6.6, respectively, as indicated. The syncytial formation was
examined under a fluorescence microscope with a FITC channel at 2 dpi. Bar
represents 25 μm.
Fig. 5. The effect of cholesterol concentration on cell fusion. (A) Sf21 cells were
infected with vAc-CHIKV-26S-Rhir-E in Sf-900 II containing 2% FCS. After 1 dpi,
the culture medium was replaced with Sf-900 II (pH 5.8) containing 2% FCS and
0.002 mg/ml to 0.2 mg/ml cholesterol as indicated. (B) The effect of a cholesterol
depleting agent on cell fusion. Sf21 cells were infected with vAc-CHIKV26S-Rhir-E
in Sf-900 II containing 2% FCS. After 1 dpi, the infected cells were treated with 4.5
mM methyl-β-cyclodextrin (MβCD) for cholesterol depletion, and then culture
medium was replaced with either Sf-900 II (pH 5.8) (middle panel) or Sf-900 II (pH
5.8) containing 0.15 mg/ml cholesterol (right panel) for replenishment.. The syncytial
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formation at 2 dpi was examined under a fluorescence microscope with a FITC
channel. Bar represents 25 μm.
Fig. 6 Time course of cell-cell fusion induced by vAc-CHIKV-26S-Rhir-E infection.
Sf21 cells were infected with vAc-CHIKV26S-Rhir-E in Sf-900 II containing 8% FCS.
After 1 dpi, the culture medium was replaced with Sf-900 II (pH 5.8) containing 2%
FCS and 0.2 mg/ml cholesterol. The cell-cell fusion was observed under a fluorescence
microscope with a bright field at the indicated times. The early fusion events were
indicated by arrows after a 30-minute incubation. Almost all Sf21 cells were fused
together after 180-minute incubation. All pictures were taken in the same field.
Fig. 7. Induction of cell fusion by expression of CHIKV 6K-E1. (A) Western blot
analysis of E1 protein in cell lysates from vAc-CHIKV-26S-Rhir-E (lane 1),
vAc-CHIKV-26S-ΔE1-Rhir-E (lane 2), vAc-CHIKV-6K-E1-Rhir-E (lane 3) and
vAc-Rir-E (lane 4) infected Sf21 cells. E1 protein was detected by rabbit anti-E1
serum staining. The molecular weights of standards (kDa) are indicated on the left, and
the E1 protein is indicated by an arrow. (B) Sf21 cells were infected with indicated
baculoviruses in Sf-900 II containing 8% FCS. After 1 dpi, medium was replaced by
Sf-900 II (pH 5.8) containing 2% FCS and 0.1 mg/ml cholesterol. Cell fusions were
examined and photographed under an inverted fluorescence microscope at 2 dpi. Bar
represents 25 μm.
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Highlights
> A cell-based assay system for Chikungunya virus induced membrane
fusion was established in baculovirus expression system. > Protein E1 of
Chikungunya virus was required for cell fusion. > Cholesterol and low pH
requirements for membrane fusion.> This cell-based system provides a
model for studying class II viral membrane fusion.
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Figure 1
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Figure 2A
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Figure 2B
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Figure(3A)
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Figure(3B)
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Figure(3C)
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Figure(3D)
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Figure 4
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Figure 5
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Figure(6)
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Figure(7A)
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Figure(7B)