sGP Serves as a Structural Protein in Ebola Virus Infection
Abstract
Background. sGP, which is perceived as nonstructural, secretory glycoprotein, shares 295 amino acids at its N-terminal with GP1,2, which include the specific residue necessary to interact with GP2. In the present study, we tested whether the sGP protein of Zaire ebolavirus (ZEBOV) could substitute for GP1 and form a complex with GP2, thus serving as a structural protein.
Methods. We expressed ZEBOV GP1,2, VP40, and NP proteins, together with sGP protein, from expression plasmids and examined the resultant virus-like particles
by using Western blot. Cells expressing GP2 in combination with either GP1 or sGP were analyzed by using flow cytometry with the KZ52 antibody, which recognizes a GP1,2
conformational epitope. A VSV pseudotype, VSVΔG*, which expresses a GFP
reporter gene instead of the G protein, was used
to produce pseudotyped viruses encoding sGP and
variants of GP to test the contribution of sGP to infectivity.
Results. Western blot and flow cytometric analyses suggested the existence of a covalently linked sGP-GP2 molecule. VSVΔG*(sGP + GP2) and VSVΔG*(GP1,2) infected Vero E6 cells and were neutralized by the KZ52 antibody. Overexpression of sGP reduced the titer of VSVΔG*(GP1,2).
Conclusions. ZEBOV sGP can substitute for GP1, forming a sGP-GP2 complex and conferring infectivity. Our studies suggest a novel role for sGP as a structural protein.
Ebola virus (EBOV) is a filamentous, enveloped, nonsegmented, negative-strand RNA virus, which, with Marburg virus, constitutes
the family Filoviridae. The fourth gene of
the EBOV genome encodes 2 glycoproteins: the secretory glycoprotein
(sGP), encoded by the predominantly
unedited transcript, and the virion envelope
glycoprotein (GP), the product of RNA editing during formation of the
mRNA [1]. The molecular ratio of unedited to edited mRNA is ∼3:1 (73% vs 27%) [1], indicating that sGP is produced more abundantly than is GP in the life cycle of EBOV.
Pre-sGP (60 kDa), the translation product of
the unedited mRNA from the fourth gene, is cleaved by furin into
secretory glycoprotein
(sGP; 50 kDa) and a delta peptide (Δ-peptide; 10–14
kDa) [2, 3]. The sGP consists of a homodimer joined in a parallel manner by 2 inter-subunit disulfide bonds at paired N-terminal and
C-terminal cysteines (C53–C53’ and C306–C306’) [4–6]. Although the N-terminal 295 amino acids of sGP are identical to those of the envelope GP, the C-terminal 69 amino acids
are unique to sGP [1, 3, 6]. The sGP homodimer is structurally and functionally distinct from the envelope GP [6].
The large amount of sGP produced by EBOV may contribute to some
infection processes in its natural hosts but may also contribute
to pathogenesis in nonnatural hosts [4].
For example, sGP may serve as a decoy ( houkutin) for EBOV-specific neutralizing and nonneutralizing antibodies [3, 7]. sGP, but not the Δ-peptide, induces the partial recovery of endothelial cell barrier function after treatment with TNF-α
[8]. sGP is also thought to interact with and inactivate human neutrophils via the IgG Fc receptor IIIb (FcγRIIIb, CD16b) [9, 10], a concept that has been challenged by some researchers [11].
The Δ-peptide is secreted in monomeric form [3]; its function is unknown.
GP is a transmembrane protein that facilitates viral receptor binding and fusion with target cells [12–14]. It comprises GP1 (140 kDa) and GP2 (25 kDa), which are proteolytically processed from the GP0 polypeptide, disulfide linked, and form the mature GP1,2 complex [2, 15]. Mutational and crystal structure analyses revealed that receptor-binding, protein stability-associated, and GP2-interacting regions exist in the N-terminal 312 amino acids of GP1 [16–20]. After cleavage by furin in the Golgi, the GP1-GP2 interaction becomes unstable and some GP1 is released as a soluble monomer [21].
The KZ52 antibody, a human monoclonal antibody established from a patient [22], bridges amino acids 42-43 of GP1 and 505-514 and 549-556 of GP2 [19] but does not recognize sGP. However, in the literature [22], immunoprecipitants with the antibody contained an additional molecule of 50–70 kDa that is unlikely to be GP1 or GP2.
On the basis of these reports, we hypothesized that sGP may substitute for GP1 and form an sGP-GP2
molecule on the virions. To test this hypothesis, we expressed Zaire
EBOV (ZEBOV) proteins from plasmids and analyzed them
by Western blot, flow cytometry, and a VSVΔG*
pseudotype system. We also used pre-sGP, which is cleaved into sGP and
the Δ-peptide
[1, 3], to investigate the influence of the Δ-peptide on the formation of sGP-GP2.
MATERIALS AND METHODS
Cell Lines and Antibodies
Human embryonic kidney 293T cells and
African green monkey kidney cells (Vero E6) were cultured in
high-glucose Dulbecco’s
modified Eagle's medium (SIGMA) supplemented
with 10% fetal calf serum and penicillin-streptomycin. The cells were
grown in
an incubator at 37°C under 5% carbon dioxide.
An affinity-purified anti-FLAG M2
monoclonal antibody, monoclonal anti-HA clone HA-7 mouse ascites fluid,
and an anti-c-myc
rabbit antibody were obtained from SIGMA. The
KZ52 human IgG antibody was kindly provided by Dr. Dennis Burton. An
anti-sGP
antibody was produced by immunization of rabbits
with peptide CLSQLYQTEPKTSVVRVRR, an amino acid sequence unique to sGP.
Anti-mouse
Alexa Fluor 488 IgG (Invitrogen) and
fluorescein-conjugated anti-human IgG F(c) (Rockland) were used for flow
cytometry. An
anti-GP monoclonal antibody (12/1.1) that
recognizes the C-terminus of GP1 [23], mouse TrueBlotTM ULTRA: horseradish peroxidase anti-mouse IgG (eBioscience), and the universal antibody in the VECTAStain
ABC kit (Vector Laboratories) were used for Western blotting.
Plasmids
To express ZEBOV proteins, cDNAs were cloned into the expression plasmid pCAGGS/MCS [24, 25]. Untranslated regions of the ZEBOV genes were deleted to minimize unexpected effects, such as mRNA splicing or the regulation
of translation by these regions. Plasmids for full-length GP1,2, sGP, and pre-sGP without tags were designated pCGP, pCsGP, and pCpresGP, respectively. For GP2 expression, the nucleotide sequence of the signal peptide of the murine Ig κ chain (METDTLLLWVLLLWVPGSTGE) was inserted at
the 5′ end of the GP2 cDNA (pCGP2). In addition, tagged plasmids were prepared as follows. A FLAG (DYKDDDDK)-tag was inserted between residues Ser (32) and
Ile (33) of the GP cDNA of pCGP to generate FLAG-tagged GP1,2 (pCGP-FLAG). To generate a FLAG/myc-tagged GP expressing plasmid (pCGP-FLAG/myc), a myc (EQKLISEEDL) tag was added to the
N-terminus of the GP2 sequence in pCGP-FLAG. FLAG-tagged GP1 was prepared by replacing the C-terminal 5 amino acids (RRTRR) of GP1 with a spacer sequence encoding TLE and by adding the FLAG-tag (pCGP1-FLAG).
The influenza virus hemagglutinin epitope (HA; YPYDVPDYA)-tagged sGP
(pCsGP-HA) and presGP (pCpresGP-HA) were prepared
by inserting the HA tag immediately downstream
of the signal sequences in pCsGP and pCpresGP, respectively.
VLP Formation and Purification
To generate Ebola virus like particles (VLPs) with or without tags, we transfected 2.4 x 107 293T cells with plasmids for the expression of GP (pCGP-FLAG/myc or pCGP; 15 μg), VP40 (pCVP40; 15 μg), and NP (pCNP: 7.5
μg) by using TransIT-293 (Takara Bio) [26–29].
In addition, to assess the effect of sGP or presGP on the glycoprotein
composition of VLPs, an empty plasmid (pCAGGS/MCS;
45 μg) or plasmids for sGP (pCsGP-HA or pCsGP;
45 μg) or presGP (pCpresGP-HA or pCpresGP; 45 μg) were cotransfected
with the
plasmids for the formation of the VLPs as
indicated above. The 1:3 ratio of GP plasmids to empty, sGP, or presGP
plasmids
was based on the ratio of the GP mRNA
transcripts to the sGP mRNA transcripts [1].
At 48 hours after transfection, the culture medium was harvested and
filtered through a 0.45 μm PVDF filter (Millipore).
The filtrate was then centrifuged through a 20%
sucrose cushion in phosphate-buffered saline (PBS) for 2 hours at 27000
rpm
(130000 g) to isolate VLPs. VLPs were
resuspended in PBS and centrifuged again for 2 hours at 27000 rpm. The
VLPs were then resuspended
in Tris-Glycine sodium dodecyl sulfate sample
buffer (Invitrogen), and protein composition was analyzed using Western
blotting.
Flow Cytometry
We transfected 293T cells with
plasmids to investigate the expression of glycoproteins on the cell
surface. The plasmids transfected
were pCAGGS/MCS, pCGP-FLAG, pCsGP-HA,
pCpresGP-HA, pCGP2, pCsGP-HA + pCGP2, pCpresGP-HA + pCGP2, pCGP1-FLAG,, and pCGP1-FLAG + pCGP2.
Cells were pelleted at 4°C for 2 minutes at 4000 rpm, then resuspended
in wash buffer (PBS with 1% fetal calf serum and 0.05%
sodium azide) and aliquoted for staining.
Glycoproteins were detected by staining cells with the KZ52 human
antibody [22]
and exposing them to a fluorescence-conjugated anti-human IgG F(c)
antibody. Cells were also stained with an anti-HA antibody
and proteins detected with an anti-mouse Alexa
Fluor 488 antibody. All staining was performed on ice for 30 minutes,
followed
by washing. For each sample, 10000 events in the
live cell gates were analyzed. Data were collected on a Becton
Dickinson
FACSCalibur and analyzed using CellQuest
software.
Vesicular Stomatitis Virus (VSVΔG*) Pseudotyped With Ebola Virus Glycoprotein
Vesicular stomatitis virus, VSVΔG*, which expresses a GFP reporter gene instead of the G protein, was used to produce VSVΔG*
pseudotyped with a different glycoprotein, as described elsewhere [12]. 293T cells were transfected with pCAGGS/MCS, pCGP-FLAG, pCGP1-FLAG and pCGP2, pCsGP-HA and pCGP2, or pCpresGP-HA and pCGP2
and then used for the preparation of VSVΔG* pseudotypes. Twenty-four
hours after transfection, cells were infected with VSVΔG*G
and after 24 hours incubation, culture fluid was
collected, centrifuged, and filtered through a 0.45-μm filter to remove
cells
and cell debris. The VSVΔG* pseudotyped viruses
were stored at -80°C until use. VSVΔG* pseudotyped with EBOV
glycoproteins
were incubated with a neutralizing anti-VSVG
antibody I1 for 30 minutes at room temperature to eliminate the effect
of VSVG
(used to prepare the initial pseudotype viruses)
on the infectivity of the VSVΔG* pseudotypes. The titers of the VSVΔG*
pseudotypes
were determined in Vero E6 in triplicate.
RESULTS
Ebola Virus GP Complexes on Purified VLPs
VLPs, purified by ultracentrifugation, were analyzed using Western blot, in which GP1 and GP2
were tagged with FLAG- and myc-epitopes, respectively. When VLPs were
prepared by expressing pCGP-FLAG/myc and pCAGGS/MCS,
pCGP-FLAG/myc and pCsGP-HA, or pCGP-FLAG/myc and
pCpresGP-HA, under non-reducing conditions, the anti-FLAG and anti-myc
antibodies
detected 150 kDa bands, corresponding to the
disulphide-bonded GP1 and GP2 complex (Figure 1A, lanes 1–6), and under reducing conditions, the anti-myc antibody detected the 25 kDa GP2 band (Figure 1A, lanes 7–9). Under nonreducing conditions, the anti-HA and anti-myc antibodies detected molecules of ∼75 kDa (Figure 1A), which did not correspond to monomers of GP1 (140 kDa), GP2 (25 kDa), sGP (50 kDa), or pre-sGP (60 kDa) (although the intensity of the band is weak for pre-sGP) (Figure 1A). The anti-HA antibody also detected a molecule of ∼100 kDa, although the band intensity was weaker for pre-sGP than for
sGP (Figure 1A). Because sGP is produced as a soluble homodimer [4, 6, 30], this result suggests that the sGP dimer does not covalently associate with VLPs. Under reducing conditions, the molecules
at 75 kDa and 100 kDa were not observed (data not shown).
Figure 1.
Composition of Ebola virus (EBOV) glycoproteins on virus-like particles (VLPs). VLPs were produced with (A) or without (B) tagged glycoproteins by cotransfection of 293T cells with plasmids. GP1 and GP2 were tagged with FLAG- and myc-epitopes, respectively, and sGP and presGP were tagged with HA-epitopes (A). VLPs without tagged proteins were analyzed by using a rabbit anti-sGP specific antibody (B). A,
lanes 1, 4, 7, 10, 13, GP-FLAG/myc; lanes 2, 5, 8, 11, 14, GP-FLAG/myc +
sGP-HA; lanes 3, 6, 9, 12, 15, GP-FLAG/myc + presGP-HA.
Lanes 1–6, 10–12 (top panel), 13–15, under non-reducing conditions; lanes 7–9, 10–12 (bottom panel), under reducing conditions. Lanes 1–3, anti-FLAG antibody; lanes 4–9, 13–15, anti-myc antibody; lanes 10–12, anti-HA antibody.
B, Lane 1, GP; lane 2, GP + sGP; lane 3, GP + presGP. Top panel, anti-GP monoclonal antibody (12/1.1); bottom panel, sGP-specific
peptide antibody.
Similar experiments were performed with untagged EBOV proteins and antibodies against GP1 and sGP. GP1,2 was again observed under nonreducing conditions with the anti-GP1 antibody (Figure 1B). The anti-sGP antibody detected 75- and 100-kDa proteins (Figure 1B), although the intensity of the band is weak for pre-sGP (Figure 1B).
These results indicate that the use of epitope tags did not cause
artifacts, and sGP was present on the purified VLPs as
75- and 100-kDa proteins. The molecules at 75
kDa and 100 kDa were not detected under reducing conditions (data not
shown).
Cell Surface Expression of EBOV Glycoproteins
The reactivities of EBOV glycoproteins expressed from plasmids on 293T cells were analyzed using flow cytometry with the human
KZ52 antibody that recognizes an epitope that spans the GP1-GP2 subunits of GP [19]. The reactivity of full-length GP1,2 (pCGP-FLAG)-expressing cells is shown in Figure 2A. Neither GP1 (pCGP1-FLAG) nor GP2 (pCGP2) reacted with this antibody (Figure 2B and C). However, cells that coexpressed GP1 and GP2 from separate plasmids reacted, indicating the formation of the epitope recognized by KZ52 (Figure 2D). Although cells expressing either sGP (pCsGP-HA) or pre-sGP (pCpresGP-HA) alone did not show any significant reactivity
(Figures 2E and 2F), cells coexpressing sGP and GP2 (pCsGP-HA and pCGP2) or pre-sGP and GP2 (pCpresGP-HA and pCGP2) reacted with the KZ52 antibody (Figures 2G and 2H). These results indicate that coexpression of the N-terminal 295 amino acids of GP (the region common to both full-length
GP and sGP) and GP2 formed the KZ52 epitope.
Figure 2.
Flow cytometric analysis of the expression of Ebola virus glycoproteins on 293T cells. The combinations of glycoproteins are
indicated. As a control, 293T cells were transfected with pCAGGS/MCS (blue).
The reactivities of Ebola virus
glycoproteins to an anti-HA antibody were also analyzed. Cells
expressing HA-tagged sGP (pCsGP-HA)
or pre-sGP (pCpresGP-HA) alone reacted with the
anti-HA antibody (Figures 2I and 2J), indicating that, although the soluble sGP homodimer was produced in the culture supernatant [4, 6, 30], a part of sGP was retained on the surface of the sGP-producing cells by an unknown mechanism. Coexpression of GP2 without the HA tag increased the reactivity with the anti-HA antibody, suggesting that GP2 could increase sGP retention on the cell surface (Figures 2K and 2L).
sGP Contribution to EBOV Infectivity and Neutralization by the KZ52 Antibody
To examine whether sGP contributes to
infectivity, 293T cells were transfected with EBOV glycoprotein
expression plasmids
and VSVΔG* pseudotypes were prepared. Plasmids
used for controls and for expression of Ebola virus glycoproteins were
as follows:
pCAGGS/MCS, pCGP-FLAG, pCGP1-FLAG, pCsGP-HA, pCpresGP-HA, pCGP2, pCGP1-FLAG + pCGP2, pCsGP-HA + pCGP2, and pCpresGP-HA + pCGP2. As shown in Figure 3, VSVΔG* pseudotyped with full-length GP1,2 (pCGP-FLAG) exhibited 4.14 x 106 IU/mL, whereas the titer for the mock-pseudotype with pCAGGS/MCS was below the detectable level (detection limit 6.67 IU/mL).
Expression of GP1 (pCGP1-FLAG), sGP (pCsGP-HA), pre-sGP (pCpresGP-HA), or GP2 (pCGP2) alone resulted in no detectable infectivity. In contrast, VSVΔG* prepared with coexpression of GP2 and GP1 exhibited infectivity of 8.05 x 105 IU/ml. Furthermore, coexpression of GP2 and either sGP or pre-sGP also resulted in infectious titers of 1.40 x 104 and 9.33 x 102 IU/mL, respectively (Figure 3). Because the KZ52 antibody recognizes a GP1,2 conformation-dependent epitope and neutralizes EBOV infectivity [19, 31], we then asked whether this antibody neutralizes the infectivities of VSVΔG* bearing GP-FLAG VSVΔG*(GP-FLAG), sGP-HA plus
GP2 VSVΔG*(sGP-HA + GP2), or presGP-HA plus GP2 VSVΔG*(presGP-HA + GP2).
These VSVΔG* pseudotypes were preincubated with the KZ52 antibody for
30 minutes at the indicated concentrations and inoculated
on Vero E6 cells. As shown in Figure 4,
a similar pattern of dose-dependent neutralization by the KZ52 antibody
was observed for all of the VSVΔG* pseudotyped viruses.
Figure 3.
Infectivities of vesicular stomatitis virus (VSVΔG*) pseudotyped with EBOV glycoproteins in Vero E6 cells. Detection limit
was 6.67 IU/mL.
Figure 4.
KZ52
neutralization of VSVΔG* pseudotyped viruses possessing EBOV
glycoproteins. Viruses were incubated with KZ52 at the indicated
concentrations prior to inoculation onto
Vero E6 cells.
Effect of sGP on the Infectivity of VSVΔG* Bearing Full-Length GP1,2
To examine the effect of
overexpression of sGP or pre-sGP on the infectivity of VSVΔG* pseudotyped
with full-length GP, pCAGGS/MCS,
pCsGP, or pCpresGP was cotransfected with the
plasmids needed to produce VSVΔG* pseudotyped with full-length GP-FLAG,
and
titers were determined. Coexpression of sGP-HA
or presGP-HA resulted in a reduction in the titer of VSVΔG* pseudotyped
with
full-length GP by 0.5 or 1.1 log units (Figure 5).
Figure 5.
The effects
of sGP on VSVΔG* pseudotypes with full-length GP. Either sGP-HA or
presGP-HA was coexpressed with full-length
GP-FLAG, and VSVΔG* pseudotypes were
prepared. The titers of each pseudotype were determined in Vero E6
cells.
DISCUSSION
Western blot analyses suggested that a molecule of ∼75 kDa that associated with VLPs was likely to be disulphide bond-linked
sGP and GP2, because the molecule reacted with anti-tag antibodies for which the epitopes, HA and myc, were fused to sGP and GP2, respectively, and the anti-sGP antibody detected the 75-kDa protein from untagged VLP (Figure 1). Moreover, the use of a reducing reagent resulted in the dissociation of the 75-kDa molecule into 50-kDa and 25-kDa bands,
corresponding to sGP and GP2, respectively (Figure 1). The KZ52 antibody showed reactivity to cells expressing both sGP and GP2 in flow cytometry and neutralizing activity against VSVΔG*(sGP-HA + GP2) and VSVΔG*(presGP-HA + GP2) (Figures 2–4), strongly suggesting that sGP and GP2 form a complex that confers infectivity and is structurally similar to full-length GP1,2. The N-terminal 295 amino acids of sGP and GP1,2 are identical and include the Cys53 residue that is necessary to form the disulphide bond with Cys609 of GP2 [1, 15]. The N-terminal 295 amino acids region contains the Cys53 residue as a part of a base subdomain, being in contact with GP2 [19], a receptor-binding site [16–18, 20] and a putative protein-stability region (amino acids 214 to 270) [16, 18]. The rest of GP1 includes the last β18 strand of the glycan cap and a mucin like domain, both of which do not interact with GP2 and can be deleted without affecting infectivity to VSVΔG* or lenti/retroviral vectors [15, 16, 19, 32, 33]. Therefore, sGP likely substitutes for the functional domains in GP1 that are necessary for the ability to pseudotype VSVΔG*.
We speculate that the sGP-GP2 complex may be synthesized as follows: sGP takes the place of GP1 and forms a disulphide bond between Cys53 of sGP and Cys609 of GP2 in the endoplasmic reticulum before GP1 and GP2 can form a disulphide bond. Formation of the disulphide bond is probably more efficient between GP1 and GP2 than between sGP and GP2, because the former pair exists on one polypeptide [1]; however, the abundance of sGP [1, 34] may make the sGP-GP2 complex possible. GP1 is then removed from sGP-GP0 by furin in the Golgi apparatus, resulting in the release of the GP1 monomer from EBOV-infected cells [21, 22]. Thus, monomeric sGP may be linked to GP2, and the heterodimer may form a heterotrimer like GP1,2 peplomer.
The infectivity of VSVΔG*(presGP-GP2) was lower than that of VSVΔG*sGP-GP2 (Figure 3), suggesting that the Δ-peptide may inhibit the formation of sGP-GP2. Further investigation is necessary to determine the function of the Δ-peptide.
The reduced infectivity of VSVΔG* pseudotyped with GP by sGP overexpression (Figure 5) suggests that sGP may interfere with the infectivity of Ebola virus. The sGP-GP2 molecule likely associates with GP1,2 molecules in the peplomer. Consequently, this heterotrimeric peplomer may result in lower infectivity. However, the net effect
of sGP on entry may be relatively small. In addition, by replacing GP1, which has a cytotoxic mucin like domain, with sGP, the cytotoxicity (CPE) of sGP-GP2 would be reduced because sGP lacks this cytotoxic region. Previously, it was reported that an EBOV mutant whose genome encodes
GP1,2, but not sGP, showed significantly increased cytotoxicity, compared with authentic EBOV, which encodes GP1,2 and sGP [35]. Our results may suggest a new role for sGP in the life cycle of EBOV.
Funding
This work was supported by Grants-in-Aid
for Specially Promoted Research and for Scientific Research; a Contract
Research
Fund for the Program of Founding Research Centers
for Emerging and Reemerging Infectious Diseases; Exploratory Research
for
Advanced Technology; the Special Coordination Funds
for Promoting Science and Technology from the Ministry of Education,
Culture,
Sports, Science, and Technology of Japan; the
Region V “Great Lakes” Regional Center of Excellence; and National
Institute
of Allergy and Infectious Diseases Public Health
Service research grants.
Inga kommentarer:
Skicka en kommentar