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Journal of Virology, February 2000, p. 1566-1571, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Assembly of Spikes into Coronavirus Particles Is
Mediated by the Carboxy-Terminal Domain of the Spike Protein
Gert-Jan
Godeke,
Cornelis
A. M.
de Haan,
John W. A.
Rossen,
Harry
Vennema, and
Peter J. M.
Rottier*
Institute of Virology, Department of
Infectious Diseases and Immunology, Faculty of Veterinary Medicine,
and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The
Netherlands
Received 21 June 1999/Accepted 19 October 1999
 |
ABSTRACT |
The type I glycoprotein S of coronavirus, trimers of which
constitute the typical viral spikes, is assembled into virions through
noncovalent interactions with the M protein. Here we demonstrate that
incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the
virus-like particle (VLP) system that we developed earlier for the
mouse hepatitis virus strain A59 (MHV-A59) and which we describe now
also for the unrelated coronavirus feline infectious peritonitis virus
(FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were
constructed, consisting of the ectodomain of the one virus and the
transmembrane and endodomain of the other. These proteins were tested
for their incorporation into VLPs of either species. They were found to
assemble only into viral particles of the species from which their
carboxy-terminal domain originated. Thus, the 64-terminal-residue
sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S
proteins appeared to cause cell fusion when expressed individually,
suggesting that they were biologically fully active. This was indeed
confirmed by incorporating one of the proteins into virions which
thereby acquired a new host cell tropism, as will be reported elsewhere.
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TEXT |
The first step in virus infection is
the binding of the virus particle to a receptor on the target cell. In
enveloped viruses, this binding is mediated by one of the viral
membrane proteins. Coronaviruses, plus-stranded RNA viruses occurring
in various mammalian and avian species including humans, usually carry
three proteins in their envelope. Most abundant is the M protein, a triple-spanning membrane glycoprotein the main function of which involves the organization of the viral envelope and the interactions with the nucleocapsid during assembly (for a review, see reference 24). Another component essential in the assembly
process is the small E protein. This protein is generally a minor
virion constituent (for a review, see reference 29).
It is largely embedded within the viral membrane, and only its
hydrophilic carboxy terminus protrudes inside the virion (M. J. B. Raamsman, J. Krijnse Locker, A. de Hooghe, A. A. F. de Vries, G. Griffiths, H. Vennema, and P. J. M. Rottier,
submitted for publication). The third envelope protein is the spike (S)
protein, a type I membrane glycoprotein, trimers of which
(8) constitute the characteristic coronavirus spikes. It is
this protein that mediates the binding of the virus to the target cell
receptor and the subsequent fusion of viral and cellular membranes
during entry (for a review, see reference 3).
Coronavirus assembly is not dependent on the S protein. Studies in
which the glycosylation and thus the proper folding of the protein were
inhibited by treatment of mouse hepatitis virus strain A59
(MHV-A59)-infected cells with tunicamycin revealed that spikeless,
noninfectious particles can be formed (12, 23). These
observations were confirmed when we (32) and others (1, 2) showed that virus-like particles (VLPs) can be assembled in
cells simply from the M and E proteins by the coexpression of their
genes; neither the S protein nor a nucleocapsid appeared to be
required. These particles, which we found to be morphologically identical to normal virus, did contain spikes if the S gene was also coexpressed.
Incorporation of spikes into coronavirus particles is effected by
interactions between the S protein and the M protein. We demonstrated
such interactions in MHV-A59-infected cells, in virions, and during
coexpression of M and S genes (7, 21, 22). In an extensive
mutagenetic analysis of the primary structure requirements of the M
protein for M-S interactions, we observed that the amino-terminal domain of M
the domain exposed on the outside of virionsis not involved (7). These observations indicate that the
association between the proteins takes place at the level of the
membrane, possibly also involving part of the M protein's
carboxy-terminal domain. For the S protein, this implies that the
interactions would be limited to the small part of the molecule
comprising the transmembrane domain and endodomain.
In order to confirm this hypothesis, we have constructed two reciprocal
chimeric S proteins composed of the S ectodomain and carboxy-terminal
domain of two unrelated coronaviruses. Our aim was to functionally test
these proteins by evaluating their assembly into VLPs derived from
these viruses. The chimeric spikes were constructed with the S genes of
MHV-A59 and of feline infectious peritonitis virus (FIPV; strain
79-1146). These viruses belong to two different groups of coronaviruses
which are genetically and serologically very divergent. For the S
proteins, the overall amino acid sequence identity is only 27%;
maximal identity (44%) occurs in the segment comprising the
transmembrane and carboxy-terminal domain. Another distinguishing
feature of these S proteins is that the MHV protein is proteolytically
cleaved during transport to the cell surface while that of FIPV is not.
Construction of chimeric S genes.
For the construction of the
proteins, we have exploited the convenient presence of a
StyI restriction site in both S genes located just at the
position which encodes the transition between the protein's ectodomain
and transmembrane domain, i.e., where the polypeptide enters the lipid
membrane. The resulting constructs are depicted in Fig.
1A. One construct (FMS) is composed of
the 1,388-amino-acid-long FIPV S ectodomain and the 64-residue
transmembrane plus endodomain from MHV-A59 S. The other one (MFS) has
the reciprocal structure and consists of 1,260 and 64 amino acids,
respectively. The amino acid sequences of the carboxy-terminal regions
of the MHV-A59 and FIPV S proteins are compared in Fig. 1B.
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FIG. 1.
(A) Spike constructs. MHV-A59 S was expressed from the
plasmid pTUMS (32), and the FIPV strain 79-1146 S protein
was expressed from pFIPVE2, which was made as follows. A 3'-terminal S
fragment was prepared by ligating the XbaI-SalI
fragment from pB1 (4) into pUC18, cutting with
AccI and SalI, and religating after filling in
with Klenow polymerase. From the resulting plasmid p3d, the
XbaI-SalI fragment was isolated and used. A
middle piece was prepared by isolating the
PstI-XbaI fragment from pB1. This fragment and
the 3' XbaI-SalI fragment were ligated into p1A
(4), which had been digested with PstI and
SalI to give pFIPVE2. Chimeric protein FMS was expressed
from pTFMS, which was constructed as follows. Plasmid p3d was digested
with HindIII, filled in with Klenow enzyme, and ligated
with BglII linkers, resulting in p3dHrB. After the plasmid
was cut with StyI and BglII, an MHV S gene
fragment was ligated into it; the fragment was prepared by digesting
the S gene, obtained as a BamHI fragment from pDGE2
(31), with StyI and taking the small fragment.
The resulting p3FM vector was cut with PstI and
SalI; into it were ligated the
XbaI-SalI fragment from p3d and the
PstI-XbaI fragment from pB1. The chimeric gene
was finally recloned as a BamHI fragment into pTUG3,
resulting in pTFMS. Chimeric protein MFS was expressed from pTMFS,
which was prepared starting with p3dHrB. This plasmid was cut with
StyI and BamHI, and a
BamHI-StyI fragment obtained from the MHV S
BamHI gene described above was ligated into it. The chimeric
S gene was recloned as a BamHI-SalI fragment into
pTUG3 cut with the same enzymes. TM, transmembrane domain; ecto,
ectodomain; endo, endodomain. (B) Carboxy-terminal sequences of the
MHV-A59 and FIPV spike proteins. The 67 terminal residues of each
protein are compared. The arrow indicates the junction point in the
chimeric S constructs.
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Expression of chimeric S proteins.
As the gene constructs were
placed in plasmids behind a bacteriophage T7 polymerase promoter
sequence, they could be tested by expression with the vaccinia virus T7
system (11). Cultures of mouse OST7-1 cells (9)
infected with vTF7-3 were transfected in parallel with the plasmids as
well as with similar plasmids containing the MHV-A59 and FIPV wild-type
S genes. Starting at 5 h postinfection (p.i.), the cells were
labeled for 1 h with 35S-amino acids. Cell lysates
were then prepared, and immunoprecipitations were carried out on two
aliquots of each lysate with the monoclonal antibodies (MAbs) WA3.10
and 23F4.5, known to recognize the ectodomain of the S protein of
MHV-A59 (33) and of FIPV (19), respectively. The
analysis of the precipitated proteins is shown in Fig.
2. The results demonstrate firstly that
the antibodies used are specific and do not cross-react: the wild-type
proteins are precipitated only by the proper MAb, not by the other.
Secondly, the analysis reveals that the chimeric proteins have the
expected properties. The MFS protein comigrates in the gel with the MHV
S protein while the mobility of the FMS construct is similar to that of
FIPV S.
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FIG. 2.
Expression of chimeric spike proteins. Parallel cultures
of OST7-1 cells in 35-mm-diameter dishes were infected with vTF7-3 and
transfected with plasmids encoding the wild-type and chimeric S
proteins described in the legend to Fig. 1. Cells were incubated at
32°C. Starting at 4.5 h p.i., they were starved for 30 min in
cysteine- and methionine-free minimal essential medium containing 10 mM
HEPES (pH 7.2) without fetal bovine serum. The medium was then replaced
by 600 µl of the same containing 100 µCi of 35S in
vitro cell labeling mix (Amersham). After a 1-h labeling period, cells
were washed with phosphate-buffered saline and solubilized in 1 ml of
lysis buffer, TES (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA)
containing 1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride.
Nuclei were removed from the cell lysates by centrifugation at
12,000 × g for 10 min at 4°C. For
immunoprecipitations, 50-µl aliquots of lysate were diluted with 1 ml
of detergent solution (50 mM Tris-HCl [pH 8.0], 62.5 mM EDTA, 0.5%
Nonidet P-40, 0.5% Na deoxycholate), and 30 µl of 10% sodium
dodecyl sulfate was added. MAbs were then added: 3 µl of hybridoma
culture supernatant WA3.10 or 23F4.5, which recognizes the S protein of
MHV ( Sm) or FIPV (Sf), respectively.
Following an overnight incubation at 4°C, immune complexes were
adsorbed for 1 h to formalin-fixed Staphylococcus
aureus cells (BRL Life Technologies) added as 45 µl of a 10%
(wt/vol) suspension. Immune complexes were collected by centrifugation
at 12,000 × g and washed three times with
radioimmunoprecipitation assay buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and
1% Na deoxycholate). Pellets were resuspended in 30 µl of Laemmli
sample buffer, heated for 2.5 min at 95°C, and analyzed by
electrophoresis in a sodium dodecyl sulfate-12.5% polyacrylamide gel
followed by fluorography. MW, molecular mass.
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Biological activity of chimeric S proteins: cell fusion.
Coronavirus S proteins undergo extensive co- and posttranslational
modifications and conformational maturation (for a review, see
reference 3). They are extensively glycosylated,
become acylated, and undergo formation of multiple intrachain disulfide bonds (20, 21). Most of these events occur during and
immediately after synthesis in the endoplasmic reticulum and are
critical for the subsequent oligomerization, assembly, and transport
processes. In infected cells, the spike complexes are incorporated into
viral particles and released with virions from the cell, but a fraction of the complexes is also transported to the plasma membrane where it
causes fusion with neighboring cells. Likewise, fusion occurs when the
S proteins are expressed individually in the proper cells.
Because the fusion phenotype of an S protein reflects its proper
folding and transport to the cell surface as well as a biological
property essential for infection, we performed fusion assays with
our
chimeric constructs. The different S genes were expressed
by using the
vaccinia virus expression system in BHK-21 cells
in which neither of
the wild-type S proteins induces fusion by
itself. Fusion was evaluated
in a coculture assay by overlaying
the cell monolayer with mouse L
cells or with feline FCWF cells.
Pictures of the results are shown in
Fig.
3. As predicted, the
controls FIPV S
(fS) and MHV-A59 S (mS) caused fusion only of
the feline and mouse
cells, respectively. The chimeric FMS protein
induced fusion of the
FCWF cells, not of the L cells, consistent
with the feline nature of
its ectodomain. The reciprocal construct
MFS, which derives its
ectodomain from MHV S, gave the opposite
results, causing fusion only
of the mouse cells. The observations
demonstrate that the chimeric
proteins are processed and transported
properly and are biologically
active.
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FIG. 3.
Fusion properties of the chimeric spike proteins.
Subconfluent monolayers of BHK-21 cells grown in 35-mm-diameter dishes
were infected with vTF7-3 and transfected with the plasmids encoding
MHV-A59 S (mS), FIPV S (fS), and the chimeric S proteins FMS and MFS.
At 8 h p.i., the cells were overlaid with either LR7 cells (mouse
L cells) or feline FCWF cells. Fusion was followed by light microscopy,
and at 24 h p.i., pictures were taken.
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Assembly of chimeric S proteins into VLPs.
As the final test
to establish whether indeed the incorporation of spikes into
coronavirus particles is determined by the carboxy-terminal domain, we
analyzed the assembly of the chimeric and wild-type S proteins into
both MHV-based and FIPV-based VLPs. Plasmids encoding the M, E, and S
proteins were transfected into OST7-1 cells that had been infected with
vTF7-3. The proteins were labeled by incubating the cells for 3 h
with 35S-amino acids. VLPs secreted into the culture medium
were purified by flotation in sucrose gradients. They were subsequently
affinity purified with MHV S- and FIPV S-specific MAbs as well as with antisera to other viral structural proteins.
The analyses of the MHV-based VLPs are shown in Fig.
4. Coexpression of all three MHV-A59
wild-type membrane proteins led
to the formation of particles that
could be affinity isolated
as expected by the MAb J1.3 against the MHV
M protein ectodomain
(
6) as well as by the MAb WA3.10
against the MHV S ectodomain
(
33) but not by the MAb 23F4.5
against the FIPV S ectodomain
(
19). Control coexpressions of
the M and S proteins were not
productive, while coexpression of M and E
yielded VLPs that could
be isolated through their M protein with MAb
J1.3 but that were
recognized by neither of the anti-S MAbs. Of the
chimeric S proteins,
only the one with the MHV-derived carboxy-terminal
domain (FMS)
was incorporated into VLPs. These particles could indeed
be collected
through their FIPV-specific S ectodomain by using the FIPV
S MAb
as well as through their M protein with the anti-M MAb and showed
the chimeric S protein having a slightly lower electrophoretic
mobility
than the MHV S protein. When the MFS protein was coexpressed
with M and
E, VLPs were produced as revealed by the anti-M MAbs,
but these could
not be isolated by the anti-S MAbs, demonstrating
the absence of S
protein. As judged from the varying intensities
of the M protein bands,
the amounts of VLPs produced seemed to
differ for the different S
proteins coexpressed. This may to some
extent be accounted for by
differences in the efficiencies with
which the different VLPs were
affinity isolated by the antibodies.
More likely, however, the
variations reflect the varying degrees
of interference of the different
S constructs with the expression
of M and E which hampered the
reproducible control of VLP production
levels. Yet, when we quantitated
the radioactivities in the M
and S proteins in the VLPs containing MHV
S and FMS, calculations
revealed that the molar ratios of M and S were
rather similar,
suggesting that the chimeric S protein is incorporated
into viral
particles with an efficiency quite similar to that of
wild-type
S protein.
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FIG. 4.
Incorporation of chimeric FMS into MHV-based VLPs.
Parallel cultures of OST7-1 cells in 35-mm-diameter dishes were
infected with vTF7-3 and transfected with different combinations of
plasmids as indicated (mS, mE, and mM represent plasmids encoding the
wild-type MHV-A59 S, E, and M proteins, respectively; FMS and MFS refer
to plasmids encoding the chimeric S proteins described in the legend to
Fig. 1). Cells were incubated at 32°C and labeled from 5 to 8 h
p.i. with 35S-amino acids (100 µCi/dish). Culture media
(0.8 ml) were harvested, cleared by low-speed centrifugation, mixed
with 2.3 ml of 67% sucrose in TM (10 mM Tris-HCl [pH 7.0], 10 mM
MgCl2), and transferred into Beckman SW50.1 ultracentrifuge
tubes. Each solution was overlaid with 1 ml of 48% sucrose, 0.5 ml of
40% sucrose, and 0.5 ml of 30% sucrose in TM, and the gradients were
centrifuged at 36,000 rpm for 43 h. After centrifugation, a
fraction consisting of the top 1 ml of each tube was collected. Virus
particles were affinity purified from 150 µl of this fraction by
addition of 25 µl of MAb J1.3 against the MHV M protein
(Mm); 10 µl of MAb WA3.10, which is directed against
an epitope in the MHV S ectodomain (Sm); or 3 µl of
MAb 23F4.5, which recognizes an epitope in the FIPV S ectodomain
(Sf). Samples were processed and analyzed as described
for Fig. 2 except that the Staphylococcus aureus immune
complexes were washed once with TM instead of three times with
radioimmunoprecipitation assay buffer. At the left of the figure, mS
and mS/gp90 indicate the positions of the uncleaved and cleaved forms
of the S protein, respectively; mM and FMS mark the positions of the M
protein and the chimeric S protein, respectively. Ab, antibody.
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So far, VLPs have been shown only for the coronaviruses MHV (
2,
5,
32) and the transmissible gastroenteritis virus
of swine
(
1). In Fig.
5, we show that
such particles can similarly
be assembled from FIPV envelope proteins.
Again, M and E are the
minimal requirements, the combination of M and S
being unproductive.
If wild-type S is coexpressed with M and E, spiked
particles which
can be affinity purified with anti-FIPV serum and with
FIPV S-specific
MAbs are formed. Coexpression of the chimeric S
proteins shows
that now only MFS, the spike protein with the
FIPV-derived carboxy
terminus, was incorporated, giving rise to
particles that could
be collected with the MHV S MAb. The reverse
construct (FMS) was
not incorporated into VLPs. When the
radioactivities in the M
and S proteins were quantitated for the VLPs
produced with wild-type
FIPV S and with chimeric MFS, it now appeared
that the latter
was significantly underrepresented. While this may
indicate that
this protein is incorporated into particles less
efficiently,
the result is, at least in part, due to the relatively
poor expression
that we observed with the MFS construct (data not
shown).
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FIG. 5.
Incorporation of chimeric MFS into FIPV-based VLPs.
Different plasmid combinations were expressed, the proteins were
labeled, and the culture media were processed, all as described for
Fig. 4. fS, fE, and fM refer to plasmids encoding the wild-type FIPV S,
E, and M proteins, respectively; FMS and MFS refer to the chimeric
constructs described in the legend to Fig. 1. The FIPV serum (G73)
was from an FIPV-infected cat. Ab, antibody.
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The combined data demonstrate that the assembly of spikes into the
coronavirus envelope is governed by the S protein's carboxy-terminal
domain. Clearly, the 64-residue segment comprising the transmembrane
and endodomain is sufficient to interact with the M protein and
to draw
the 1,308 (MHV)- or 1,433 (FIPV)-residue-long mature (i.e.,
devoid of
its predicted cleaved signal sequence) protein into
particles. It will
now be interesting to investigate whether this
segment is required in
its entirety or whether the functional
domain can be narrowed down
further. In this respect, it is of
note that quite substantial homology
occurs among transmembrane
domains of coronavirus S proteins,
particularly on the amino-terminal
side of the transmembrane domain
where a highly conserved 8-residue
sequence (KWPWYVWL) occurs (Fig.
1B). In contrast, besides the
generally high cysteine content little
similarity exists in the
endodomain.
Although for several enveloped viruses a role of the membrane-anchoring
and/or cytoplasmic domain has been implicated in the
incorporation of
membrane proteins, no general conclusions can
yet be drawn. Quite
inconsistent observations were, for instance,
made with well-studied
proteins such as the influenza virus hemagglutinin
(
10,
13,
18) and the rhabdovirus G protein (
14-17,
25-28).
As
an illustration, incorporation of G protein (
25) or of
heterologous
membrane proteins (
26,
28) into the vesicular
stomatitis virus
envelope appeared to occur nonspecifically, i.e., with
efficiencies
independent of the nature of the transmembrane and
cytoplasmic
domains, while for the efficient assembly of foreign
membrane
proteins into the rabies virus envelope the autologous G tail
was required (
15,
27). A fair comparison with the
coronavirus
S protein is, however, difficult to make, as for many of
these
viruses an interaction between these proteins
through their
cytoplasmic
domain
and the viral core is important (
17) or
essential (
30,
34). For coronaviruses, such interactions are
not essential:
particle formation is nucleocapsid independent and
occurs irrespective
of the presence of S protein (
12,
23,
32; also the present
paper).
The chimeric coronavirus S proteins appeared to be biologically active,
causing fusion of reporter cells in a coculture assay
(Fig.
3). This
indicated that such proteins might mediate infection
when incorporated
into coronavirions. We have therefore introduced
the FMS gene construct
into the MHV-A59 genome by targeted RNA
recombination, giving rise to a
murine coronavirus that, by virtue
of its FIPV-derived spike
ectodomain, is unable to infect murine
cells but has acquired the
property to infect and multiply in
feline cells (L. Kuo, G.-J. Godeke,
M. J. B. Raamsman, P. S. Masters,
and P. J. M. Rottier, unpublished data). Not only do these observations
confirm the
results presented in this paper, the recombinant chimeric
MHV also
provides a powerful new tool to introduce mutations into
the
3'-terminal genomic domain encoding the structural proteins.
By using
as a recombination partner a donor RNA construct that
will restore the
wild-type S gene, isolation of mutants can simply
be done by selecting
for growth on murine
cells.
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ACKNOWLEDGMENTS |
We are grateful to Rhône Mérieux (Lyon, France) for
providing MAb 23F4.5 and to John Fleming (University of Wisconsin) for the MAb WA3.10.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Virology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands. Phone: 31-30-2532462. Fax:
31-30-2536723. E-mail: P.Rottier{at}vet.uu.nl.
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Journal of Virology, February 2000, p. 1566-1571, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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