The Missing Link in Coronavirus Assembly
RETENTION OF THE AVIAN CORONAVIRUS INFECTIOUS BRONCHITIS VIRUS
ENVELOPE PROTEIN IN THE PRE-GOLGI COMPARTMENTS AND PHYSICAL INTERACTION
BETWEEN THE ENVELOPE AND MEMBRANE PROTEINS*
K. P.
Lim and
D. X.
Liu
From the Institute of Molecular Agrobiology, The National
University of Singapore, 1 Research Link, Singapore 117604
Received for publication, October 25, 2000, and in revised form, January 29, 2001
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ABSTRACT |
One missing link in the coronavirus assembly is
the physical interaction between two crucial structural proteins, the
membrane (M) and envelope (E) proteins. In this study, we demonstrate
that the coronavirus infectious bronchitis virus E can
physically interact, via a putative peripheral domain, with M. Deletion
of this domain resulted in a drastic reduction in the incorporation of
M into virus-like particles. Immunofluorescent staining of cells
coexpressing M and E supports that E interacts with M and relocates M
to the same subcellular compartments that E resides in. E was retained in the pre-Golgi membranes, prior to being translocated to the Golgi
apparatus and the secretory vesicles; M was observed to exhibit similar
localization and translocation profiles as E when coexpressed with E. Deletion studies identified the C-terminal 6-residue RDKLYS as the
endoplasmic reticulum retention signal of E, and site-directed
mutagenesis of the
4 lysine residue to glutamine resulted in the
accumulation of E in the Golgi apparatus. The third domain of E that
plays a crucial role in virus budding is a putative transmembrane
domain present at the N-terminal region, because deletion of the domain
resulted in a free distribution of the mutant protein and in
dysfunctional viral assembly.
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INTRODUCTION |
Morphogenesis and assembly of mammalian enveloped RNA viruses are
complex processes. During these processes, the viral core, consisting
of viral RNA and core proteins, becomes wrapped in a membranous
structure (viral envelope) derived from host cell membranes to form
virion particles. The assembly process, which is referred to as
budding, usually occurs at either the plasma or intracellular
membranes. The formation of viral core, envelope, and the assembly of
virus particles would involve interaction between viral proteins and
host membrane components, among viral structural proteins and between
viral proteins and viral RNA. Characterization of these events at the
molecular level has been greatly facilitated by the understanding that
viral structural proteins may contain all of the necessary information
to dictate the assembly process. In fact, it was observed that
coexpression of viral structural proteins would result in the formation
of virus-like particles
(VLPs)1 in many different
viral systems (1-9). In this study, we exploit the coronavirus VLP
system to study the interaction between two structural proteins during
coronavirus assembly and the implication of this interaction in the
assembly and release of coronavirus particles.
Coronavirus is the largest RNA virus known so far. It has a
positive-sense, single-strand RNA genome of 27-30 kilobases in length.
Despite of the huge genome size, coronavirus typically contains four
structural proteins, i.e. a type I spike (S) glycoprotein required for infectivity, a phosphorylated nucleocapsid (N) protein that interacts with the viral genome to form a helical core, a major
type III integral membrane (M) protein, and a minor type III envelope
(E) protein (10-15). A fifth protein, the hemagglutinin esterase
glycoprotein (HE) is found in some but not all coronaviruses as short
spikes (16). Extensive cellular studies on porcine coronavirus
transmissible gastroenteritis virus (TGEV), murine coronavirus mouse
hepatitis virus (MHV), and avian coronavirus infectious bronchitis
virus (IBV) have demonstrated that coronaviruses assemble at the
pre-Golgi membranes of the intermediate compartment (IC) early in
infection and in the rough endoplasmic reticulum (ER) at late times of
the infection (17-20). Unlike most other enveloped RNA viruses,
coronaviruses employ a nucleocapsid-independent strategy to drive
assembly and budding (2). Coexpression of both M and E in intact cells
was initially shown to be required for inducing the formation of VLPs,
which are similar in size and appearance as the authentic MHV virions
(2). More recently, it was demonstrated that expression of E alone
resulted in the release of E-containing vesicles (15, 21). These
particles were referred to as VLPs (15). The crucial role of E in viral assembly was also indicated by other studies on MHV and TGEV (23, 24).
Previously, E was shown to be an integral membrane protein (22, 15). It
expresses on the surface of the infected cell (25), in the ER and the
IC compartment (22) and in the Golgi complex (15). In this study, the
subcellular localization and the intracellular translocation of IBV E
were studied in detail in intact cells by using specific organelle
markers. Indirect immunofluorescence showed that the protein resided
temporarily in the pre-Golgi compartments consisting of the ER and IC
membranes for up to 7 h post-transfection, before it progressed
down the secretory pathway. The signal that determines the temporal
retention of E in the pre-Golgi compartments was mapped to the
C-terminal extreme 6-residue RDKLYS, which may resemble the well
characterized di-lysine ER targeting motif for membrane proteins.
Interestingly, coexpression of E with IBV M, a typical Golgi-localizing
protein, showed temporal retention of M in the pre-Golgi compartments. In fact, M was shown to be colocalized and cotranslocated to the same
subcellular compartments when coexpressed with E in a time course
experiment, suggesting a strong physical interaction between the two
proteins. Coimmunoprecipitation and deletion analysis revealed that the
two proteins could indeed form a heterogeneous complex and that a
putative peripheral domain was required for the interaction with M. Deletion of this domain significantly reduced the assembly of M into
VLPs. Furthermore, the membrane anchorage of E was demonstrated to be
contributed by a stretch of hydrophobic residues at the N-terminal
region of the protein and to be essential for the formation of VLP,
because deletion of this putative transmembrane domain resulted in the
relocation of the mutant protein to the cytosol and led to a drastic
reduction in the release of VLPs. This study reveals that IBV E protein plays a fundamental role in the assembly of viral particles.
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EXPERIMENTAL PROCEDURES |
Viruses and Cells--
The egg-adapted Beaudette strain of IBV
(VR-22) was obtained from the American Type Culture Collection and was
adapted to Vero cells as described previously (26). Vero cells and
Cos-7 cells were grown at 37 °C in 5% CO2 and
maintained in Glasgow's modified Eagle's medium supplemented with
10% fetal calf serum.
Transient Expression of IBV Sequence in Cos-7 Cells Using a
Vaccinia Virus-T7 Expression System--
IBV sequences were placed
under the control of a T7 promoter and transiently expressed in
mammalian cells using the system described by Fuerst et al.
(27). Briefly, 60-80% confluent monolayers of Cos-7 cells grown on
35-mm dishes (Falcon) were infected with 10 plaque-forming units/cell
of a recombinant vaccinia virus (vTF7-3) that expresses bacteriophage
T7 RNA polymerase. The cells were then transfected with 5 µg of
plasmid DNA (purified by Qiagen plasmid Midi kits) mixed with
Lipofectin transfection reagent according to the instructions of the
manufacturer (Life Technologies, Inc.). After incubation at 37 °C,
5% CO2 for 5 h, the cells were washed twice with
methionine-free medium and labeled with 25 µCi/ml [35S]methionine. The radiolabeled cells and culture media
were then harvested at 18 h post-transfection.
SDS-Polyacrylamide Gel Electrophoresis--
Electrophoresis of
viral polypeptides was performed on SDS-17.5% polyacrylamide gels
(28). The 35S-labeled polypeptides were detected by
autoradiography of the dried gels.
Polymerase Chain Reaction--
Complementary DNA templates for
PCR were prepared from purified IBV virion RNA by using a first strand
cDNA synthesis kit (Roche Molecular Biochemicals). Amplification of
the respective template DNAs with appropriate primers was performed
with Pfu DNA polymerase (Stratagene) under the standard
buffer conditions with 2 mM MgCl2. The reaction
conditions used were 30 cycles of 95 °C for 45 s, X °C for
45 s, and 72 °C for X min. The annealing temperature
(55 °C) and the extension time (4 min) were subjected to adjustments
according to the melting temperature of the primers employed and the
length of PCR fragments synthesized.
Radioimmunoprecipitation--
Media of transfected Cos-7 cells
were collected and mixed with 5× RIPA buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate,
0.5% Nonidet P-40, 0.05% SDS) and precleared by centrifugation at
4,000 × g for 30 min at 4 °C in a
microcentrifuge. Cells were lysed with 1× RIPA buffer and
precleared by centrifugation at 12,000 rpm. Immunoprecipitation with
anti-E and anti-M rabbit polyclonal antisera (14, 29) and anti-T7
monoclonal antiserum (Novagen) was carried out as previously described
(30).
Immunofluorescence and Confocal Microscopy--
IBV sequences
were transiently expressed in Cos-7 cells grown on 4-well chamber
slides (IWAKI). At 5 h post-transfection (or otherwise stated),
cells were rinsed with phosphate-buffered saline and subjected to
fixation using 4% paraformaldehyde for 15 min and permeabilized with
0.2% Triton X-100. Fluorescence staining was performed by
incubating cells with either an antibody or a mixture of both primary
antibodies (rabbit anti-M (1:30) or mouse anti-T7 (1:200)) for 1 h
at room temperature, followed by FITC- or tetramethyl rhodamine
isocyanate-conjugated secondary antibodies for 1 h at 4 °C.
Goat anti-rabbit antibody was used at 1:400 (Sigma) and goat anti-mouse
IgG at 1:20 (DAKO). Images were viewed and collected with a Zeiss
confocal microscope connected to a Bio-Rad MRC1024 laser scanner.
Construction of Plasmids--
Plasmid pIBVM-1, which covers the
IBV sequence between nucleotides 24498 and 25159, was constructed by
cloning a PvuII/SacI-digested PCR fragment into
PvuII/SacI digested pKTO vector (31). The PCR
fragment was generated using primers LDX59
(5'-CAGCAACAGCTGAAGATGCCCAACG-3') and LDX60
(5'-CTACACACGAGCTCTTATGTGTAAAGA-3').
Plasmid pIBVE was constructed as follows. A 735-base pair fragment,
obtained by PCR using LDX55
(5'-GATTGTTCAGGCCATGGTGAATTTATTGAA-3') and XIANG8
(5'-GCACCATTGGCACACTC-3'), was digested with NcoI and BamHI and ligated into
NcoI/BamHI-digested pKTO, resulting in a plasmid
containing the IBV sequence between nucleotides 24205 and 24795. Plasmid pT7E was constructed by fusing the 11-amino acid T7 tag
(MASMTGGQQMG) to the N terminus of E.
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RESULTS |
Subcellular Localization of IBV E in Cells Overexpressing the
Protein--
In a previous study using immunoaffinity-purified
antibodies specific for IBV E, the protein was demonstrated to be
localized to intracellular membrane structures as well as on cell
surface in IBV-infected cells (25). Although some evidence of
polarization of the fluorescence into structures resembling the Golgi
apparatus was observed, the reticular staining patterns suggest that
the majority of the protein may be localized to pre-Golgi compartments at the time of observation (25). More recently, Raamsman et al. (22) reported that E colocalizes with Rab-1, a marker for the
IC and the ER, supporting that the protein may be localized to the
pre-Golgi compartments. However, Corse and Machamer (15) have recently
presented data showing that IBV E may be colocalized with M to the
Golgi apparatus. To address this issue further, IBV E was fused to an
11-amino acid T7 Tag (Novagen), and a highly specific monoclonal
antibody against the T7 tag was used to study the subcellular
distribution of E in intact cells. This strategy would also facilitate
dual labeling of cells expressing both M and E in subsequent studies.
Subcellular localization of E in Cos-7 cells was examined by indirect
immunofluorescence microscopy. A reticular staining pattern (Fig.
1A) colocalizes with the
staining profile of R6 (rhodamine B hexyl-ester chloride, Molecular
Probes) (Fig. 1B), a short chain carbocyanine dye known to
stain specifically the ER of mammalian cells, as indicated by the
merged image (Fig. 1C). In contrast, IBV M, which was
previously shown to be localized to the Golgi apparatus (32), does not
colocalize with the R6 marker (Fig. 1, D-F); instead, it
coaligns with the staining pattern of the fluorescence vital dye BODIPY
TR-ceramide (Molecular Probes) (Fig. 1, G-I). This dye was
shown to stain the Golgi apparatus specifically (Molecular
Probes).

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Fig. 1.
Subcellular localization of IBV E and M
proteins in transfected Cos-7 cells. The T7-tagged E and M
expressed in Cos-7 cells were detected using monoclonal anti-T7
(A) and polyclonal anti-M antibodies (D and
G), respectively. The proteins were then labeled with the
FITC-conjugated secondary antibodies. Immunofluorescent staining of E
(A) and M (D and G) gives a reticular
staining pattern and a perinuclear, Golgi-like staining pattern,
respectively. B and E refer to cells stained with
R6, a dye for the ER, and H refers to a cell stained with
BODIPY TR-ceramide (TR-C), a vital dye for the Golgi
apparatus. The green images represent the FITC-derived green
fluorescence, and red images represent the rhodamine and
Texas Red-derived red fluorescence. Colocalization of viral
proteins with the organelle markers is represented by the
yellow region within each cell in the merged images
(C, F, and I). The fluorescence was
viewed using a confocal scanning Zeiss microscope.
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To further clarify the subcellular distribution pattern of E,
transfected cells were incubated in the presence of 100 µg/ml of
cycloheximide for 30 min at 3.5 h post-transfection to stop new
protein synthesis. The expressed viral proteins were then chased and
fixed at 4, 7, 10, and 16 h post-transfection, respectively, and
the subcellular localization patterns were viewed by indirect immunofluorescent staining. E was observed to display the reticular staining profiles overlapping with the R6 staining patterns up to
7 h post-transfection (Fig. 2,
A-F). A more perinuclear staining pattern was observed at
10 h post-transfection (Fig. 2G). It overlaps with the
staining pattern of the fluorescence vital dye BODIPY TR-ceramide (Fig.
2, G-I). Granular fluorescent aggregates scattering in the
cytoplasm of the transfected cells were observed at 16 h
post-transfection (Fig. 2J). This fluorescence pattern
overlaps with the staining pattern of LysoTrackerTM Red
DND-99 (Molecular Probes), a biotinylated acidotropic probe that stains
acidic compartments including lysosomes, trans-Golgi vesicles, and
secretory vesicles.

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Fig. 2.
Subcellular localization and
translocation of E protein. Cos-7 cells expressing E were
incubated in the presence of 100 µg/ml cycloheximide for 30 min at
3.5 h post-transfection. The cells were fixed at 4, 7, 10, and
16 h and subjected to indirect immunofluorescent staining with
anti-T7 monoclonal antibody followed by incubating with FITC-conjugated
anti-mouse antiserum. Immunostaining of E is presented in A,
D, G, and J. B and
E show the ER staining with R6; H shows the
BODIPY TR-ceramide (TR-C) staining pattern; and K
shows the LysoTrackerTM Red DND-99 (LT)
staining pattern. The green images represent the
FITC-derived green fluorescence, and red images represent
the Rhodamine and Texas Red-derived red fluorescence. Colocalization of
viral proteins with the organelle markers is represented by the
yellow region within each cell in the merged images
(C, F, I, and L).
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The Effects of Deletion on the Subcellular Distribution of IBV E
and Delineation of Its ER Retention Signal--
The amino acid
sequence of E was then subjected to computer analysis using the PSORT
program for prediction of subcellular localization signals (33) and the
TMPRED proteomics tools for prediction of transmembrane regions and
membrane topology of proteins (34). As shown in Fig.
3a, a putative transmembrane
domain is predicted for residues between 17 and 33, a putative
peripheral domain is located between amino acids 37 to 53, and a
potential ER retention signal is predicted to be present at the
C-terminal extreme end of the protein.

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Fig. 3.
a, diagram indicating the presence of
four putative domains in IBV E. E adopts a type III topology. The
boxed regions refer to the putative domains. I
refers to a potential N-linked glycosylation site which is not utilized
(15), II refers to a putative transmembrane domain between
residues 17 and 33, III refers to a putative peripheral
domain located between amino acids 37 and 53, and IV refers
to a potential ER retention signal. b, summary of the
effects of deletion and mutation on the subcellular localization of IBV
E, the interaction with M, VLP release, and the incorporation of M into
VLPs. The 109 amino acids of E are represented by white
boxes, and the putative transmembrane (TM) and
peripheral domains are indicated in gray boxes. The deleted
regions are represented in black boxes, and a single
mutation is represented by a stippled box. RDKLYS
stands for the cytoplasmic tail sequence of E containing the potential
ER retention signal. Also included is a summary of the position of
deletion, subcellular localization of each mutant,
coimmunoprecipitation with M, VLP release, and the incorporation of M
into the wild type and mutant E-induced VLPs.
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To determine the effects of each putative domain on the subcellular
distribution of E, several deletion constructs were constructed. E
1
deletes the N-terminal sequence from amino acids 1 to 14, E
2 deletes
the putative transmembrane domain (residues 18-33), E
3 contains a
deletion of residues 33-51 in the putative peripheral membrane domain,
E
4 deletes amino acids 50-64, E
5 deletes amino acids 67-83, and
E
6 deletes 26 residues from the C-terminal end (thus removing the
potential ER retention signal) (Fig. 3b). The effects of
each deletion on the subcellular distribution of E are summarized in
Fig. 3b. As shown in Fig. 4,
the two deletions that resulted in an altered subcellular distribution
profile of E are E
2 and E
6. Deletion of the putative
transmembrane domain (E
2) resulted in a diffuse staining pattern
(Fig. 4G), which does not overlap with the staining pattern
of R6 (Fig. 4, H and I). Deletion of the
C-terminal 26 residues led to the detection of the mutant protein with
a staining profile similar to that of M (Fig. 4, S-U). This
result indicates that the C-terminal region of E may contain sufficient
information for its ER retention. Other deletion constructs including
deletion of ~95% of the putative peripheral domain of E rendered no
obvious effects on the subcellular localization of E (Fig. 4).

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Fig. 4.
The effects of deletions on the subcellular
localization of E protein. Cos-7 cells expressing the wild type
and mutant E were fixed at 7 h post-transfection. The expressed
proteins were detected with anti-T7 monoclonal antibody against the T7
tag fused to the N terminus of the wild type E (A), E 1
(D), E 2 (G), E 3 (J), E 4
(M), E 5 (P), and E 6 (S).
B, E, H, K, N,
Q, and T refer to cells stained with R6. The
green images represent the FITC-derived green fluorescence,
and red images represent the Rhodamine and Texas Red-derived
red fluorescence. The merged images (C, F, I, L, O, R, and
U; yellow) represent colocalization of proteins
with the ER marker.
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Further deletion of the C-terminal most six amino acids was
carried out, giving rise to E
8. Similar to E
6, expression of E
8 showed that the protein exhibits a Golgi-like staining profile (Fig. 5A), which does not
overlap with the R6 staining pattern (Fig. 5, B and
C). Instead, it overlaps with the staining pattern of dye
BODIPY TR-ceramide (Fig. 5, D-F), indicating that the
mutant protein may be localized to the Golgi apparatus. Furthermore, when the
4 lysine residue was mutated to a nonconservative glutamine, the mutant protein accumulates at the Golgi apparatus, as it
colocalizes with the staining pattern of dye BODIPY TR-ceramide (Fig.
5, G and I).

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Fig. 5.
Subcellular localization of E proteins
lacking the putative ER retention signal. Cos-7 cells transiently
expressing E 8 (A and D) and E(K N)
(G) were fixed at 7 h post-transfection. Proteins are
detected with anti-T7 monoclonal antibody. B refers to cells
stained with R6, and E and H show cells stained
with BODIPY TR-ceramide (TR-C). The green images
represent the FITC-derived green fluorescence, and red
images represent the Rhodamine and Texas Red-derived red fluorescence.
The merged images (yellow) represent colocalization of
proteins with the organelle markers (C, F, and
I).
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Physical Interaction of IBV E with M and Temporary Retention of M
in the ER via the Interaction--
Because E has been shown to be the
sole component for VLP production (15), it was speculated that
incorporation of other viral structural proteins into VLPs would
involve the interaction between E and those proteins. In fact, Maeda
et al. (21) demonstrated that anti-E antibody is able to
coimmunoprecipitate structural proteins E, M, and N in MHV-infected
cells, indicating the interactions of E with other structural proteins.
Because M was also shown to be an essential component for VLP
formation, we tested the interaction between E and M by coexpressing
them in Cos-7 cells. Fig. 6 refers to
coimmunoprecipitation of cells expressing both proteins. Fig.
6a shows that M alone cannot be precipitated by anti-T7
antiserum (lane 6). In the presence of E (lane
4), a protein species corresponding to the pre-Golgi form of M was
precipitated (lane 5). This form of M is likely to be a
mixture of unglycosylated and pre-Golgi modified M and was referred to
as pre-Golgi form of M in this report. Similarly, E cannot be
immunoprecipitated by anti-M when expressed alone (lane 1).
However, when M was coexpressed, E could be precipitated by anti-M
(lane 2). Similar forms of the physical interaction between
E and M were observed in IBV-infected Vero cells. As shown in Fig.
6b, E and both pre- and post-Golgi forms of M were
coimmunoprecipitated by anti-M (lane 1) and anti-E (lane 5) antisera, respectively, in cell lysates prepared
from IBV-infected Vero cells harvested at 18 h post-infection.
Coimmunoprecipitation of both proteins were also observed from the
viral particles released into the culture medium (lanes 2 and 6).

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Fig. 6.
a, coimmunoprecipitation of M and E
proteins in Cos-7 cells expressing the two proteins. Cos-7 cells
expressing E (lanes 1 and 4), M (lanes
3 and 6), or coexpressing both proteins (lanes
2 and 5) were lysed and subjected to
immunoprecipitation before analysis on SDS-17.5% polyacrylamide gel.
The expressed proteins were immunoprecipitated with anti-M (lanes
1-3) and anti-T7 (lanes 4-6) antisera, respectively.
Numbers on the left indicate molecular masses in
kilodaltons. b, coimmunoprecipitation of M and E proteins in
IBV-infected Vero cells. Cell lysates were prepared from Vero cells
harvested at 18 h post-infection, and the collected culture media
were precleared by centrifugation. The viral proteins in the cell
lysates (lanes 1, 3, 5, and
7) and in the culture medium (lanes 2,
4, 6, and 8) were immunoprecipitated
with anti-M (lanes 1-4) and anti-E (lanes 5-8),
before analysis on SDS-17.5% polyacrylamide gel. The upper
part of the left panel was prepared from a gel exposed
for 1 day, and the lower part was prepared from the same gel
exposed for 3 days. Numbers on the left indicate
molecular masses in kilodaltons.
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To view this form of interaction in intact cells, M and E proteins were
coexpressed in Cos-7 cells. At 3.5 h post-transfection, new
protein synthesis was stopped by incubation of the cells in the
presence of 100 µg/ml of cycloheximide for 30 min. The expressed viral proteins were then chased and fixed at 4, 7, 10, and 16 h
post-transfection, respectively, and were viewed by immunofluorescent staining. Representative cells are shown in Fig.
7. E was observed to show a reticular
staining pattern similar to that in Figs. 1A and
2D up to 7 h post-transfection (Fig. 7, A
and D). At 10 and 16 h post-transfection, the protein
accumulates in the Golgi apparatus and the secretory vesicles,
respectively (Fig. 7, G-J), as shown in Fig. 2
(G and J). Interestingly, dual labeling of M in
cells coexpressing both E and M showed that M colocalizes with E to the
ER up to 7 h post-transfection (Fig. 7, A-F). M was
then translocated with E to the Golgi apparatus and the secretory vesicles at 10 and 16 h post-transfection, respectively (Fig. 7,
G-L). These results suggest that interaction of E with M
may occur in intact cells, because E can retain and translocate M to
the compartments it resides in. The detection of both E and M in the
ER, the Golgi and the secretory vesicles may represent the route that
M-E protein complexes take to exit the host cells.

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Fig. 7.
The effects of coexpression of E with M on
the subcellular localization of M. M was coexpressed with E in
Cos-7 cells, incubated in the presence of 100 µg/ml of cycloheximide
for 30 min, and subjected to indirect immunofluorescent staining. The
cells were fixed at 4 (A-C), 7 (D-F), 10 (G-I), and 16 (J-L) h, respectively, and the
subcellular localization of proteins were examined by dual labeling
with a mixture of anti-T7 (mouse) and anti-M (rabbit) antisera,
followed by incubating with a mixture of FITC-conjugated anti-mouse and
tetramethyl rhodamine isocyanate-conjugated anti-rabbit antisera.
The staining patterns of E are indicated by the green images
(A, D, G, and J), and the
staining patterns of M are indicated by the red images
(B, E, H, K, N,
and Q). M and P refer to cells
expressing E 2 and E 7, respectively. Merged images
(yellow) represent colocalization of the two proteins
(C, F, I, L, O,
and R).
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To further confirm this observation, M was coexpressed with E
2 and
E
7, respectively, and the cells were stained at 7 h
post-transfection. E
7 contains a deletion of residues 37-57, the
putative peripheral domain. Once again, E
2 exhibits a diffuse
staining pattern (Fig. 7M). Instead of the typical Golgi
localization profile (Fig. 1D), coexpression of E
2 with M
forces M to adapt the same diffuse staining pattern of E
2 (Fig. 7,
M-O). Coexpression of E
7 and M showed a reticular
staining pattern of E
7 (Fig. 7P), similar to the staining
pattern of E
3, which contains a deletion considerably overlapping
with that in E
7 (Fig. 4J). However, the staining pattern
of M did not overlap with that of E
7 (Fig. 7, P-R). As can be seen, the majority of M was accumulated in the Golgi region, with a faint reticular staining (Fig. 7Q).
Subcellular Localization of M and E in IBV-infected Vero
Cells--
The subcellular distribution patterns of E and M in
IBV-infected cells were analyzed by indirect immunofluorescence with
polyclonal antisera against E and M, respectively. At 4.5 h
post-infection, Vero cells were incubated in the presence of 100 µg/ml of cycloheximide for 30 min to stop new protein synthesis and
were chased and fixed at 5, 7, 9, and 12 h post-infection,
respectively. Both E and M were observed to display the reticular
staining profiles overlapping with the R6 staining patterns up to
7 h post-infection (Fig. 8, A-F and M-R). E and M proteins were then
observed to accumulate at the perinuclear region at 10 h
post-infection (Fig. 8, G and S), overlapping
with the staining pattern of the fluorescence vital dye BODIPY
TR-ceramide (Fig. 8, G-I and S-U). Granular
fluorescent aggregates of both E and M were detected in the cytoplasm
at 12 h post-infection (Fig. 8, J and V),
overlapping with the staining pattern of LysoTrackerTM Red
DND-99 (Molecular Probes) (Fig. 8, J-L and
V-X). These distribution patterns resemble the staining
patterns observed in cells coexpressing the T7-tagged E and M,
suggesting that the T7 tag does not obviously affect the subcellular
distribution patterns of E. However, we were unable to dual label the
same cells for detailed analysis of the distribution profiles of the
two proteins, because both anti-E and anti-M were raised in
rabbits.

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Fig. 8.
Subcellular localization of IBV E and M
proteins in IBV infected cells. Vero cells infected with IBV were
incubated in the presence of 100 µg/ml cycloheximide for 30 min at
4.5 h post-infection and subjected to indirect immunofluorescent
staining. The cells were fixed at 5 (A-C and
M-O), 7 (D-F and P-R), 9 (G-I and S-U), and 12 (J-L and
V-X) h post-infection, respectively. Viral proteins were
detected with anti-E (A, D, G, and
J) and anti-M (M, P, S, and
V) antisera. B, E, N, and
Q refer to ER staining with R6; H and
T show cells stained with BODIPY TR-ceramide
(TR-C); and K and W show cells stained
with LysoTrackerTM Red DND-99 (LT). The
green images represent the FITC-derived green fluorescence,
and red images represent the rhodamine and Texas Red-derived
red fluorescence. The merged images (yellow) represent
colocalization of the proteins with the organelle markers
(C, F, I, L, O,
R, U, and X).
|
|
Determination of the IBV E Sequences Responsible for the Physical
Interaction with M--
To determine the domain(s) required for the
interaction with M, immunoprecipitation of lysates prepared from cells
coexpressing M and an E deletion mutant was performed. Most mutants can
be coimmunoprecipitated with M, except for E
3 and E
7 (Fig.
9). As shown in Fig. 9, only trace
amounts of E
3 and E
7 were coimmunoprecipitated by anti-M
(lanes 4 and 15), and no M was
coimmunoprecipitated by anti-T7 (lanes 11 and
16). Taken together with the colocalization data present in
Fig. 7P-R, these results suggest that the predicted peripheral domain
(amino acids 37-57) may be responsible for the interaction of E with
M.

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|
Fig. 9.
Coimmunoprecipitation of M protein and E
deletion mutants from transfected Cos-7 cells. Cos-7 cells were
transfected with plasmids expressing M and the wild type and mutant E,
as indicated above each lane. The cells were lysed and subjected to
immunoprecipitation before analysis with SDS-17.5% polyacrylamide gel.
Lanes 1-7 and 15 refer to products precipitated
with anti-M, and lanes 8-14 and 16 refer to
products precipitated with anti-T7. Numbers on the
left indicate molecular masses in kilodaltons.
|
|
The Effects of Deletion of IBV E on the Release of VLPs and on the
Assembly of M into VLPs--
The effects of deletion of E on the
release of VLPs were tested by expression of E and the deletion
constructs in Cos-7 cells and by detection of the expressed proteins in
the culture medium. As shown in Fig.
10a, the wild type and
deletion constructs E
2 and E
7 were efficiently detected in
lysates prepared from cells transfected with the corresponding
constructs (lanes 6-8). The wild type and E
7 were also
efficiently detected from the culture media by immunoprecipitation with
anti-T7 antibody (Fig. 10a, lanes 14 and
16), indicating that VLPs were efficiently released from cells expressing the two constructs. Because E
7 contains a deletion of the putative peripheral domain of E, this result implies that this
putative domain may be not essential for the release of VLPs. Interestingly, no release of VLPs was observed from cells transfected with E
2 (Fig. 10a, lane 15), suggesting that
the transmembrane domain may be essential for the release of VLPs.
Because deletion of the putative transmembrane domain resulted in the
detection of E in the cytoplasm (Fig. 4G), these results
demonstrate that insertion of E into the intracellular membrane
structures is essential for the release of VLPs. As a control
experiment, M was detected in transfected cells by immunoprecipitation
with anti-M (Fig. 10a, lane 1); however, it was
undetectable in the culture media (Fig. 10a, lane
2). The effects of other deletions on the release of VLPs are
summarized in Fig. 3b.

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[in this window]
[in a new window]
|
Fig. 10.
a, the effects of deletion of the
putative transmembrane and the peripheral membrane domains of E on the
release of VLPs. Cos-7 cells were transfected with plasmids as
indicated above each lane. Cell lysates were prepared and subjected to
immunoprecipitation with anti-M (lanes 1-4) and anti-T7
(lanes 5-8), and protein expression efficiency was analyzed
on SDS-17.5% polyacrylamide gel. The release of VLP was analyzed by
immunoprecipitation of the culture medium from each transfection with
anti-M (lanes 9-12) and anti-T7 (lanes 13-16)
after the medium was centrifuged at low speed (4,000 × g) to preclear the cell debris. The numbers
between the two panels indicate molecular masses in kilodaltons.
b, the effects of deletion of the putative transmembrane and
the peripheral membrane domains of E on the incorporation of M into
VLPs. Cos-7 cells were transfected with plasmids as indicated above
each lane. Cell lysates were prepared and subjected to
immunoprecipitation with anti-M (lanes 1-3) and anti-T7
(lanes 4-6), and protein expression efficiency was analyzed
on SDS-17.5% polyacrylamide gel. The release of VLP was analyzed by
immunoprecipitation of the culture medium from each transfection with
anti-M (lanes 7-9) and anti-T7 (lanes 10-12)
after the medium was centrifuged at low speed (4,000 × g) to preclear the cell debris. The numbers
between the two panels indicate molecular masses in kilodaltons.
|
|
We next tested the effects of the deletions of the putative
transmembrane domain and the peripheral domain on the release of VLPs
and on the assembly of M into VLPs by coexpression of E and the
deletion mutants with M in Cos-7 cells. Efficient expression of M and E
constructs were observed in cells transfected with the corresponding
constructs (Fig. 10b, lanes 1-6). In the culture medium, wild type E was readily immunoprecipitated by either anti-M or
anti-T7 when coexpressed with M (Fig. 10b, lanes
7 and 10). Anti-M was also able to immunoprecipitate at
least two different types of M from cells coexpressing M and the wild
type E (Fig. 10b, lane 7), whereas anti-T7 could
predominantly immunoprecipitate the pre-Golgi form of M only (Fig.
10b, lane 10). Once again, neither E
2 nor M
was detectable by either antiserum in the culture medium when two
proteins were coexpressed (Fig. 10b, lanes 8 and
11). Coexpression of E
7 and M resulted in the detection
of only E
7 from the culture medium by anti-T7 (Fig. 10b,
lane 12), whereas anti-M could weakly coimmunoprecipitate
both types of M and trace amounts of E
7 (Fig. 10b,
lane 9). These results suggest that a strong physical
interaction between M and E via the putative peripheral domain of E
protein may facilitate the incorporation of the pre-Golgi form of M
into VLPs. The detection of both types of M in E
7-induced VLPs
suggested that although the ability of E
7 to interact with M was
severely diminished, E
7-induced VLPs could passively incorporate M
when they form at the pre-Golgi membranes and progress through the
Golgi complex for maturation.
 |
DISCUSSION |
Increasing evidence has shown that coronavirus E protein may play
a pivotal role in viral morphogenesis and virion assembly. Direct
evidence of the involvement of E in coronavirus morphogenesis is
derived from a recent study using clustered charged-to-alanine mutagenesis and targeted RNA recombination to mutate the hydrophilic tail of E (23). One of the two mutant viruses generated exhibits aberrant morphology, with many virions showing pinched and elongated shapes (23). The essential role of E in virion assembly was revealed by
studies showing the formation and release of VLPs from cells expressing
E alone (21, 15). It suggests that E may be the driving force in
determining the budding process, including selection of the budding
site, the formation of viral envelope, and the assembly of virion
particles. In this study, we provide evidence that IBV E resides in the
pre-Golgi compartments consisting of the ER and IC membranes for up to
7 h post-transfection. The signal determined that this
localization pattern was mapped to a di-lysine-like ER targeting signal
located in the C-terminal extreme 6-residue RDKLYS. The protein was
able to retain M in the same compartments by forming a heterogeneous
complex with M, thereby facilitating the assembly of M into VLPs.
Coronavirus M protein, being the most abundant membrane component of
the coronavirus virion, is able to laterally interact with itself to
form homogeneous complexes (35). It could also form heterogeneous
complexes with S, HE, and N proteins (36-38). The interaction of these
structural proteins with M may facilitate their assembly into the
virion particles (2, 36, 39). However, M must also interact with E to
be assembled into the virion, as expression of M alone could not induce
the formation of VLP. The demonstration of strong physical interaction
between IBV E and M via the putative peripheral domain of E in this
report connects the missing link between M and the E-induced envelope
components. By this interaction, E provides a temporary anchor to
relocate M in the pre-Golgi compartments, where it "prepares" the
membranes for budding (22). Meanwhile, a certain proportion of M may be passively incorporated into virions during envelopment, because considerable amounts of M were detected in VLPs induced by coexpression of E
7 and M. Upon accumulation of sufficient essential structural proteins, VLPs assemble and bud at the budding site, transport through
a functional Golgi stack, and are released out of the host cells by the
exocytic pathway. These VLP assembly and release processes, as revealed
by the immunofluorescence microscopy and time course experiments,
resemble the assembly and maturation processes of the coronavirus
virion as demonstrated by extensive cellular studies on
coronavirus-infected cells (17-20, 22, 40, 41).
IBV E was previously shown to be an integral protein with an
Nexo-Cendo orientation (15). The putative
peripheral domain is therefore exposed on the cytoplasmic face of the
intracellular membranes and is required for interaction with M. In a
recent study, the C-terminal cytoplasmic tail of MHV M was shown to
have more detrimental effects on VLP assembly and release than domains within its N-terminal region (42). One possible explanation is that
this region may be required for the interaction with E, because it is
exposed on the same cytoplasmic face as the putative peripheral domain
of E.
In a study on the intracellular assembly of TGEV (17), virions
formed in the perinuclear region were observed to be large and have a
clear center (immature virion), dissimilar to the smaller extracellular
particles (mature virion) containing compact internal cores with
polygonal contours. The mature virions were observed only at or after
the trans-Golgi network, indicating that maturation starts in the late
Golgi compartments. The molecular mechanisms underlining this
maturation process are unknown, but one reason may be the
differential glycosylation of M, as suggested by Risco et
al. (43). The coimmunoprecipitation of E with the pre-Golgi form
but not the post-Golgi form of M supports that E interacts with M in
the pre-Golgi compartments. Because this interaction facilitates the
assembly of M into the virion, it is likely that the immature virions
may contain mostly the pre-Golgi form of M. How and where do the
immature virions acquire the post-Golgi form of M? One possibility is
that the pre-Golgi form of M that has already assembled into the virion
undergoes modification when the particle goes through the Golgi
apparatus. The occurrence and significance of this modification in the
maturation of the immature virion are yet to be understood.
Alternatively, incorporation of M carrying complex sugar chains into
the immature virion may occur when the particle goes through the Golgi
complex, contributing to the maturation of the virus. In this case, the
incorporation of M must be independent of E, because the post-Golgi
form of M does not interact with E. The observation that certain
amounts of M was still incorporated into the E
7-induced VLPs
supports that M could be assembled into the virion by mechanisms
independent of E.
The deletion analysis mapped the ER retention signal of E to the
C-terminal extreme 6-residue RDKLYS, a di-lysine-like ER targeting
motif. Many membrane proteins carry a di-lysine signal in their
cytosolic domain that confers localization of a membrane protein to the
ER. For example, a type I ER membrane protein encoded by the
E19 gene of adenovirus 3 contains a 6-residue di-lysine motif DEKKMP located at the extreme C terminus that was necessary and
sufficient for retention of the protein to the ER of mammalian cells
(44). Mutagenesis study showed that the lysine residue at the
4
position of E is likely to be crucial, because a single mutation
resulted in the accumulation of the protein in the Golgi complex. To
support this observation, sequence analysis of IBV isolates showed that
this residue is conserved in six out of seven strains, despite
considerable variation in the total number of amino acid residues and
the primary sequences of E among different isolates (14, 45, 46).
Likewise, the leucine residue at the
3 position was also conserved in
these six isolates (14, 45, 46). However, the di-lysine ER retention
signal for all ER resident membrane proteins known so far contains a
lysine residue at the
3 position (47). Mutation of this residue
destroyed the targeting motif. We do not know whether the change to a
leucine residue at this crucial position for IBV E might suggest a
different targeting mechanism. Interestingly, neither the
3 leucine
nor the
4 lysine residues were found to be conserved in the
counterpart E protein sequences of other coronaviruses, including MHV,
human coronavirus, and TGEV (23, 48, 49). Because MHV E was also localized to the ER and IC, a different retention signal may exist in
this protein.
Further assessment of the di-lysine-like RDKLYS motif on the retention
of E is complicated by the fact that E is retained only temporarily in
the pre-Golgi membranes of the ER and IC. Immunofluorescence microscopy
and time course experiments present in this report demonstrated that E
was retained in the pre-Golgi membranes for up to 7 h before it
was translocated to the Golgi apparatus and down the secretory pathway.
Is this temporary retention of E due to the weakness of the
di-lysine-like RDKLYS signal or due to the formation of VLPs that
triggers the translocation of the protein? It is very likely that the
latter is the case. After assembling into VLPs, the protein was
translocated to the Golgi apparatus together with VLPs for the
maturation and finally release of VLPs out of the cells. Systematic
mutation of E to create a mutant E protein that maintains the same
subcellular distribution pattern as the wild type protein but loses the
ability to induce the formation of VLPs would be of help to study this
retention signal further.
 |
ACKNOWLEDGEMENTS |
We thank Lisa F. P. Ng and H. Y. Xu
for excellent technical assistance.
 |
FOOTNOTES |
*
This work is supported by a grant from the National Science
and Technology Board of Singapore.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
65-8727468; Fax: 65-8727007; E-mail: liudx@ima.org.sg.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009731200
 |
ABBREVIATIONS |
The abbreviations used are:
VLP, virus-like
particle;
TGEV, transmissible gastroenteritis virus;
MHV, mouse
hepatitis virus;
IBV, infectious bronchitis virus;
IC, intermediate
compartment;
ER, endoplasmic reticulum;
PCR, polymerase chain
reaction;
FITC, fluorescein isothiocyanate.
 |
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