From the Institut für Virologie,
Philipps-Universität Marburg, D-35037 Marburg, Germany and the
§ Institut für Virologie, Tierärztliche
Hochschule Hannover, D-30559 Hannover, Germany
Received for publication, November 8, 2000, and in revised form, February 26, 2001
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ABSTRACT |
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As we have shown previously, release of measles
virus (MV) from polarized epithelial cells is not determined by the
viral envelope proteins H and F. Although virus budding is
restricted to the apical surfaces, both proteins were abundantly
expressed on the basolateral surface of Madin-Darby canine
kidney cells. In this report, we provide evidence that the
basolateral expression of the viral proteins is of biological
importance for the MV infection of polarized epithelial cells. We
demonstrate that both MV glycoproteins possess a basolateral targeting
signal that is dependent upon the unique tyrosine in the cytoplasmic
tails. These tyrosines are shown to be also part of an endocytosis
signal. In MV-infected cells, internalization of the glycoproteins was
not observed, indicating that recognition of the endocytosis signals is
disturbed by viral factors. In contrast, basolateral transport was not
substantially hindered, resulting in efficient cell-to-cell fusion of
polarized Madin-Darby canine kidney cells. Thus, recognition of the
signals for endocytosis and polarized transport is differently
regulated in infected cells. Mutation of the basolateral sorting signal in one of the MV glycoproteins prevented fusion of polarized cells. These results suggest that basolateral expression of the MV
glycoproteins favors virus spread in epithelia.
Epithelial cells play crucial roles in diverse processes such as
water and ion balance, secretion, adsorption of nutrients, and signal
transduction. The polarized nature of these cells is central to their
function. The plasma membrane of these cells is divided into an apical
and a basolateral domain that have different lipid and protein
compositions (for review, see Ref. 1). Sorting of membrane proteins was
shown to occur in the trans-Golgi network and to depend on
special targeting signals. The most thoroughly investigated model for
epithelial polarity is the Madin-Darby canine kidney
(MDCK)1 continuous cell line.
Several viral glycoproteins have been shown to be sorted to either the
apical or basolateral surfaces (2). Since glycoproteins of enveloped
viruses perform critical functions during viral assembly and serve as
main targets of humoral immune responses, polarized expression of these
proteins affects the viral life cycle. The viral glycoproteins studied
in most detail are the vesicular stomatitis virus G protein and the
influenza virus hemagglutinin (HA). Vesicular stomatitis virus G
protein was found to contain basolateral sorting information that is
critically dependent upon the presence of a tyrosine in the cytoplasmic
tail (3). The signal responsible for the apical transport of HA is
contained within the extracellular or transmembrane portion of the
molecule (4-6). Vesicular stomatitis virus was shown to be released
mainly from the basolateral side of infected MDCK cells, whereas
influenza virus almost exclusively buds from the apical cell surface
(7). These data were the basis for the view that budding of enveloped
viruses occurs only at the site at which their envelope proteins are
mostly concentrated (2, 8, 9).
However, recent data on virus release and glycoprotein sorting
indicated that this model is not valid for measles virus (10, 11).
Measles virus (MV) is a member of the Paramyxoviridae family and
possesses a negative-stranded RNA genome encoding for six structural
proteins. The nucleoprotein together with the phosphoprotein, the viral
polymerase, and the genomic RNA form the ribonucleoprotein complex that
assembles in the cytoplasm of infected cells. The matrix protein (M
protein), synthesized in the cytoplasm, mediates the contact of the
ribonucleoproteins with viral envelope proteins initiating virus
budding at the plasma membrane (12, 13). Hemagglutinin (H protein), the
envelope protein responsible for the binding of the virus to the cell
surface receptor (14-16), is a type II integral membrane protein with
a C-terminal ectodomain. The second envelope protein, the fusion
protein (F protein), is a type I glycoprotein possessing an N-terminal
ectodomain that has to be cleaved into the F1/F2 subunits to mediate
pH-independent fusion of virus with the plasma membrane (17). We have
previously shown that both proteins are abundantly expressed on the
basolateral side of polarized MDCK cells, although virus budding is
restricted to the apical cell surfaces (10). This finding was recently confirmed using another polarized cell line, CaCo2 cells (11). Furthermore, we have demonstrated that upon stable expression in MDCK
cells, both H and F proteins are targeted almost completely to the
basolateral surface (10). Thus, targeting information resides in the
proteins itself. Since MV glycoproteins do not determine the site of
virus budding, MV must have developed a maturation strategy different
from those of viruses such as influenza virus or vesicular stomatitis
virus. Most recent work has shown that MV M protein may specify apical
virus release because it is predominantly localized at the apical
plasma membrane of polarized MDCK2 and CaCo2 cells
(11).
The aim of this study was to analyze the signals responsible for the
basolateral sorting of H and F proteins to elucidate the importance of
the basolateral glycoprotein expression for the pathogenesis of measles
virus infection. We show that polarized targeting of both proteins is
dependent upon a tyrosine residue in the cytoplasmic tails. The same
tyrosines are shown to be part of an endocytosis signal. Since the
signals in the F protein cytoplasmic tail appeared to be more
efficiently recognized than those of the H protein, they were
transferred to another protein. Chimeric proteins from MV F protein and
influenza virus HA revealed that the signals residing in MV F protein
can only partly override the targeting signals of an apical protein
that is not internalized. To analyze the biological importance of the
signals residing in the cytoplasmic tails of the MV envelope proteins,
the polarized transport and endocytosis of the glycoproteins in
infected cells were monitored. Endocytosis, but not polarized
transport, appeared to be inhibited. Fusion of polarized MDCK cells was
observed only when basolateral sorting signals of both glycoproteins
were intact. This result led us to suggest that endocytosis interferes
with the assembly of new viruses at the plasma membrane, whereas
basolateral expression of the MV glycoproteins favors virus spread in
epithelial cells.
Cell Culture--
MDCK cells (strain II) were grown in Eagle's
minimal essential medium (Life Technologies, Inc.) supplemented with
10% fetal calf serum (Life Technologies, Inc.), 100 units/ml
penicillin, and 100 µg of streptomycin. For studies of cell polarity,
tissue culture-treated 0.4-µm pore size Transwell polycarbonate
filters (Costar Corp., Cambridge, MA) were used. Cells were seeded 5 days before experiments (2 × 106 cells/24-mm unit).
The polarity was determined by measurement of the transepithelial
resistance using a Millipore ERS instrument. MDCK cells formed a tight
monolayer with an electrical resistance of 1000-2500 ohms × cm2. The different patterns of surface proteins on the
apical and basolateral membranes of the polarized cell line were
controlled by surface biotinylation.
Plasmid Constructs and Stable Expression in MDCK
Cells--
Cloning of the viral glycoprotein (H and F protein) genes
into the expression vector pCG under the control of the cytomegalovirus early promotor has been described previously (18). The glycoprotein mutants were prepared by introduction of site-specific mutations with
the complementary primers F549Y/A 1 (5'-GGAACATCAAAATCCGCTGTAAGGTCGCTCTGATCC-3') and
F549Y/A 2 (5'-GGATCAGAGCGACCTTACAGCGGATTTTGATGTTCC-3') and H12Y/A 1 (5'-CGGATAAATGCCTTCGCCAAAGATAACCCC-3')
and H12Y/A 2 (5'-GGGGTTATCTTTGGCGAAGGCATTTATCCG-3') in the
double-stranded pCG plasmids using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The chimeras used in this
study are marked by their composition (ectodomain-transmembrane domain-cytoplasmic tail) as H7-H7-F, H7-F-H7, and H7-F-F. H7 represents the respective domain of the influenza virus hemagglutinin, and F
represents the respective domain of the measles virus fusion protein.
The recombinant proteins H7-H7-F and H7-F-F were generated by a
recombinant polymerase chain reaction technique (19) using synthetic
oligonucleotides and pSG5new-HAmut7 and pCG-F as templates. The mutant
hemagglutinin HAmut7 of the influenza A virus has been described
elsewhere (20). The internal sense primers were
5'-CTTGTTTTCATATGTGTGAGGGGGCGTTGTAATAAA-3' (H7-H7-F) and
5'-GAGTAGTGGCTACAAAGATGGTTTATCGAGCACTAGC-3' (H7-F-F), and the internal
antisense primers were
5'-TTTATTACAACGCCCCCTCACACATATGAAAACAAG-3' (H7-H7-F) and
5'-GCTAGTGCTCGATAAACCATCTTTGTAGCCACTACTC-3'(H7-F-F). The external
primers were 5'-ATACTTATGATCACAGCAAAT-3' (sense) and
5'-GGCGGCGCGCAGATCTTGTGTTTCAAGAGTTGTAGAGG-3' (antisense). pSG5new-H7-F-F and pSG5new-HAmut7 were used to produce H7-F-H7 by the
same recombinant polymerase chain reaction technique with the internal
primers 5'-GCTTTAATATGTTGCTGCAAGAACGGAAACATGCGG-3' (sense) and
5'-CCGCATGTTTCCGTTCTTGCAGCAACATATTAAAGC-3' (antisense) and the external
primers 5'-ATACTTATGATCACAGCAAAT-3' (sense) and 5'-GGCGGCGCGCACTAGTAATAAACAAGTTCTGC-3' (antisense). The sequences of the plasmid constructs were confirmed by dideoxy sequencing. For
stable expression, MDCK cells were cotransfected with either of the
expression plasmids and the neomycin resistance-conferring plasmid pIGI
at a ratio of 1:10 using the cationic lipid transfection reagent
LipofectAMINE 2000 (Life Technologies, Inc.) according to the protocol
of the manufacturer. Cells were screened for Geneticin resistance by
addition of 1.0 mg of Geneticin (Calbiochem)/ml medium. The selected
cell clones were screened for expression of foreign proteins by
immunofluorescence analysis.
Surface Immunofluorescence Analysis--
MDCK cells stably
expressing parental proteins (FEdm and HEdm) or
tyrosine mutants (F549Y/A and H12Y/A) were
grown on glass coverslips and incubated with the monoclonal antibody
(mAb) A504 (directed against MV F protein; kindly provided by J. Schneider-Schaulies) or mAb 8905 (directed against MV H protein;
Chemicon, Temecula, CA) for 60 min at 4 °C without prior fixation.
The primary antibodies were detected using an FITC-labeled goat
anti-mouse IgG (Dako Corp.) for 45 min at 4 °C. The samples
were mounted in Mowiol and 10% 1,4-diazabicyclo(2,2,2)octane.
The coverslips were viewed and photographed with a Zeiss Axiophot
microscope equipped with UV optics.
Surface Biotinylation and Immunoprecipitation--
Filter-grown
MDCK cells were biotinylated essentially as described by Lisanti
et al. (21). Cells were washed three times with cold
phosphate-buffered saline (PBS) containing 0.1 mM
CaCl2 and 1 mM MgCl2. The apical or
basolateral cell surface was incubated twice for 20 min at 4 °C with
2 mg/ml sulfo-N-hydroxysuccinimidobiotin (Pierce) by adding
1 ml of the biotinylating reagent to the respective filter chamber. The
same volume of PBS containing 0.1 M glycine was placed into
the opposite filter chamber. After biotinylation, cells were washed
once with cold PBS containing 0.1 M glycine and three times
with cold PBS. Cells were lysed in 0.5 ml of radio immunoprecipitation
assay buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
0.15 M NaCl, 10 mM EDTA, 10 mM
iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 50 units/ml aprotinin, and 20 mM Tris-HCl, pH 8.5), followed
by centrifugation for 20 min at 100,000 × g. The
supernatant was immunoprecipitated with mAb A504 (directed against MV F
protein), mAb 8905 (directed against MV H protein), or mAb 2A11-H7
(directed against the H7 subtype of the influenza A virus
hemagglutinin; kindly provided by W. Garten) at a final dilution of
1:100. After addition of 40 µl of a suspension of protein A-Sepharose
CL-4B (Sigma) coated with rabbit anti-mouse IgG (Dako Corp.),
immunocomplexes were washed three times with radio immunoprecipitation
assay buffer and suspended in reducing (MV H protein) or nonreducing
(MV F and recombinant proteins) sample buffer for SDS-polyacrylamide gel electrophoresis. Following separation on a 10% polyacrylamide gel,
proteins were blotted onto nitrocellulose by the semidry blot
technique. After blocking of nonspecific binding sites by 5% nonfat
dry milk in PBS, blots were incubated for 45 min at 4 °C with
streptavidin-biotinylated horseradish peroxidase complex (Amersham
Pharmacia Biotech, Uppsala, Sweden) diluted 1:2000 in PBS containing
0.1% Tween 20. Biotinylated proteins were detected with the enhanced
chemiluminescence system (Amersham Pharmacia Biotech) by exposure to
Eastman Kodak XAR autoradiography film.
Antibody Uptake Assay--
MDCK cells were grown on coverslips
and transfected with either the parental (pCG-FEdm and
pCG-HEdm) or mutant (pCG-F549Y/A and
pCG-H12Y/A) protein-expressing plasmids alone or
cotransfected with plasmids containing parental proteins using the
transfection reagent LipofectAMINE 2000. A total amount of 1 µg of
DNA was used. Since infection of confluent MDCK cells has been shown to be very inefficient (10), virus was added to the cells directly after
seeding. At 7 h post-infection, the virus-containing growth medium
was replaced by fresh minimal essential medium containing 10% fetal
calf serum. At 20 h post-transfection or 30 h post-infection, surface-expressed protein was labeled with mAb A504 (directed against
MV F protein) or mAb 8905 (directed against MV H protein). After
incubation for 60 min on ice, the antibody was removed by washing with
PBS, and the cells were incubated with cell culture medium for 15 min
at 37 °C to allow endocytosis of the antigen-antibody complexes.
Internalization was stopped by rapid cooling on ice. Surface-bound
antibodies were detected by incubation with rhodamine-conjugated goat
anti-mouse Fab fragments (Dianova) at a dilution of 1:100 in PBS
for 60 min on ice. After washing with PBS, the cells were fixed and
permeabilized for 5 min at Biotin Internalization Assay--
The experiment was mainly
performed as described by Graeve et al. (22). MDCK cells
stably expressing either one of the parental or one of the mutant
proteins were grown on 6-cm plastic dishes and incubated twice for 20 min at 4 °C with a 2 mg/ml concentration of the
non-membrane-permeating, cleavable biotin derivative
sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (Pierce).
Following washing with cold PBS containing 0.1 M glycine
and several washings with cold PBS, cells were incubated with cell
culture medium for 0, 5, 15, or 30 min at either 4 or 37 °C to allow
endocytosis of the labeled proteins. After internalization was stopped
by rapid cooling on ice, cells were incubated three times for 20 min at
4 °C with 50 mM 2-mercaptoethanesulfonic acid (Sigma) in 50 mM Tris-HCl, pH 8.7, 100 mM NaCl, and 2.5 mM CaCl2. Biotin exposed at the cell surface
was thereby cleaved. After thorough rinsing with PBS containing 20 mM Hepes, cells were lysed in radio immunoprecipitation
assay buffer, and proteins were immunoprecipitated as described above.
Biotinylated proteins were separated on a 10% SDS-polyacrylamide gel
under nonreducing conditions, transferred to nitrocellulose, and
detected as described above. The internalization rate was determined by
densitometric quantification.
Cell Fusion Assays--
MDCK cells were seeded in 35-mm diameter
wells to reach 90-95% confluence 1 day after plating. The MV
glycoprotein-expressing plasmids pCG-FEdm and
pCG-HEdm were transfected in the absence or presence of
either the corresponding parental protein-expressing plasmids
(pCG-FEdm and pCG-HEdm) or the mutant
protein-expressing plasmids (pCG-F549Y/A and
pCG-H12Y/A) using the cationic lipid transfection reagent
LipofectAMINE 2000. A total amount of 5 µg of DNA was used. At
20 h post-transfection, the transiently expressing cells were
fixed with ethanol and stained with 1:10 diluted Giemsa staining
solution (Merck, Darmstadt, Germany).
To analyze the biological activity of the MV glycoproteins in polarized
MDCK cells, MDCK cells stably expressing either MV FEdm or
MV F549Y/A were seeded on coverslips at high density. At
24 h post-seeding, cells were transfected with
pCG-HEdm or pCG-H12Y/A, respectively. For
comparison, MDCK cells were infected as described above. At 7 h
post-transfection or post-infection, transfection complexes or
virus-containing growth medium was removed, and cells were incubated
for 24 h in Dulbecco's modified essential medium either with or
without calcium. Since the maintenance of tight junctions requires
calcium, the lack of calcium abolishes cell polarity. To visualize
syncytium formation in polarized or non-polarized cells, immunostaining
was performed. After fixation and permeabilization at Both H12Y/A and F549Y/A Mutant Proteins Are
Transported to the Cell Surface and Are Biologically
Active--
Cytoplasmic tyrosines have been identified as important
constituents of many, but not all, basolateral sorting signals. The cytoplasmic tails of both MV glycoproteins contain a single tyrosine at
position 12 in the H protein and at position 549 in the F protein. To
determine if these tyrosines residues contribute to a basolateral targeting signal, mutants were constructed in which amino acid 12 of
the H protein and amino acid 549 of the F protein were changed to an
alanine. The sequences of parental proteins and mutants H12Y/A and F549Y/A (tyrosine mutants) are shown
in Fig. 1A. Successful use of
site-specific mutagenesis to identify amino acids serving as
recognition signals during intracellular transport requires that local
changes do not disturb other, perhaps distant, parts of the protein,
resulting in inefficient transport or loss of function. Without prior
fixation, surfaces of transiently expressing MDCK cells were incubated
with anti-H protein (mAb 8905) or anti-F protein (A504) antibodies and
an FITC-conjugated secondary antibody. The surface immunofluorescence
in Fig. 1B shows that the exchange of the tyrosine did not
prevent surface transport. A common assay for testing the biological
function of H and F proteins is to analyze cells coexpressing H and F
proteins for their ability to induce syncytium formation. Only
fusion-competent F proteins in combination with H proteins that bind to
MV receptors and that can interact with the F protein support
virus-to-cell or cell-to-cell fusion (23). To analyze the mutants for
syncytium formation, subconfluent MDCK cells coexpressing either
parental HEdm or H12Y/A in combination with
parental FEdm or F549Y/A were fixed at 20 h post-transfection and stained with Giemsa staining solution. As shown
in Fig. 1C, H or F proteins expressed alone did not induce fusion, whereas syncytium formation was observed in cells expressing different combinations of parental and mutant proteins. This result indicates that tyrosine mutants were not affected in their ability to
mediate fusion of non-polarized cells.
Both Tyrosine Mutants Are Re-targeted in Polarized Epithelial
Cells--
To analyze the polarized transport of membrane proteins,
stably expressing cell lines have to be established. For this purpose, plasmids expressing either the parental HEdm or
FEdm protein or tyrosine mutants were transfected into MDCK
cells, and neomycin-resistant transfectants were selected using G418.
To monitor the targeting of the expressed proteins, the cells were
cultured on permeable filter supports, where they form polarized
monolayers. Cells grown on filters were cooled to 4 °C, and either
the apical or basolateral surface proteins were labeled by adding the
non-membrane-permeating reagent
sulfo-N-hydroxysuccinimidobiotin to the respective filter chamber. The cells were lysed, and H or F proteins were
immunoprecipitated by specific antibodies (mAb 8905 or A504). After
separation on a 10% SDS-polyacrylamide gel (reducing conditions for H
proteins and nonreducing conditions for F proteins), proteins were
transferred to nitrocellulose. Biotin-labeled proteins were detected
with peroxidase-conjugated streptavidin. As shown in Fig.
2 (lanes b), efficient
biotinylation of both parental proteins was obtained only after
labeling the cells from the basolateral side (HEdm, 95%;
and FEdm, >99%). This confirms our previous results that both MV glycoproteins are predominantly expressed on the basolateral surface of polarized MDCK cells. In contrast, strong labeling of both
tyrosine mutants was detected only after apical surface biotinylation
(F549Y/A and H12Y/A
panels, lanes a). Only a minor band was found
after basolateral labeling (lanes b). The protein
distribution was further confirmed by surface immunofluorescence of
filter-grown cells using a confocal laser scanning microscope (data not
shown). The redirection of the mutant proteins to the apical cell
membrane indicates that both MV glycoproteins contain a
tyrosine-dependent basolateral sorting signal.
The Tyrosine Responsible for Basolateral Transport of H and F
Proteins Also Mediates Endocytosis--
In certain cases, the
cytoplasmic signal responsible for efficient localization to coated
pits may be very similar to that responsible for basolateral targeting
(24-26). To determine if MV glycoproteins also possess an endocytosis
signal, we performed an antibody uptake experiment. Without prior
fixation, H or F proteins on the surface of stably expressing cells
were labeled with the respective antibodies at 4 °C and warmed to
37 °C for 15 min to allow endocytosis to occur. Surface-bound
antibodies were detected by incubation of the living cells with a
rhodamine-conjugated second antibody at 4 °C. After permeabilization
of the cells, internalized H or F protein-antibody complexes were
detected with FITC-conjugated second antibody. In Fig.
3A, the result of the double
immunofluorescence (surface and intracellular) is
shown. Cells expressing parental HEdm and FEdm
proteins showed both surface fluorescence and numerous small
fluorescent intracellular vesicles distributed throughout the cell.
Both MV glycoproteins were internalized during 15 min at 37 °C,
indicating that both proteins possess an endocytosis signal. Since the
antibody uptake experiment is only a qualitative test, we performed a
biotin internalization assay (see "Experimental
Procedures"). With this assay, it can be determined at what rate MV
proteins are internalized and whether mutation of tyrosine 12 in the H
protein and tyrosine 549 in the F protein affects endocytosis. In Fig.
3C, quantitation of the blots shown in Fig. 3B
revealed that the FEdm protein was endocytosed at a higher
rate than the HEdm protein (3 versus 2.5%/min).
Since the antibody uptake experiment is only a qualitative assay, this difference could not be detected. The amount of intracellular F protein
increased with increasing incubation at 37 °C. In contrast, the
amount of intracellular H protein decreased after 15 min of endocytosis. The same reduction was also observed when the total amount
(surface and intracellular H proteins) was determined (data not shown).
Thus, the decrease was due to intracellular degradation. The
endocytosis rate of the tyrosine mutants was significantly lower than
that of parental proteins, indicating that the tyrosines responsible
for the basolateral transport are also responsible for endocytosis of H
and F proteins. Tyrosine mutants are internalized at a slow rate
(<1%/min) that is more characteristic of bulk uptake during membrane
turnover than of active clustering into endocytotic vesicles.
Construction and Analysis of Chimeric Proteins from MV F Protein
and the H7 Protein of Influenza Virus--
In infected MDCK cells, the
F protein was found to be targeted to the basolateral surface, as found
in cells singly expressing FEdm (10). Basolateral transport
of the H protein was found to be less stringent in infected cells.
Furthermore, endocytosis of MV F protein was more efficient than was
internalization of the H protein. Taken together, the F protein
cytoplasmic tail appeared to have stronger transport signals. To know
whether this signal functions as an autonomous sorting sequence, we
wanted to determine whether it is able to redirect an apical membrane protein to the basolateral cell surface. For this purpose, we chose the
hemagglutinin (H7) protein of fowl plaque virus, an avian influenza A
virus. The apical localization of this protein is well documented (7,
27). Furthermore, it was shown that it is excluded from coated pits
(28). Therefore, foreign sequences introduced into the H7 protein can
be analyzed not only for basolateral targeting information, but also
for their ability to mediate endocytosis through coated pits. For the
construction of chimeras, an H7 mutant was used in which the
recognition site for proteolytic cleavage by furin-like enzymes was
destroyed. As we described previously (29), establishment of cells
stably expressing the H7 protein is facilitated when the protein is not
cleaved intracellularly and thus is unable to mediate cell-to-cell
fusion. The chimeric proteins used in this study are shown in Fig.
4A. In the H7-H7-F chimera,
the 11-amino acid tail of the H7 protein was replaced with the 33 amino
acids of the cytoplasmic portion of MV F protein. In the H7-F-F
chimera, the transmembrane and cytoplasmic domains of the H7 protein
were exchanged with the respective domains of MV F protein. In the
H7-F-H7 chimera, only the transmembrane domain of the H7 protein was
replaced with the corresponding portion of MV F protein. For all
constructs, stably expressing MDCK cell lines were established. The
surface-expressed chimeric proteins were analyzed whether they were
present on the apical or basolateral domain of the plasma membrane. For
this purpose, cells were cultured on filters and subjected to
domain-specific biotinylation as described above for the tyrosine
mutants. Following immunoprecipitation with mAb 2A11-H7, the proteins
were analyzed by SDS-polyacrylamide gel electrophoresis, and
biotinylated proteins were detected with peroxidase-conjugated
streptavidin. As shown in Fig. 4B, the H7 wild-type protein
(>99%) as well as H7-H7-F (98%) were predominantly expressed on the
apical cell surface of MDCK cells. H7-F-H7 was found almost equally
distributed on both cell surfaces (60% apical and 40% basolateral).
Only when both the cytoplasmic tail and the transmembrane domain of MV
F protein were transferred to the luminal domain of the H7 protein
(H7-F-F) the protein was re-targeted to the basolateral side (>95%).
This result indicates that the cytoplasmic tail containing the tyrosine
critical for the basolateral transport of MV F protein is not
sufficient to redirect the apical H7 protein. The additional
replacement of the transmembrane domain appeared to be required either
for the recognition of the basolateral targeting signal in the F
protein cytoplasmic tail or to provide a second basolateral
targeting signal. To assay how far the endocytosis signal can be
transferred from the F to H7 protein, cells stably expressing chimeric
molecules were analyzed by a biotin internalization assay in which
endocytosis was allowed to occur for 5-25 min. After
immunoprecipitation using an H7 protein-specific antibody, the samples
were separated by SDS gel electrophoresis, and endocytosed biotinylated
protein was detected after blotting onto nitrocellulose with
peroxidase-conjugated streptavidin. In Fig. 4C,
quantification of the endocytosis is shown. As described by Lazarovits
et al. (28) for the H2 hemagglutinin, the H7 protein was not
internalized during 25 min at 37 °C. In contrast, endocytosis of all
chimeras could be measured; but none of the proteins was internalized
at a rate significantly higher than that of F549Y/A or the
bulk of the plasma membrane. Uptake of the H7 chimera containing both the MV F transmembrane and cytoplasmic domains (H7-F-F) was found to be
only slightly enhanced in comparison with H7-F-H7 and H7-H7-F. This
result indicates that the endocytosis signal in the F protein cytoplasmic tail is not recognized in the H7 protein context even if
the transmembrane domain of the F protein is present at the same
time.
Apical Expression of the MV Glycoproteins Prevents Syncytium
Formation in Epithelial Cells--
To elucidate the importance of the
basolateral sorting of glycoproteins H and F for MV spread, we analyzed
whether inactivation of the targeting signal affects the biological
activity of MV glycoproteins in polarized MDCK cells. In polarized cell
monolayers, only lateral cell membranes can fuse. Therefore, fusogenic
proteins must be expressed on the basolateral cell surface to induce
fusion (30). Apically expressed proteins are unable to mediate fusion. Thus, we examined whether replacement of the tyrosines in the cytoplasmic tails of H and F proteins affects the syncytium formation in polarized cell monolayers. For this purpose, cells stably expressing either FEdm or F549Y/A were grown to
over-confluency. The cells that had already established a polarized
phenotype were then transfected either with the parental
HEdm protein or with the mutant H12Y/A protein.
As a control, MDCK cells were infected with MV. At 7 h
post-infection or post-transfection, the culture medium was removed,
and cells were grown for 24 h either in normal growth medium or in
calcium-depleted medium. In the absence of calcium, tight junctions
could not be maintained or formed, resulting in non-polarized cells in
which apical and basolateral surfaces were no longer separated. At
30 h post-transfection or post-infection, cells were fixed and
stained with an H protein-specific antibody and an FITC-conjugated
secondary antibody. Immunofluorescence was used to monitor cell-to-cell
fusion since syncytium formation in over-confluent MDCK cell monolayers
is difficult to detect by staining with Giemsa staining solution. As
expected, all samples demonstrated large syncytia in the absence of
calcium (Fig. 5, Endocytosis of the MV Glycoproteins Is Inhibited in Virus-infected
Cells--
The basolateral sorting signals were shown to be functional
in virus-infected MDCK cells (10). To analyze whether endocytosis signals function also in infected cells, we analyzed the endocytosis of
the MV glycoproteins by an antibody uptake experiment as described above. None of the glycoproteins was found to be endocytosed to a
detectable amount (Fig. 6A).
This observation indicates that internalization of H and F proteins is
prevented in infected cells. In contrast, cells cotransfected with
plasmids containing HEdm and FEdm showed
internalization of both glycoproteins (Fig. 6B). Since
internalization was also observed in coexpressing cells having already
formed syncytia, lack of endocytosis in infected cells cannot be
explained by general cell damage induced by cell fusion. This is
further supported by the observation that neither endocytosis of a
fluid-phase marker (FITC-dextran) nor endocytosis of a constitutive
cellular membrane protein (CD4613F/Y) (25) is affected in
virus-infected cells (data not shown).
We have shown here that both MV envelope proteins possess
targeting signals that critically depend upon a tyrosine residue in the
cytoplasmic tails. Stable expression of the MV glycoproteins in MDCK
cells showed that the tyrosine residues are involved both in
basolateral transport and in endocytosis. In MV-infected cells, recognition of the sorting signals of F and H proteins is differently regulated. Whereas endocytosis appeared to be abolished, basolateral transport was not substantially prevented; and thus, efficient fusion
of polarized epithelia was observed. This suggests that the basolateral
signals have biological importance for virus replication in epithelia.
Both MV Glycoproteins Possess Overlapping Signals for Endocytosis
and Basolateral Transport--
Most cellular transmembrane proteins
are internalized together with the bulk of the plasma membrane; others
are destined to be rapidly endocytosed and are concentrated in
clathrin-coated pits (31-33). Rapid internalization is known to be
mediated by the cytoplasmic domain containing a structural motif that
includes one or more aromatic residues, generally a tyrosine. In
polarized cells, proteins containing an endocytosis signal are
generally transported to the basolateral membrane. Motifs responsible
for internalization and polarized targeting appear to share common Sorting Signals in the F Protein Cytoplasmic Tail Lack
Functionality in HA-F Protein Chimeras--
The properties of
influenza virus HA completely differ from those of the MV
glycoproteins. HA is preferentially expressed on the apical surface of
epithelial cells and is internalized 40 times more slowly than is the
bulk of the plasma membrane (33). A mutant that is rapidly endocytosed
and targeted to the basolateral cell surface could be generated by
replacement of cysteine 543 with a tyrosine residue in the short
cytoplasmic tail of HA (HA-Y543) (38, 39). Just as the MV
glycoprotein sorting signals, the YRIC motif from the HA-Y543 mutant
does not follow the YXXØ or NPXY pattern. MV F
protein and HA-Y543 have an additional feature in common. In both
proteins, the tyrosine responsible for endocytosis and basolateral
transport is located at position Endocytosis, but Not Basolateral Targeting, of MV Glycoproteins Is
Prevented in Virus-infected Cells--
Given the importance of
cell-associated viral glycoproteins in mediating cytopathic effects and
as targets of host immune responses, endocytosis of MV envelope
proteins may be a regulatory mechanism to prevent extensive expression
of viral antigen on the cell surface. A likely explanation for the lack
of endocytosis in MV-infected cells is that interaction of the H and F
protein cytoplasmic tails with the viral matrix protein initiating
assembly of new virions (13, 41) prevents the interaction with cellular adaptor complexes. We propose that MV has developed a system that removes those H and F proteins from the cell surface that are not
associated with the M protein and are therefore not destined for
incorporation into virions. This view is supported by our observation3 that both MV
glycoproteins were efficiently endocytosed in cells infected with
recombinant MV lacking the M protein (MV
In contrast to endocytosis, we found the basolateral transport of MV
glycoproteins not to be severely impaired in infected cells. We have
reported previously that both glycoproteins are expressed on the
basolateral surface of infected MDCK cells (10). Here we show that
cell-to-cell fusion of polarized MDCK cells occurs both in
virus-infected cells and in cells coexpressing H and F proteins in the
absence of other viral proteins. The ability to mediate fusion
critically depends upon intact targeting signals in both proteins. We
propose that the presence of H and F proteins on the basolateral
surface promotes spread of virus infection from cell to cell. Although
it has been recently reported that the M protein can partially
re-target the glycoproteins to the apical membrane of polarized CaCo2
cells (11), a sufficient amount of glycoprotein appeared to reach the
basolateral membrane to induce fusion.
In summary, MV regulates the recognition of its signals for endocytosis
and basolateral targeting by differential interaction with other viral
components. In this way, MV glycoproteins can have a
tyrosine-dependent endocytosis and sorting signal that permits polarized transport, but prevents internalization of
glycoproteins during infection of epithelial cells. As virus release
was shown to occur only at the apical cell surface, basolateral
glycoprotein expression may be required for MV to spread from the
primarily infected epithelia to underlying tissues by direct
cell-to-cell fusion. In the absence of viral core proteins,
glycoprotein expression on the cell surface is down-regulated by
endocytosis, thereby preventing the exposure of viral antigens on the
cell surface. Although just one component of the complex biology of the
virus, it is clear from the experiments described here that the
tyrosine-dependent signals contribute to the cytopathic
properties of MV in culture and may also be important for the
pathogenesis in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C with methanol/acetone (1:1).
Internalized antibodies were detected with FITC-labeled goat anti-mouse
IgG at a dilution of 1:100. The samples were mounted in Mowiol
containing 10% 1,4-diazabicyclo(2,2,2)octane and analyzed using a
Zeiss Axiophot microscope equipped with UV optics.
20 °C with
methanol/acetone (1:1), cells were incubated with monoclonal antibody
8905 (directed against MV H protein) and an FITC-labeled goat
anti-rabbit IgG (Dako Corp.). The samples were mounted and analyzed as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, sequences of the cytoplasmic domains
of parental and mutant H and F proteins. Protein sequences are shown in
single-letter code. The vertical lines separate the
transmembrane sequences from those predicted to be in the cytoplasm.
TM, transmembrane domain; CD, cytoplasmic domain.
B, surface expression of parental and mutant glycoproteins.
MDCK cells transiently expressing either the parental (HEdm
and FEdm) or mutant (H12Y/A and
F549Y/A) proteins were incubated with either anti-H or
anti-F protein monoclonal antibodies at 4 °C without prior fixation.
Surface-bound antibodies were detected with FITC-conjugated anti-mouse
immunoglobulins. C, fusion activity of parental and mutant
proteins. HEdm and FEdm were transiently
expressed in the absence or presence of either the corresponding
parental protein (FEdm and HEdm) or mutant
protein (F549Y/A and H12Y/A). At 20 h
post-transfection, cells were fixed and stained with Giemsa staining
solution.
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Fig. 2.
Surface distribution of parental and mutant
proteins. MDCK cells stably expressing HEdm,
H12Y/A, FEdm, or F549Y/A were grown
on filters and surface-biotinylated from either the apical (lanes
a) or basolateral (lanes b) side. After cell lysis,
proteins were immunoprecipitated with an H or F protein-specific
monoclonal antibody. Precipitates were analyzed by SDS gel
electrophoresis, transferred to nitrocellulose, and probed with
peroxidase-conjugated streptavidin.
View larger version (40K):
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Fig. 3.
A, internalization of antibodies bound
to MV glycoproteins. Living MDCK cells expressing either
HEdm or FEdm were incubated with an H or F
protein-specific antibody at 4 °C. After 60 min, cells were warmed
to 37 °C to allow endocytosis to occur. After 15 min, surface-bound
antibodies were detected by incubation at 4 °C with a
rhodamine-conjugated anti-mouse serum (surface panels).
After permeabilization of the cells, internalized antibodies were
stained with an FITC-conjugated anti-mouse serum (intracellular
panels). B, endocytosis of parental and mutant
proteins. MDCK cells stably expressing HEdm,
H12Y/A, FEdm, or F549Y/A were
surface-labeled with sulfosuccinimidyl
2-(biotinamido)ethyl-1,3'-dithiopropionate at 4 °C. Cells were
shifted to 37 °C for the times indicated to allow endocytosis to
occur. Subsequently, cell-surface proteins were either reduced with
2-mercaptoethanesulfonic acid (MESNA) at 4 °C (+) or left
untreated ( ). After cell lysis, proteins were immunoprecipitated with
an H or F protein-specific antibody. The precipitates were separated by
SDS gel electrophoresis under nonreducing conditions and transferred to
nitrocellulose. Biotinylated proteins were detected with
peroxidase-conjugated streptavidin. C, rate of
internalization of parental and mutant proteins. The percentage of
internalized protein measured in the experiment shown in Fig.
3B is plotted as a function of the time that cells were
incubated at 37 °C before reduction with 2-mercaptoethanesulfonic
acid in the cold.
, FEdm;
, F549Y/A;
,
HEdm;
, H12Y/A.
View larger version (26K):
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Fig. 4.
A, diagram of the chimeric and parental
MV F and influenza H7 proteins. MV F protein is shown in gray
boxes, and the H7 protein is shown in white boxes. The
luminal (LD), transmembrane (TM), and cytoplasmic
(CD) domains are not drawn to scale. B,
apical/basolateral distribution of the chimeric proteins on the surface
of MDCK cells. MDCK cells stably expressing H7, H7-H7-F, H7-F-H7, or
H7-F-F were grown on filters and surface-biotinylated from either the
apical (lanes a) or basolateral (lanes b) side.
Cells were lysed, and proteins were immunoprecipitated with an H7
protein-specific antibody. Precipitates were separated on a 10% SDS
gel, blotted onto nitrocellulose, and probed with peroxidase-conjugated
streptavidin. C, internalization rate of chimeric proteins.
Stably expressing MDCK cells were surface-biotinylated. The
internalization assay was performed as described in the legend to Fig.
3. The percentages of endocytosed H7 protein ( ), H7-H7-F (
),
H7-F-H7 (
), and H7-F-F (
) are depicted.
Ca
panels). In contrast, fusion of polarized cells cultured in normal growth medium (+Ca panels) was observed only when
parental HEdm and FEdm proteins were
coexpressed either in transfected or infected cells (MV and
FEdm+HEdm panels). In
polarized MDCK cells expressing a tyrosine mutant in combination with a
parental protein (F549Y/A+HEdm
and FEdm+H12Y/A panels), fusion was largely prevented. This result indicates
that the presence of a basolateral targeting signal in the cytoplasmic tails of H and F proteins has significant consequences for cell-to-cell fusion in polarized cells and thus for the phenotype of MV-infected cells.
View larger version (71K):
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Fig. 5.
Syncytium formation in polarized MDCK
cells. Transient coexpression of glycoproteins H and F was
achieved either after infection with MV or after transfection. For
transfection, confluent MDCK cells stably expressing FEdm
were transfected with a plasmid encoding for either the parental
HEdm protein
(FEdm+HEdm panels) or
the mutant H12Y/A protein
(FEdm+H12Y/A panels).
Cells stably expressing mutant F549Y/A were transfected
with DNA encoding for HEdm
(F549Y/A+HEdm panels).
At 7 h post-transfection or post-infection, culture media were
exchanged with either calcium-containing (+Ca panels) or
calcium-deficient ( Ca panels) medium. After
incubation for 24 h, syncytia were visualized by indirect
immunofluorescence using an anti-H protein monoclonal antibody and an
FITC-conjugated second antibody.
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Fig. 6.
Endocytosis of MV glycoproteins in infected
or coexpressing cells. At 30 h post-infection, MV-infected
cells (A) or at 20 h post-transfection, cells
transfected with pCG-HEdm and pCG-FEdm
(B) were incubated with an H protein-specific
(HEdm panels) or an F protein-specific
(FEdm panels) antibody at 4 °C. After
60 min, endocytosis was allowed to proceed for 15 min at 37 °C.
Surface-bound antibodies were detected by incubation at 4 °C with a
rhodamine-conjugated anti-mouse serum (surface panels).
Internalized antibodies were stained after permeabilization of the
cells with an FITC-conjugated anti-mouse serum (intracellular
panels). Arrows indicate syncytia.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-turn structures with consensus sequences involving a 4-amino acid
motif: either YXXØ (where X is any amino acid
and Ø is an amino acid with a bulky hydrophobic side chain) or
NPXY (for review, see Ref. 26). It has been shown that
interaction with specific recognition molecules, the adaptor complexes,
leads to a selective recruitment of cargo proteins into either
endosomes or lysosomes or to the basolateral cell membrane (Refs. 26
and references therein). Even when basolateral sorting signals that
depend on a tyrosine overlap or are collinear with internalization
signals, the two sorting processes are sensitive to different
characteristics of the sequence (34, 35). Furthermore, the position of
the signal in the cytosolic domain and the presence of other signals in
this domain may contribute to differential interactions with the
adaptor complexes (29). Both MV glycoproteins contain a tyrosine in the
cytoplasmic tail. Since the sequences around do not properly fit the
YXXØ or NPXY motif, they possess only
"degenerated" signals. Recognition of such signals cannot be
predicted with confidence. Examples are known of basolateral proteins
that appeared to possess a "tyrosine" internalization signal, but
that have been shown to function poorly or not at all (3, 34, 36, 37).
Thus, signals for polarized transport and endocytosis do not
necessarily overlap. Here, we showed that in the MV glycoproteins, they
do. Mutation of the critical tyrosines abolished basolateral transport
and rapid internalization, suggesting that the sequences around the
tyrosines are involved in forming a
-turn.
5 from the cytoplasmic end of the
proteins. Nevertheless, transfer of the F protein cytoplasmic tail with
a tyrosine at position
5 to the HA protein (H7-H7-F) resulted neither
in efficient endocytosis nor in re-targeting to the basolateral
membrane. The lack of functionality cannot be explained by a more
degenerated signal (YXXS in H7-H7-F versus YXXC in HA-Y543) since an HA mutant (HA-Y543/S546)
with a serine instead of cysteine (YXXS) was endocytosed
efficiently (40). This indicates that other information than only a
tyrosine in the correct position is required to override the intrinsic
properties of HA. HA was only re-targeted by MV F protein sequences
when both the transmembrane and cytosolic portions of the F protein were transferred to the luminal portion of HA (H7-F-F). From this it
follows that the tyrosine-dependent targeting signal in the F protein cytoplasmic tail either is overridden by a counteracting apical signal in the HA transmembrane domain or requires an additional supporting signal in the transmembrane region of the F protein. This
view is supported by the observation that the F protein transmembrane domain alone (H7-F-H7) could not redirect HA to the basolateral surface, although it abolished the strict apical localization of the
chimera. In contrast to basolateral transport, the F protein cytosolic
portion was unable to mediate efficient endocytosis even in the
presence of the F protein transmembrane domain. Thus, we conclude that
the internalization signal in the F protein cytoplasmic tail functions
only in the context of the authentic protein.
M) (12). Similar mechanisms
to regulate the expression of viral envelope proteins by interaction
with viral core proteins have been proposed for Sendai virus and the
human immunodeficiency virus (42, 43). In contrast, glycoproteins from
the simian immunodeficiency virus and simian virus 5 have been found to
be internalized even in the presence of viral core proteins (44, 45).
This points out that the biological relevance of endocytosis signals in
viral envelope proteins cannot be predicted, but has to be analyzed in
the context of virus infection.
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ACKNOWLEDGEMENTS |
---|
We thank J. and S. Schneider-Schaulies, W. Garten, and R. Cattaneo for kindly providing monoclonal antibodies and cloned MV genes.
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FOOTNOTES |
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* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to A. M.).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: Inst. für Virologie, Robert-Koch-Str. 17, D-35037 Marburg, Germany. Tel.: 49-6421-2865146; Fax: 49-6421-2868962; E-mail: maisner@mailer.uni-marburg.de.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M010183200
2 P. Riedl, M. Moll, H.-D. Klenk, and A. Maisner, submitted for publication.
3 M. Moll, and A. Maisner, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: MDCK, Madin-Darby canine kidney; HA, influenza virus hemagglutinin; MV, measles virus; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.
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