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Journal of Virology, December 2001, p. 11384-11391, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11384-11391.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Matrix and Fusion Proteins in Budding of
Sendai Virus
Toru
Takimoto,1,*
K. Gopal
Murti,1
Tatiana
Bousse,1
Ruth Ann
Scroggs,1 and
Allen
Portner1,2
Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
38105,1 and Department of Pathology, The
Health Science Center, University of Tennessee
Memphis, Memphis,
Tennessee 381632
Received 31 May 2001/Accepted 5 September 2001
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ABSTRACT |
Paramyxoviruses are assembled at the surface of infected cells,
where virions are formed by the process of budding. We investigated the
roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that
contained M, as was previously observed with human parainfluenza virus
type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA
caused the release of virus-like particles bearing surface F, although
their release was less efficient than that of particles bearing M
protein. Cells that expressed only hemagglutinin-neuraminidase (HN)
released no HN-containing vesicles. Coexpression of M and F proteins
enhanced the release of F protein by a factor greater than 4. The
virus-like particles containing F and M were found in different density
gradient fractions of the media of cells that coexpressed M and F, a
finding that suggests that the two proteins formed separate vesicles
and did not interact directly. Vesicles released by M or F proteins
also contained cellular actin; therefore, actin may be involved in the
budding process induced by viral M or F proteins. Deletion of
C-terminal residues of M protein, which has a sequence similar to that
of an actin-binding domain, significantly reduced release of the
particles into medium. Site-directed mutagenesis of the cytoplasmic
tail of F revealed two regions that affect the efficiency of budding:
one domain comprising five consecutive amino acids conserved in SV and
hPIV1 and one domain that is similar to the actin-binding domain
required for budding induced by M protein. Our results indicate that
both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.
 |
INTRODUCTION |
Enveloped viruses possess a membrane
that encases the viral nucleocapsid or core, which contains the viral
genome. The membrane and its viral glycoproteins are acquired during
the final stage of viral assembly and budding. Sendai virus (SV), a
prototype of the Paramyxoviridae, is a pleomorphic enveloped
virus whose membrane has the hemagglutinin-neuraminidase (HN) and
fusion (F) glycoproteins embedded on its outer surface and matrix (M)
protein embedded on its inner surface. The M proteins interact
with each other at the inner surface of the lipid bilayer to form a
sheet that interacts with viral nucleocapsid and glycoproteins
(26). The M protein can promote vesiculation of the
membrane and release of M-containing particles into the extracellular
medium without the aid of other viral proteins (16, 19).
When M is coexpressed with homologous viral nucleoprotein (NP),
vesicles containing M and nucleocapsid-like structures are produced
(6). It is therefore believed that the M protein
orchestrates the budding of paramyxovirus by acting as a bridge between
the nucleocapsid and the plasma membrane.
There is also evidence that the cytoplasmic domains of spike
glycoproteins play a crucial role in virus budding and assembly at the
plasma membrane. For example, the particle formation of a rabies virus
mutant that is deficient for glycoprotein G was enhanced 6- and 30-fold
in the presence of tailless G or G, respectively (19).
Also, the budding of influenza virus encoding tailless hemagglutinin
and neuraminidase proteins was found to be significantly impaired (15). In paramyxoviruses, truncation of the
cytoplasmic tail of HN of simian virus 5 or SV HN or F caused
inefficient release of progeny virus particles from infected cells
(11, 32).
The cellular cytoskeleton has also been reported to play an important
role in the assembly of paramyxoviruses. Cytoskeletal components appear
to be directly involved in the transport of viral glycoproteins to the
assembly site (10). Cellular actin has been found in
purified preparations of paramyxoviruses (17, 23, 38),
rabies virus (21), and human immunodeficiency virus (24). The M1 protein of influenza virus colocalizes with
the actin cytoskeleton in vivo (4), and paramyxovirus M
protein and human immunodeficiency virus Gag protein bind directly to filamentous actin (F-actin) (12, 29). Despite these
findings, however, the role of actin in virus assembly and budding is
not well understood.
In this study, we investigated the ability of individually expressed SV
membrane proteins (M, F, and HN) to induce the formation of vesicles
that contain the respective proteins. Expression of SV M induced the
production of M-bearing virus-like particles, and expression of SV F
induced the production of virus-like particles bearing F spikes at
their surface. By analyzing mutant M and F proteins, we identified the
domains required for their induction of budding.
 |
MATERIALS AND METHODS |
Cells and viruses.
We cultured 293T cells (9)
in Dulbecco's modified Eagle's medium with 10% fetal calf serum. SV
was propagated in 10-day-old chicken embryonated eggs.
cDNA clones.
The M and F genes of SV
were cloned from viral RNAs by using the Titan RT-PCR system (Roche
Molecular Biochemicals, Indianapolis, Ind.). The primers were specific
for noncoding regions of the genes. The cDNAs were cloned into the
transient expression vector pCAGGS (22), and the plasmids
containing the SV M and F genes were designated
pCAGGS-SVM and pCAGGS-SVF, respectively. The creation of pCAGGS vectors
for the SV HN gene and human parainfluenza virus type 1 (hPIV1) M gene has been described elsewhere (6,
36). Mutant cDNA clones which express deletion mutants of the SV
and hPIV1 M proteins were constructed by inserting a stop codon into the respective M genes with the Transformer site-directed
mutagenesis kit (Clontech, Palo Alto, Calif.).
Mutant SV F genes were generated by using the same kit.
Detection of release of vesicles into culture medium and
immunoprecipitation of the expressed proteins.
We used 16 µg of
Lipofectamine (Life Technologies, Grand Island, N.Y.) and 2 µg of
expression vectors encoding M, F, or
HN genes to transfect 293T cells growing in six-well plates.
To obtain cells that coexpressed two proteins, we used 1 µg of each
expression vector for transfection. Cells were cultured for 24 h
and then labeled overnight with 100 µCi of
[35S]Trans-Label (ICN, Costa Mesa, Calif.) at
33°C. The culture medium was briefly clarified, and vesicles released
into the medium were then purified by ultracentrifugation at
190,000 × g for 2 h at 4°C through 4 ml of 30%
glycerol in phosphate-buffered saline (PBS). Vesicles were resuspended
in Laemmli reducing sample buffer and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
To quantify the expression of proteins in the transfected cells, we
first washed the cells with PBS and lysed them with 1
ml of TNE buffer
(10 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40,
1 mM EDTA). The
lysates were then clarified by centrifugation
at 15,000 ×
g for 10 min, and the supernatants were analyzed by
immunoprecipitation. Two microliters of a cocktail of monoclonal
antibodies (MAbs) to SV M, F, or HN were incubated with 20 µl
of
Dynabeads (Dynal, Lake Success, N.Y.) in TNE buffer at 4°C
for 30 min. The MAb-Dynabead complexes were washed with TNE buffer
and
incubated with 100 µl of cell lysate in TNE buffer at 4°C
for 30 min. The immunocomplexes were washed with TNE buffer and
analyzed by
SDS-PAGE. Proteins were quantified by using a STORM
860 imaging system
(Amersham Pharmacia Biotech, Piscataway, N.J.).
Cell surface expression of F protein.
Expression of F
protein at the cell surface was quantified by cell surface
enzyme-linked immunosorbent assay (3). Cells were
incubated with the cocktail of anti-SV F MAbs and then with horseradish
peroxidase-conjugated sheep anti-mouse immunoglobulin G (Bio-Rad,
Hercules, Calif.) in PBS containing 0.1% bovine serum albumin. The
cells were then incubated with the substrate
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). The absorbance
of the solution at 405 nm was determined by spectrophotometry.
Electron microscopy.
Vesicles in the culture
supernatants of transfected cells were prepared for analysis as
described previously (6). Briefly, 293T cells were
transfected with expression vectors containing M or
F cDNAs as described above and cultured for 2 days in
Opti-MEM (Life Technologies, Rockville, Md.). The culture medium was
then clarified by centrifugation at 15,000 × g for 10 min, and the supernatants were concentrated by centrifugation through a
Centricon 100 filter (Millipore, Bedford, Mass.). The vesicles in the
concentrated medium were adsorbed to carbon-coated grids, negatively
stained with 1% aqueous uranyl acetate, and examined with a Phillips
301 electron microscope operated at 60 kV.
Analysis of proteins in density gradient fractions.
293T
cells were infected with SV at an multiplicity of infection of 5 or
were transfected with expression vectors containing M,
F, or HN cDNAs and then were metabolically
labeled, as described above. Culture supernatants (1 ml each) were
centrifuged through 10 ml of a 5 to 40% sucrose gradient in PBS. Eight
equal fractions were then taken from the top of each sucrose layer. An
aliquot of each fraction was removed for density measurement by
refractometry (Bausch & Lomb, Rochester, N.Y.). The remainder of each
fraction was diluted with 3 ml of PBS and centrifuged at 190,000 × g for 2 h at 4°C. The pellets were resuspended in
Laemmli reducing sample buffer and analyzed by SDS-PAGE.
Western blot detection of actin in vesicles.
After
transfection with expression vectors encoding SV M or F, the cells were
metabolically labeled with [35S]Cys and
[35S]Met, and the proteins released into
culture medium were fractionated by ultracentrifugation through a 5 to
40% sucrose gradient. Proteins in the eight fractions collected from
each sample were subjected to electrophoresis and transferred to
Immobilon-P membranes (Millipore). The membranes were then incubated
with anti-actin MAb1501 (Chemicon, Temecula, Calif.). After reaction
with goat anti-mouse immunoglobulin G conjugated to horseradish
peroxidase (Bio-Rad), membranes were treated with SuperSignal West Pico
chemiluminescent substrate (Pierce, Rockford, Ill.). After
chemiluminescent signals dissipated, the membrane was exposed to Kodak
BioMax X-ray film overnight to detect 35S-labeled proteins.
 |
RESULTS |
Protein-induced release of virus-like particles into culture
medium.
We previously reported that expression of hPIV1 M protein
from cDNA induces the release of virus-like particles into culture medium, a finding that indicates that M protein plays a role in the
budding of PIV (6). To further characterize the process of
virus particle formation, we investigated the roles of the HN, F, and M
proteins in the budding process of SV. We transfected cells with
expression vectors encoding the M, F, or HN proteins and assessed their
induction of virus-like particle formation. After transfection, the
cells were metabolically labeled with [35S]Cys
and [35S]Met, and the proteins released into
culture medium were purified by ultracentrifugation through 30%
glycerol and analyzed by SDS-PAGE. As shown in Fig.
1, SV M expressed from cDNA was detected
in the culture medium, as hPIV1 M had been previously (6).
SV F protein expressed from cDNA was also detected in the culture
medium. Cells expressing SV HN, however, did not release the HN protein
into the medium, although HN was expressed abundantly in the cells.

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FIG. 1.
Release of viral proteins into culture medium. Cells
were transfected with cDNAs encoding SV M, F, or HN and were labeled
with [35S]Met and [35S]Cys for 16 h.
(A) Protein expression in transfected cells. Cells were lysed in 1 ml
of TNE buffer, and 100 µl of the clarified lysate was used for
immunoprecipitation with specific MAbs. (B) Proteins released into
culture medium were collected, purified through 30% glycerol in PBS,
and analyzed by SDS-PAGE. The upper bands observed in the material of
the M vesicles are charge-induced M aggregates (28). The
arrows indicate cellular actin.
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We next determined whether the F and M proteins released into the
medium were included in the plasma membrane-derived vesicles
or were
protein aggregates. The culture media of cells expressing
M or F were
concentrated by membrane filtration, and the samples
were examined by
electron microscopy (Fig.
2). The medium
of cells
expressing M protein contained a number of virus-like
particles
that were 50 to 150 nm in diameter (Fig.
2A), as were those
found
in the medium of cells expressing hPIV1 M (
6). The
medium of
cells expressing F protein contained round, virus-like
particles
bearing spikes at the surface (Fig.
2B and C). These
particles
were 30 to 100 nm in diameter, which is about one-third the
standard
size of SV. These results indicate that SV F protein and SV M
protein can induce budding that releases virus-like particles
into
culture medium.

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FIG. 2.
Electron micrographs of virus-like particles released
into medium. Supernatants from cultures of cells transfected with SV
M (A) or SV F (B and C) cDNAs were
concentrated by membrane filtration. The samples were adsorbed to
carbon-coated grids, negatively stained, and examined by electron
microscopy. Bars, 86 nm.
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|
Density gradient analysis of virus-like particles released into
medium.
We further characterized the virus-like particles released
from cells expressing M or F by centrifugation of the culture media on
a 5 to 40% sucrose density gradient. Eight fractions were collected from each sample, and the proteins in each fraction were analyzed by
SDS-PAGE. The proteins released from SV-infected cells were assayed for
comparison. The structural proteins derived from whole SV virions were
recovered mainly from the fractions whose densities were between 1.14 and 1.17 g/ml (Fig. 3, fractions 6 to 8).
Fractions 4 and 5 contained the M and F proteins but very little of the other structural proteins (P, HN, NP, or L). This result
suggests that the SV-infected cells released vesicles that contained
only M, only F, or both M and F, in addition to whole virus particles.

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FIG. 3.
Density gradient analysis of the vesicles released from
cells expressing viral proteins. Cells infected with SV or transfected
with cDNAs encoding M, F, or HN alone or in combination were labeled
with [35S]Met and [35S]Cys. The clarified
cell culture medium was centrifuged through a 5 to 40% sucrose
gradient. Eight fractions were collected, and the proteins in each
fraction were analyzed by SDS-PAGE.
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In the culture supernatant of cells transfected with the SV M
expression vector, most of the M protein was detected in the
fractions
whose densities were between 1.09 and 1.11 g/ml (Fig.
3, fractions 4 and 5). F-containing vesicles released from cells
transfected with SV F
cDNA were detected mainly in the fractions
with densities of 1.11 to
1.14 g/ml (Fig.
3, fractions 5 and 6).
These results and the electron
microscopic findings indicate that
the M or F proteins released into
medium were incorporated into
the lipid
membrane.
Coexpression of M and F proteins significantly enhances the release
of vesicles containing M and F.
We next compared the efficiency
with which budding was induced by coexpression of M and F proteins.
Released vesicles containing radiolabeled proteins were purified by
centrifugation through 30% glycerol (Fig.
4A). Radiolabeled proteins in cell
lysates were purified by immunoprecipitation (Fig. 4B). After SDS-PAGE analysis, the M and F proteins in each sample were quantified by using
the PhosphorImager system, and the proportions of the synthesized
proteins released into the medium were calculated. M protein was
released from cells expressing M protein much more efficiently than F
was released from F-expressing cells (52 versus 5%) (Fig. 4C).
Interestingly, the release of F protein into culture medium was
significantly enhanced in cells that coexpressed M and F proteins (Fig.
4A, lanes 3 and 5). Quantification of the proteins revealed that cells
that coexpressed F and M released F protein with an efficiency about
4.2 times that of cells expressing F alone and released M with an
efficiency about 1.4 times that of cells expressing M alone (Fig. 4C).
Although SV HN was not released into medium when expressed alone, HN
was detected in the media of cells that coexpressed HN and M (Fig. 4A,
lane 6). HN was not released from cells that coexpressed F and HN (Fig. 4A, lane 7). However, because coexpression of F resulted in the downregulation of HN expression (Fig. 4B, lane 10), we cannot evaluate
the role of coexpression of F in the budding of HN-containing vesicles.

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FIG. 4.
Efficiency of protein release from transfected cells.
Cells transfected with expression vectors containing M,
F, or HN cDNAs alone or in combination
were labeled with [35S]Met and [35S]Cys.
(A) Vesicles released into the medium were purified and analyzed by
SDS-PAGE. (B) Protein expression in cells was analyzed by
immunoprecipitation and SDS-PAGE. (C) Proteins in cell lysates and in
culture medium were quantified from SDS-PAGE gels (A and B) by using a
STORM 860 imaging system, and the efficiency of protein release was
calculated. Each value represents the mean of three independent
experiments.
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To determine whether the enhanced release of coexpressed viral membrane
proteins might have been caused by their direct interaction,
we
analyzed the distribution of the coexpressed proteins in density
fractions to investigate whether they had been included in the
same
vesicles or in separate vesicles (Fig.
3, bottom two panels).
Most of
the F-containing vesicles released from cells coexpressing
F and M were
recovered from fractions 5 and 6, the fractions that
contained F
vesicles released from cells that expressed only F.
Similarly, the
M-containing vesicles were recovered mainly from
fractions 4 and 5, which were the same fractions that contained
M released from cells
expressing only M protein. Although some
M- or F-containing vesicles
overlap in fraction 5, the distribution
patterns of M- or F-containing
vesicles obtained from coexpressing
cells were not different from the
samples obtained from cells
transfected with M or F cDNA separately.
These results suggest
that vesicles containing M and F were released
separately from
cells that coexpressed the two proteins, while we
cannot exclude
the possibility of the presence of the vesicles
containing both
M and F in fraction 5. The same result was obtained
with cells
expressing both HN and M proteins. The distribution of HN
was
similar to that of F protein and not to that of M protein (Fig.
3,
bottom
panel).
Cellular actin is involved in the formation of particles.
Purified virus-like particles induced by expression of M or F proteins
included several proteins derived from transfected cells. One of the
proteins (Fig. 1) migrated at the position of 43 kDa, which is similar
to that of the cellular actin molecule. The SV virion is known to
contain cellular actin (17), which is hypothesized to play
a role in paramyxovirus replication and assembly (7, 8,
14). Therefore, we next investigated whether the virus-like
particles containing M or F protein contained actin as well. Cells
transfected with SV M or SV F expression vectors were labeled with
[35S]Cys and [35S]Met,
and the culture supernatant was fractionated by ultracentrifugation on
a 5 to 40% sucrose gradient. Each fraction was assayed by Western blotting with an anti-actin MAb, and the membrane was exposed to film
overnight to detect the radiolabeled M or F proteins. Cellular actin
was detected in fractions 4, 5, and 6, a distribution that was
correlated well with that of M protein (Fig.
5). The same result was observed in the
supernatant of cells expressing F: most of the F protein and actin was
found in fractions 5 and 6. These results indicate that actin was
included in the vesicles that contained M or F and are consistent with
the requirement of actin for the formation of the virus-like particles.

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FIG. 5.
Cellular actin was present in vesicles that contained M
or F. Cells expressing SV M or F protein were labeled with
[35S]Met and [35S]Cys, and the clarified
cell culture supernatants were centrifuged on 5 to 40% sucrose
gradients. Eight density fractions were collected, and Western
blots of proteins in each fraction were analyzed with an anti-actin
MAb. After chemiluminescent signals dissipated, the membrane was
exposed to X-ray film overnight to detect 35S-labeled
proteins.
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The C-terminal region of SV M is required for particle
formation.
To gain insight into the budding process induced by
viral proteins, we next identified the domain of the M protein required for budding. SV M contains a C-terminal sequence similar to that of an
actin-binding domain (KLKK motif), and the sequence is conserved in SV
and hPIV1 (Fig. 6A). Because the KLKK
motif of thymosin
4 was identified by mutational studies as an
important residue in actin binding (39), we investigated
whether the C-terminal sequence that contains the KIRK sequence is
required for the budding induced by M protein. A mutant SV M(
5) cDNA
that encodes M protein lacking the C-terminal five amino acids was
constructed and expressed in cells as described above. The deletion of
the C-terminal five amino acids did not affect the level of protein
expression in the cells, but the release of the mutant M(
5) into
culture medium was significantly less than that of SV M (Fig. 6B). A
membrane flotation assay showed no difference between the two proteins in their membrane association (data not shown). It should be noted, however, that a membrane flotation assay provides association not only
with the plasma membrane but with all the membranes in transfected
cells.

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FIG. 6.
C-terminal regions of SV and hPIV1 M proteins are
required for the release of M-containing vesicles. (A) Sequences of the
C-terminal region of SV and hPIV1 M proteins. (B and C) Release of the
deletion mutant of SV M (B) or of a series of deletion mutants of hPIV1
M (C) into the culture medium was determined as described in the legend
to Fig. 1. Expression of M proteins in the transfected cells was
quantified by immunoprecipitation with an anti-M MAb.
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The M protein of hPIV1 can induce the budding of virus-like particles
(
6), and it contains a C-terminal sequence like that
of
SV. To confirm the role of C-terminal residues in budding,
we made a
series of cDNAs encoding deletion mutants of hPIV1 M
and assessed the
release of the encoded M proteins into culture
medium. Deletion of one
or two C-terminal amino acids did not
diminish the protein's release
into medium. However, deletion
of three to five C-terminal amino acids
significantly reduced
the release of protein (Fig.
6C). These results
indicate that
the C-terminal regions of SV and hPIV1 M proteins include
residues
essential to the budding of M-containing
vesicles.
Amino acids in the cytoplasmic domain of SV F are required for
particle formation.
To identify residues of the cytoplasmic domain
of SV F protein that are necessary for particle formation, we made
cDNAs that encoded SV F with deletions or site-directed mutations.
Figure 7A shows the sequence identity of
the cytoplasmic domains of SV and hPIV1 F proteins. Although the
overall homology of these two virus F proteins is very high (68%)
(20), amino acids in the cytoplasmic domain of F are not
highly conserved in the two viruses. However, there are five
consecutive amino acids (TYTLE) that are conserved between the two F
proteins, suggesting that these residues play an essential role in the
life cycles of the closely related viruses. hPIV3 and bovine PIV3 also
share a five-residue sequence (PYVLT) in the cytoplasmic tail of F. Two
amino acids (YxL) are conserved between the F proteins of all
the respiroviruses (Fig. 7A). To determine the role of these residues
in the production of F-containing vesicles, we made two deletion
mutants, SVF546 and SVF541, that encode SV F proteins that lack 19 or
24 C-terminal residues, respectively. We also created a mutant SV F
(SVFYLAA) in which alanine was substituted for Y543 and L545. SVF546,
which contained the conserved sequence (TYTLE), was easily detected in
the culture medium of cells transfected with the SVF546 cDNA, whereas
SVF541 was barely detectable. The release of SVFYLAA into culture
medium was also significantly reduced. These mutants were expressed at
the cell surface at wild-type levels (Fig. 7C). These findings suggest
that the five consecutive amino acids conserved in SV F and hPIV1 F are
required for the production of F-containing vesicles.

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FIG. 7.
Identification of SV F residues that affect budding of
F-containing vesicles. (A) Sequence of the F protein cytoplasmic
domain. SV and hPIV1 F share five consecutive amino acids (TYTLE).
Sequences of the F mutants used are also shown. (B) Release of the SV F
mutants into the medium was assessed as described in the legend to Fig.
1. (C) Surface expression of the mutant F proteins by transfected cells
was quantified by cell-surface enzyme-linked immunosorbent assay with a
cocktail of anti-F MAbs.
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Results of deletion mutant analysis of M protein indicated that the
C-terminal domain composed of basic-hydrophobic-basic-basic
amino acids
plays a role in the budding induced by M protein.
The cytoplasmic
domain of SV F contains a similar motif just beneath
the membrane
anchor region (Fig.
7A). To evaluate the role of
this motif, we created
a mutant in which alanine is substituted
for the four residues, 524 to
527. We also created a mutant that
has alanines at three residues (525 to 527) and left residue 524
unchanged, because a positively charged
residue flanking the hydrophobic
domain is important in maintaining the
correct orientation of
the glycoprotein in the membrane
(
25). The expression of these
mutants at the cell surface
was comparable to that of wild-type
F (Fig.
7C). However, the
efficiency with which the mutant proteins
were released into medium was
reduced to 13% (F525-7A) and 19%
(F524-7A) of that of wild-type F
(Fig.
7B). This finding, together
with the results of studies of M
deletion mutants, suggests that
the domain composed of basic and
hydrophobic residues is required
for the efficient budding of
virus-like particles induced by M
and F
proteins.
 |
DISCUSSION |
Viral M protein localizes at the inner surface of the plasma
membrane of infected cell and plays a central role in viral assembly and budding. When expressed alone, M proteins of hPIV1, vesicular stomatitis virus, and influenza virus can induce the formation and
release of M-containing particles into culture medium (6, 13,
16). When M is coexpressed with homologous NP, the vesicles contain both M and a nucleocapsid-like structure composed of NP (6). M protein is therefore considered to be the central
organizer in virus assembly. Induction of the formation of virus-like
particles by SV M protein, which is highly homologous to hPIV1 M (87%
identity) (28), indicates that M drives the budding
process in SV.
We found that SV F protein alone can also induce the budding and
release of virus-like particles that carry F spikes. F protein appears
to participate actively in the assembly of measles virus and SV
particles. Most measles viruses isolated from the brains of patients
with subacute sclerosing panencephalitis are defective in producing
infectious virus and have defects in the M gene, the
F gene, or both. In many viruses isolated from these
patients, the cytoplasmic domain of F is shortened, elongated, or
markedly altered (5, 31); these findings suggest that the
cytoplasmic domain of F protein is required for the assembly of measles
virus. Furthermore, in a study of recombinant SV carrying deletions in the cytoplasmic tail of F, a specific sequence in the cytoplasmic tail
between residues 538 and 550 was shown to affect virus particle production (11). The TYTLE sequence (residues 542 to 546) in the F cytoplasmic domain, which we found to be required for
the budding of F-containing vesicles, lies between residues 538 and 550. The virus particle production of their mutant SV from infected cells was about half as efficient as that of the wild type, which was
not significant, considering our result with mutant F541 that showed no
release of F vesicles (Fig. 7B). However, their mutant SV contained all
the other structural proteins intact, including M. Therefore, it is not
surprising that particle formation by the mutant SV was not
significantly impaired because M can induce budding much more
efficiently than F (Fig. 4). In our present study, we showed the
importance of the TYTLE domain in the budding induced by F protein.
This is also supported by the fact that the TYTLE sequence is conserved
between SV and hPIV1 F proteins although the overall homology of the F
cytoplasmic tails is very low (Fig. 7A).
No HN protein was released into the culture medium of cells expressing
HN alone, despite the nearly equivalent expression of HN and F
proteins. This result indicates that HN alone is unable to induce the
budding of HN-containing vesicles. Coexpression of M with HN, however,
resulted in the release of HN into medium; thus, coexpressed M protein
may supply the machinery required to produce HN-containing vesicles.
Because HN and M were found in fractions of different densities, it is
clear that HN was not released because of direct physical interaction
with M. Instead, it is likely that a function of M triggers the budding
of vesicles containing HN.
Virions that contain no HN have been efficiently produced at a
nonpermissive temperature from cells infected with a
temperature-sensitive SV mutant (ts271) (27, 35), and
recombinant SV that contains no HN gene has been recovered
from cDNA (18), indicating that HN is not essential for
the production of virions. However, characterization of recombinant SV
or simian virus 5 with truncated HN cytoplasmic tails showed that the
cytoplasmic tail sequence affects the budding efficiency of the
mutant viruses (11, 32). In cells infected with
recombinant simian virus 5 that expressed a truncated HN, viral
proteins failed to accumulate at presumptive budding sites; therefore,
the HN cytoplasmic tail may be involved in the formation of budding
complexes required for the efficient budding of simian virus 5. In
virus-infected cells, the cytoplasmic tail of HN may enhance the
efficiency of budding by participating in the budding complex that
includes M and F.
Direct interaction between SV M and F proteins has been reported. One
group, who treated cells expressing SV M and F with detergent, reported
that M protein interacts specifically with both the transmembrane
domain and the cytoplasmic tail of F (1). They also
observed specific colocalization of M with F at the plasma membrane by
using confocal microscopy. Another group of investigators reported that
coexpression of F and M reinforced the association of M with the
membrane, a finding that suggests direct interaction between F and M
(30); however, these results have not been confirmed
(34). We have shown that when M was coexpressed with F,
the release of vesicles containing M and F was strongly enhanced.
However, sucrose gradient analysis suggested that the M- and
F-containing vesicles were produced separately. These apparently
contradictory findings may reflect a weak physical interaction between
M and F in the absence of other viral proteins or an interaction
between M and F that interferes with the machinery that drives the
budding of vesicles that contain both proteins.
Interestingly, vesicles released from cells expressing SV M or F
contained cellular actin in addition to these viral proteins. This
finding supports the hypothesis that actin is involved in virus
budding. Interaction between virus and actin is suggested by the
presence of actin in highly purified virions. Paramyxoviruses were
among the first viruses shown to contain actin (17, 23, 38). The electron microscopic observation of actin filaments in
association with budding measles virus suggests the involvement of
actin in paramyxovirus assembly (2). Cytochalasin B, an agent that disrupts actin microfilaments, completely inhibited the
release of measles virions, a finding that suggests that actin is
essential for virus budding (33). Furthermore, there is
evidence of direct binding between actin and the M proteins of
Newcastle disease virus and SV (12). Our findings with
deletion mutants of SV and hPIV1 M indicate that C-terminal residues
are required for the budding of M-containing vesicles. Interestingly,
the residues we found to be required for budding are similar to the
actin-binding domain (KLKK motif) identified in several F-actin-binding
proteins, such as thymosin
4, villin, and vasodilator-stimulated
phosphoprotein (37, 39). M protein contains the domain
only at the C-terminus of the molecule. Remarkably, SV F contains a
similar sequence in its cytoplasmic tail, and mutation of these
residues affected the efficiency of budding. We do not have biochemical
evidence to indicate whether M and F proteins directly bind to actin
through these domains. However, our mutational analysis together with previous reports at least suggests the possible role of cellular actin
in the process of virus budding. Further characterization of the
interaction between actin and M and/or F proteins will be required to
unveil the role of actin in the budding process and the mechanism of
the final steps of virus assembly.
 |
ACKNOWLEDGMENTS |
This work was supported by grants AI-11949 and AI-38956 from the
National Institute of Allergy and Infectious Diseases, by Cancer
Center Support (CORE) grant CA-21765 from the National Cancer
Institute, and by the American Lebanese Syrian Associated Charities (ALSAC).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3438. Fax:
(901) 523-2622. E-mail: toru.takimoto{at}stjude.org
 |
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Journal of Virology, December 2001, p. 11384-11391, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11384-11391.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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