Laboratory of Viral Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes
of Health, Bethesda, Maryland 20892-0445
The products of the A33R and A36R genes of vaccinia virus are
incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with H332PO4 were
immunoprecipitated with antibodies against the A33R protein, two
prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins,
respectively. The immunoprecipitated complex contained disulfide-bonded
dimers of A33R protein that were noncovalently linked to A36R protein.
Biochemical analysis indicated that the two proteins were
phosphorylated predominantly on serine residues, with lesser amounts on
threonines. The A36R protein was also phosphorylated on tyrosine, as
determined by specific binding to an anti-phosphotyrosine antibody.
Serine phosphorylation and A33R-A36R protein complex formation occurred
even when virus assembly was blocked at an early stage with the drug
rifampin. Tyrosine phosphorylation was selectively reduced in cells
infected with F13L or A34R gene deletion mutants that were impaired in
the membrane-wrapping step of IEV formation. In addition, tyrosine
phosphorylation was specifically inhibited in cells infected with an
A33R deletion mutant that still formed IEV. Immunofluorescence and
immunoelectron microscopy indicated that in the absence of the A33R
protein, the A36R protein was localized in Golgi membranes but not in
IEV. In the absence of the A36R protein, however, the A33R protein
still localized to IEV membranes. These studies together with others
suggest that the A33R protein guides the A36R protein to the IEV
membrane, where it subsequently becomes tyrosine phosphorylated as a
signal for actin tail formation.
 |
INTRODUCTION |
Vaccinia virus, the prototypic
member of the poxvirus family, is a complex enveloped DNA virus with
antigenically distinct intracellular and extracellular infectious forms
(1, 5). Although the majority of intracellular mature
virions (IMV) reside in the cytoplasm until cell lysis, some are
wrapped by additional membranes and transported to the cell surface.
Two related types of extracellular virions have been identified:
adherent cell-associated enveloped virions (CEV), which are needed for
efficient cell-to-cell spread (4), and released
extracellular enveloped virions (EEV), which promote long range
dissemination (19).
Although the mechanism of formation of IMV is still poorly understood,
considerable progress has been made in identifying the cellular
membranes and viral proteins required for extracellular virion
formation. The double membranes that wrap IMV to form intracellular enveloped virions (IEV) are derived from late Golgi or endosomal cisternae (15, 25). IEV are transported to the cell
periphery, where they acquire actin tails, similar to those made by
other intracellular pathogens such as Listeria and
Shigella spp., and form the tips of protruding microvilli
(6, 14, 16, 27). Virions are externalized at the plasma
membrane, apparently losing the outermost of the two IEV-specific
membranes in the process.
Six proteins, encoded by the F13L, B5R, A33R, A34R, A36R, and A56R open
reading frames (ORFs), have been identified as constituents of the IEV
or EEV membrane (8, 10, 17, 18, 20, 22, 29). Deletion of
any of these ORFs, except for A56R, which encodes the viral
hemagglutinin, yields a mutant with a small-plaque phenotype. Mutants
with deleted F13L or B5R (3, 11, 30) ORFs have defects in
membrane wrapping that lead to reduced production of CEV and EEV. The
small-plaque phenotypes of A33R, A34R, or A36R deletion mutants,
however, correlated with defects in actin tail formation rather than
with reduced production of EEV (21, 24, 31, 33). The
latter results indicated that actin tails were important for virus
spread rather than for the egress of vaccinia virus.
Based on the above observations, we proposed that the A33R, A34R, and
A36R proteins interact to form a platform for nucleation of actin tails
(33). While we were carrying out experiments to further
investigate this hypothesis, evidence for physical associations among
EEV proteins and between tyrosine-phosphorylated A36R protein and the
cellular adapter protein Nck, leading to the recruitment of N-WASP to
the site of actin assembly, was reported (12, 23). Here,
we confirm interactions between the A33R and A36R proteins and
demonstrate that both are phosphorylated predominantly on serine
residues and that viral membrane localization and tyrosine phosphorylation of the A36R protein are dependent on expression of the
A33R protein.
| |
MATERIALS AND METHODS |
Cells and viruses.
Cells and virus were propagated as
previously described (9). Unless otherwise specified, HeLa
cells were infected with trypsin-treated vaccinia virus stocks at a
multiplicity of 10 PFU in medium containing 2.5% fetal bovine serum.
Antibodies.
Rabbit antiserum 
-A36RC was raised to a
C-terminal peptide of A36R (EHDDIESSVVSLV) coupled to keyhole limpet
hemocyanin. Monoclonal antibody (MAb) 4 against the A33R protein
(-A33R) was a kind gift of L. Payne, MAb 19C2 (25)
against the B5R protein and polyclonal antiserum against the F13L and
B5R proteins were generous gifts from G. Hiller, and MAb PY99 against
phosphotyrosine was purchased from Santa Cruz Laboratories.
Immunoprecipitations.
Cells were labeled with 50 to 100 µCi of [35S]methionine (Amersham) per ml of
methionine-free medium or with 100 to 200 µCi of
H332PO4 (ICN Biochemicals) per ml
of phosphate-free medium for the indicated times. The cells were then
incubated for 20 min in half-strength phosphate-buffered saline (PBS)
containing 1% NP-40 supplemented with protease (complete protease
inhibitor tablets; Roche Molecular Biochemicals) and phosphatase
(sodium fluoride and sodium vanadate; Sigma-Aldrich) inhibitors. The
lysates were cleared by centrifugation at 30,000 rpm for 60 min in a
Beckman 42.2 Ti rotor and were incubated first with preimmune serum and
then with protein A-Sepharose. Proteins were incubated with either
-A36RC or PY99. The antigen-antibody complexes were bound to protein
A-Sepharose beads, washed three times with half-strength PBS containing
1% NP-40 and protease inhibitors, resuspended in Laemmli sample buffer
in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol
(DTT), and boiled for 3 min before application to an SDS-polyacrylamide
gel. After electrophoresis, the gels were dried and subjected to autoradiography.
Phosphoamino acid analysis.
BS-C-1 cells were infected with
vaccinia virus and metabolically labeled with
H332PO4 as described above. After
immunoprecipitation with -A33R or -A36RC, the phosphoproteins
were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and
transferred to a polyvinylidene difluoride membrane (Millipore).
The band of interest was excised, and the protein was hydrolyzed in 6 M
HCl for 60 min at 110°C. The hydrolysate was dried under a vacuum,
resuspended, and analyzed by two-dimensional thin-layer electrophoresis
(26).
Immunofluorescence.
At 8 h after infection, HeLa cells
were fixed in 3% paraformaldehyde and stained with -A36RC followed
by rhodamine-conjugated anti-rabbit immunoglobulin G (IgG) (Dako
Corp.). Cells were incubated with rat MAb 19C2 against the B5R protein
followed by fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG
(Dako Corp.). Finally, cells were stained with 5 µg of Hoechst 33258 (Pierce)/ml, washed, and mounted in Fluoromount G (Southern
Biotechnology Associates) before observation by confocal microscopy.
Images were collected on a Leica model TCS NT laser scanning confocal
microscope with an attached UV laser; each channel was collected
separately and then merged.
Electron microscopy.
Infected BS-C-1 cells, grown in 60-mm
dishes, were fixed with paraformaldehyde and cryosectioned as
previously described (32). Thawed cryosections were
incubated with either -A36RC, -A33R, or an antibody to the F13L
protein. Sections were washed and incubated with protein A conjugated
to 10-nm colloidal gold (Department of Cell Biology, Utrecht University
School of Medicine, Utrecht, The Netherlands). Immunostained sections
were viewed using a Philips CM100 transmission electron microscope.
| |
RESULTS |
Coimmunoprecipitation and phosphorylation of the A33R and A36R
proteins.
Recent studies indicating that some IMV membrane
proteins were phosphorylated on serine, threonine, and tyrosine
residues (2, 7, 28) prompted us to determine whether EEV
proteins are also phosphorylated. Initially, we focused on the A33R
protein because of its relatively long cytoplasmic tail
(22). Cells were infected with wild-type (WR) or mutant
vaccinia viruses and labeled from 8 to 24 h with
H332PO4. NP-40 detergent lysates of
these cells were incubated with a mouse MAb (-A33R) to the A33R
protein followed by protein A-Sepharose. The bound proteins were
analyzed by SDS-PAGE and subjected to autoradiography. From
32P-labeled extracts of cells infected with WR, -A33R
immunoprecipitated a species of 28 to 30 kDa (referred to below as a
28-kDa band) with the expected molecular mass of the A33R protein (Fig.
1A). Unexpectedly, a more highly
32P-labeled protein of approximately 50 kDa coprecipitated
with the A33R protein. Neither protein was immunoprecipitated from uninfected cells or from cells infected with an A33R deletion mutant
(v
A33R; Fig. 1A). We considered that the 50-kDa protein might be a
dimer of the A33R protein (22) that was unusually resistant to reducing agents and SDS or another cellular or viral protein that interacted with the A33R protein. With regard to the
latter possibility, the A36R protein seemed a likely candidate because
of its size. However, the A36R protein had been classified as a type 2 membrane protein with an N-terminal hydrophobic domain (18) and therefore had no predicted cytoplasmic sites of
phosphorylation. Nevertheless, the 32P-labeled 50-kDa
protein was not precipitated from lysates of cells infected with the
A36R deletion mutant vA36R (Fig. 1A).
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 1.
SDS-PAGE analysis of metabolically labeled proteins from
vaccinia virus-infected cells. BS-C-1 cells were uninfected (U) or were
infected with vaccinia virus WR (W) or recombinant vaccinia viruses
from which the A33R ORF (A33) or the A36R ORF (A36) had been
deleted. After 8 h, infected and uninfected cells were labeled
with H332PO4 (A, C, and D) or
[35S]methionine (B, C, and D) for 18 h. Lysates were
immunoprecipitated with either -A33R (-A33) or -A36RC
(-A36). In panel D, cells were infected in the absence ( ) or
presence (+) of rifampin (Rif). Immunoprecipitated proteins were run on
SDS-PAGE gels under reducing conditions (A, B, and D) or nonreducing
conditions (C) and visualized by autoradiography. Masses of marker
proteins (in kilodaltons) are given on the left. Arrows point to the
28-kDa A33R or the 50-kDa A36R protein.
|
|
To more directly identify the 50-kDa protein, rabbit polyclonal
antiserum -A36RC directed against a C-terminal peptide of the A36R
protein was used. This antibody brought down a 32P-labeled
50-kDa band from extracts of cells infected with WR or vA33R but not
from cells infected with vA36R or from uninfected cells, confirming
its identity as the product of the A36R gene (Fig. 1A). Although
immunoprecipitation of the 50-kDa band by -A36RC seemed efficient,
coprecipitation of the 32P-labeled 28-kDa band was not
detected (Fig. 1A). The latter result could be due to excess
uncomplexed 50-kDa protein and the much lower 32P labeling
of A33R protein compared to A36R protein. As will be shown below,
-A36RC did coprecipitate [35S]methionine-labeled A36R
and A33R proteins. Thus, these data indicated that
32P-labeled 28- and 50-kDa bands were derived from the A33R
and A36R ORFs, respectively, and that the A36R and A33R proteins
coimmunoprecipitated when a MAb to the latter was used. The
phosphorylation of the A36R protein meant that it was unlikely to have
a type 2 membrane topology. Evidence that the A36R protein is, in fact,
a type 1b membrane protein with a long cytoplasmic tail was recently
reported (23).
Parallel experiments, in which the infected cells were incubated with
[35S]methionine, were also carried out. Because of the
inhibition of host protein synthesis, only viral proteins should be
labeled during an 8- to 24-h period. A predominant 28-kDa band was seen when extracts of [35S]methionine-labeled cells that had
been infected with WR were immunoprecipitated with -A33R (Fig. 1B).
The labeled 50-kDa species was faint compared to the 28-kDa band.
However, neither the 28- nor the 50-kDa 35S-labeled band
was immunoprecipitated with -A33R when the extracts were from
uninfected cells or cells infected with vA33R, and only the 28-kDa
band was observed when the extracts were from cells infected with
vA36R (Fig. 1B). The difference between the 32P and
35S labeling of the A33R and A36R bands could not be
explained by the relative numbers of methionines in the two proteins,
because the A33R ORF contains six residues compared to five for the
A36R ORF. Therefore, the difference in labeling signified that the A36R
protein is more highly phosphorylated than the A33R protein.
Immunoprecipitation of extracts of the
[35S]methionine-labeled cells was also carried out with
-A36RC. Apparently due to the greater labeling of the A33R protein
with [35S]methionine compared to
H332PO4, coprecipitation of the
A33R and A36R proteins from extracts of cells infected with WR was
clearly seen (Fig. 1B). As expected, the 28-kDa band was not
immunoprecipitated from uninfected cells or from cells infected with
vA33R, and neither the 50- nor the 28-kDa band was
immunoprecipitated from cells infected with vA36R (Fig. 1B). In some
individual experiments, however, we did not achieve efficient
coprecipitation of the A33R protein with the A36R antibody.
The 32P- and 35S-labeled proteins from cells
infected with WR were also analyzed by SDS-PAGE under nonreducing
conditions. The major labeled protein that was immunoprecipitated with
either -A33R or -A36RC migrated as a diffuse band of
approximately 50 kDa (Fig. 1C), consistent with dimeric A33R
(22) and monomeric A36R.
To determine whether IEV membrane formation was required for the
interaction and phosphorylation of A33R and A36R, we immunoprecipitated extracts of cells that had been infected with WR and labeled with H332PO4 or
[35S]methionine in the presence of rifampin, an inhibitor
of virus assembly (13). Although rifampin caused an
overall decrease in 32P and 35S labeling,
-A33R precipitated phosphorylated proteins corresponding in size to
A33R and A36R even in the presence of the drug (Fig. 1D), indicating
that some complex formation and phosphorylation could occur in the
absence of assembly.
Taken together, our data demonstrated that the A33R and A36R proteins
formed a complex and were both phosphorylated. In addition, phosphorylation of each occurred independently of the other, as indicated by labeling of the proteins in cells infected by deletion mutants, and did not require virus assembly.
Phosphoamino acid analysis of the A33R and A36R proteins.
The
32P-labeled A33R and A36R proteins were purified by
immunoprecipitation with specific antibodies, followed by SDS-PAGE. The
proteins were transferred to a membrane, and the labeled bands were
excised and acid hydrolyzed. The phosphoamino acids were resolved by
two-dimensional thin-layer electrophoresis and detected by
autoradiography. Major and minor spots from the A33R protein comigrated
with phosphoserine and phosphothreonine standards, respectively (Fig.
2). Additional radioactive spots
represented incompletely hydrolyzed peptides and inorganic phosphate.
Of the radioactivity in these phosphoamino acids, 93% was
phosphoserine and 7% phosphothreonine. Phosphoserine was also the
major hydrolysis product of 32P-labeled A36R protein (Fig.
2). Overexposure of the thin-layer plate revealed a trace amount of
material that comigrated with the phosphothreonine marker (data not
shown). However, no phosphotyrosine was detected by this procedure in
either the A33R or the A36R protein.
View larger version (64K):
[in this window]
[in a new window]
|
FIG. 2.
Phosphoamino acid analysis of vaccinia virus A33R and
A36R proteins. BS-C-1 cells were infected with vaccinia virus WR and
labeled overnight with H332PO4.
After cell lysis and incubation with -A33R or -A36RC, the
proteins were resolved by SDS-PAGE and transferred to a membrane. The
labeled protein was excised and hydrolyzed with HCl, and phosphoamino
acid standards were added. Standards and sample were analyzed by
two-dimensional thin-layer electrophoresis. Standards were visualized
with ninhydrin, and the plates were autoradiographed. Positions of
phosphoserine (Pser), phosphothreonine (Pthr), and phosphotyrosine
(Ptyr) standards, as well as Pi and the origin (O), are
marked.
|
|
The A36R protein is also phosphorylated on tyrosine.
In a
previous study, we had determined that the major phosphorylated amino
acids in the vaccinia virus A17L IMV membrane protein were
phosphothreonine and phosphoserine (2). Although
phosphotyrosine was not abundant enough to detect by chemical analysis,
its presence was established by using a phosphotyrosine antibody to
precipitate 32P-labeled proteins. The phosphotyrosine
antibody had also precipitated additional uncharacterized minor bands,
one of which was approximately 50 kDa (2). Because
phosphotyrosine antibodies exhibit some selectivity for neighboring
amino acids, we tested several anti-phosphotyrosine antibodies and
selected PY99 because it consistently precipitated a
32P-labeled 50-kDa protein from extracts of cells infected
with vaccinia virus but not from control uninfected cells. We had noted that the A36R proteins from the WR and IHDJ strains had slightly different electrophoretic mobilities, and we took advantage of this to
help with the identification. As shown earlier, -A36RC precipitated
a phosphorylated protein of 50 kDa from cells infected with WR or
vA33R but not from vA36R-infected cells (Fig.
3A). However, a slightly faster migrating
protein was obtained from cells infected with the IHDJ strain of
vaccinia virus (Fig. 3A). When the 32P-labeled proteins
from the same extracts were precipitated with the anti-phosphotyrosine
antibody PY99 and analyzed by SDS-PAGE, a major band of approximately
50 kDa was detected in extracts from cells infected with WR and a
slightly faster migrating one from cells infected with IHDJ (Fig. 3A).
Moreover, the phosphotyrosine band was not discerned in lanes
containing material from uninfected cells or from cells infected with
vA36R, consistent with its being a product of the A36R gene (Fig.
3A). Interestingly, only a faint 50-kDa phosphotyrosine band was
detected in extracts from cells infected with vA33R (Fig. 3A). This
contrasted with the prominent 50-kDa band seen when
32P-labeled proteins from cells infected with vA33R were
precipitated with -A36RC (Fig. 3A), suggesting that A33R expression
is important for tyrosine phosphorylation but not serine
phosphorylation. A 25-kDa band, corresponding to
tyrosine-phosphorylated A17L protein, was detected in some experiments
but apparently was poorly recognized by the PY99 antibody (data not
shown).
View larger version (86K):
[in this window]
[in a new window]
|
FIG. 3.
SDS-PAGE analysis of tyrosine-phosphorylated proteins
from infected cells. BS-C-1 cells were uninfected (U) or were infected
with vaccinia virus strain WR (W) or IHDJ (I) or with a recombinant
vaccinia virus from which the A33R ORF (A33) or the A36R ORF
(A36) had been deleted. Cells were then labeled with
H332PO4. (A) Lysates were
immunoprecipitated with either the anti-phosphotyrosine antibody PY99
or -A36RC and were analyzed by SDS-PAGE. (B) An equivalent portion
of the material immunoprecipitated by PY99 was eluted from the protein
A-coupled antibody with cold phosphotyrosine and a high salt
concentration. This postelution fraction was diluted and
immunoprecipitated with either -A36RC or antibody to the N-terminal
region of A17L (anti-A17LN). The washed samples were analyzed by
SDS-PAGE and autoradiography. The masses (in kilodaltons) and positions
of migration of markers are indicated on the left.
|
|
Further experiments were performed to confirm that the 50-kDa protein
that bound to the PY99 antibody contained phosphotyrosine and was the
A36R protein. Extracts of 32P-labeled cells were bound to
the PY99 antibody, complexed to protein A-Sepharose, and specifically
eluted with phosphotyrosine. The proteins that were eluted with
phosphotyrosine were immunoprecipitated with -A36RC or an antibody
to the A17L protein and were also analyzed by SDS-PAGE (Fig. 3B).
Significantly, from cells infected with WR or IHDJ, the
32P-labeled 50-kDa species that was eluted from PY99 bound
to -A36RC, whereas virtually no signal was detected from cells
infected with vA36R or vA33R (Fig. 3B). The anti-A17LN antibody
served as a negative control; the 50-kDa phosphoprotein was not
precipitated, although a faint 25-kDa band was detected in the lanes
containing proteins from infected cells.
Differential regulation of serine and tyrosine
phosphorylation of the A36R protein.
The difference between the
total 32P labeling and the phosphotyrosine labeling of the
A36R protein in cells infected with vA33R suggested that serine and
tyrosine phosphorylation were regulated differently. To investigate
this further, cells were infected with vF13L, a mutant with a defect
in wrapping IMV that produces few EEV; vA34R, which produces few IEV
but increased numbers of EEV; and vA33R, which accumulates
incompletely wrapped IEV but increased numbers of EEV. Extracts of
cells labeled with H332PO4 were
immunoprecipitated with -A33R or -A36RC to determine serine
phosphorylation, or with PY-99 to analyze tyrosine phosphorylation. -A33R immunoprecipitated 32P-labeled 28-kDa proteins
from cells infected with all mutants except vA33R and
32P-labeled 50-kDa proteins from cells infected with all
mutants except vA33R and vA36R (Fig.
4). Thus, neither the A34R nor the F13L
protein was needed for either the serine phosphorylation of the A36R
protein or its association with the A33R protein. -A36RC
immunoprecipitated a phosphorylated 50-kDa protein from cells infected
with all mutants except vA36R, indicating that none of the other EEV
proteins were required for serine phosphorylation (Fig. 4). Quite
different results were obtained, however, with the phosphotyrosine
antibody PY99. A strongly labeled band was obtained only with cells
infected with WR (Fig. 4). Except for vA36R, vA33R had the
greatest defect, with no detectable phosphotyrosine-labeled 50-kDa band
(Fig. 4). In addition, only faint bands were obtained from cells
infected with vA34R or vF13L (Fig. 4).
View larger version (97K):
[in this window]
[in a new window]
|
FIG. 4.
SDS-PAGE analysis of proteins that were
immunoprecipitated from lysates of cells infected with vaccinia virus
deletion mutants. BS-C-1 cells were infected with vaccinia virus WR or
the recombinant vaccinia virus vA33, vA34, vA36, or vF13L
and then labeled with H332PO4 for
18 h. Lysates were immunoprecipitated with -A33R, -A36RC, or
the phosphotyrosine antibody PY99 and then analyzed by SDS-PAGE and
autoradiography. The masses (in kilodaltons) and migration positions of
markers are indicated on the left.
|
|
Localization of the A36R protein in infected cells.
At this
point, we considered the possibility that the A33R protein might be
necessary for targeting the A36R protein to the IEV membrane, where
tyrosine phosphorylation occurs. To investigate this question, we first
compared the intracellular localization of the A36R protein in cells
infected with WR or vA33R by confocal microscopy. Another EEV
protein, B5R, served as a marker for Golgi and viral membranes. Cells
were incubated with -A36RC followed by a rhodamine-conjugated
anti-rabbit IgG and with a MAb against B5R followed by
fluorescein-conjugated anti-rat IgG. In the WR-infected cells, both
antibodies gave bright juxtanuclear Golgi membrane staining as well as
a pattern of punctate staining within the cytoplasm and bright areas at
the tips of cells that may represent accumulations of IEV (Fig.
5). By merging the images of WR-infected cells, we observed considerable colocalization of the two proteins. In
cells infected with vA33R, however, the colocalization was mainly
limited to the Golgi regions (Fig. 5).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 5.
Localization of the A36R protein in infected cells by
indirect immunofluorescence. HeLa cells grown on coverslips were
infected with vaccinia virus WR or recombinant vA33R. Fixed,
permeabilized cells were stained sequentially with -A36RC (-A36R)
followed by tetramethyl rhodamine isocyanate (TRITC)-conjugated
anti-rabbit antisera and with MAb 19C against B5R (-B5R) followed by
an FITC-conjugated anti-rat antibody. Fluorescence signals were
collected independently using a Leica model TCS NT laser scanning
confocal microscope.
|
|
Higher-resolution studies were carried out by immunoelectron
microscopy. In agreement with the immunofluorescence data, there was
similar labeling of Golgi membranes by -A36RC in cells infected with
vA33R or WR (data not shown). In contrast, the labeling of the
partially wrapped IEV in vA33R-infected cells was sparse, while that
of IEV formed in WR-infected cells was more intense (Fig.
6). The specificity of this effect was
demonstrated by the labeling of the partially wrapped IEV in
A33R-infected cells by an antibody to F13L (Fig. 6). Although the
A33R protein appears to be necessary for incorporation of the A36R
protein into IEV, the opposite was not true. There was a similar
distribution of labeling with the -A33R antibody on IEV formed in
cells infected with WR or vA36R (Fig. 6).
View larger version (162K):
[in this window]
[in a new window]
|
FIG. 6.
Immunolabeling of IEV in infected cells. Thawed
cryosections of BS-C-1 cells infected with WR, vA33R, or vA36R
were labeled with -A33R, -F13L, or -A36RC (-A36R) followed
by 10-nm protein A-gold.
|
|
We also noticed a difference in the labeling of the IEV membranes by
antibodies to A36R and F13L in cells infected with WR. The latter
appeared to label the two membranes similarly, whereas the former
primarily labeled the outer membrane. This result is in agreement with
recent data of van Eijl et al. (29), who reported, contrary to previous data, that the A36R protein is present in IEV but
not in EEV. The latter result was confirmed by counting the number of
gold grains on virus particles in thin sections of WR-infected cells
incubated with anti-F13L, -A33R, or -A36RC (Table
1). With anti-F13L or -A33R, similar
percentages of IEV, CEV, and EEV were labeled, although the absolute
numbers differed either because of the amount of protein or because of
the avidity of the antibody. However, despite respectable labeling of
IEV with -A36RC, there was scarcely any labeling of CEV and EEV
(Table 1). These data support the conclusion that the A36R protein is IEV specific, although biochemical studies are needed to exclude the
possibility that the reactivity of the A36R protein was masked in the
extracellular viral forms.
| |
DISCUSSION |
Previous studies from our laboratory and others had shown that the
A33R, A34R, and A36R proteins are required for the formation of actin
tails on the IEV (21, 24, 31, 33). Recent data, published
during the course of the present investigation, suggested that the
nucleation of actin tails involves the association of tyrosine-phosphorylated A36R protein with the adapter protein Nck and
the recruitment of N-WASP (12). The role of the A33R proteins in this process, however, was unknown. Here we have shown that
(i) the A33R and A36R proteins are phosphorylated predominantly on
serine residues; (ii) serine phosphorylation of A33R and that of A36R
occur independently of each other and do not require virus assembly;
(iii) disulfide-bonded A33R dimers form a noncovalent complex with the
A36R protein, and this also does not require virus assembly; (iv)
tyrosine phosphorylation of the A36R protein is dependent on the
presence of the A33R protein and is greatly reduced in the absence of
the A34R and F13L EEV membrane proteins; (v) the A33R protein is
necessary for the localization of the A36R protein but not the B5R or
F13L protein in IEV membranes; and (vi) the A36R protein is not needed
for the localization of the A33R protein in IEV membranes. Taken
together, these data suggest that one function of the A33R protein is
to guide the A36R protein to the IEV membrane, where tyrosine
phosphorylation can occur. This does not, however, exclude the
possibility that the A33R protein also has a more direct role in actin
tail formation.
Our finding that the A36R protein is highly phosphorylated on serine
residues was initially surprising, since the protein had been
classified as a type 2 membrane protein with virtually no cytoplasmic
tail (18). However, there is now agreement that the A36R
protein is a type 1b protein with a long cytoplasmic tail but little or
no extracellular domain (23). The low level of
phosphorylation on tyrosine relative to serine is precisely what one
would expect if phosphotyrosine were the signal for nucleation of actin
tails. The bulk of the A36R protein, which is associated with the
endoplasmic reticulum, the Golgi network, or IEV that have not yet
acquired actin tails, would be phosphorylated on serine but not
tyrosine. Recently, van Eijl et al. (29) reported that the
A36R protein is targeted to the outer IEV membrane and is removed
during fusion with the plasma membrane. We also noted that the antibody
to the A36R protein did not label EEV and CEV, and labeling of IEV was
predominantly observed on the outer IEV membrane. How this asymmetry is
created is unclear, especially since the A33R and A36R proteins
interact with each other even in the absence of morphogenesis. This
immunogold labeling pattern suggested to van Eijl et al.
(29) that actin tail formation occurs at the plasma
membrane. A three-dimensional analysis of confocal images indicated
that actin tails are mostly near the periphery of the cell, although it
was not possible to ascertain whether they are all associated with the
plasma membrane (E. J. Wolffe, unpublished data). The formation of
actin tails at the plasma membrane would be consistent with evidence
for the tyrosine phosphorylation of A36R by Src family kinases
(12).
We thank Norman Cooper for preparing cells and Owen Schwartz for
assistance with the confocal microscopy.
| 1.
|
Appleyard, G.,
A. J. Hapel, and E. A. Boulter.
1971.
An antigenic difference between intracellular and extracellular rabbitpox virus.
J. Gen. Virol.
13:9-17[Abstract/Free Full Text].
|
| 2.
|
Betakova, T.,
E. J. Wolffe, and B. Moss.
1999.
Regulation of vaccinia virus morphogenesis: phosphorylation of the A14L and A17L membrane proteins and C-terminal truncation of the A17L protein are dependent on the F10L protein kinase.
J. Virol.
73:3534-3543[Abstract/Free Full Text].
|
| 3.
|
Blasco, R., and B. Moss.
1991.
Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein.
J. Virol.
65:5910-5920[Abstract/Free Full Text].
|
| 4.
|
Blasco, R., and B. Moss.
1992.
Role of cell-associated enveloped vaccinia virus in cell-to-cell spread.
J. Virol.
66:4170-4179[Abstract/Free Full Text].
|
| 5.
|
Boulter, E. A., and G. Appleyard.
1973.
Differences between extracellular and intracellular forms of poxvirus and their implications.
Prog. Med. Virol.
16:86-108[Medline].
|
| 6.
|
Cudmore, S.,
P. Cossart,
G. Griffiths, and M. Way.
1995.
Actin-based motility of vaccinia virus.
Nature
378:636-638[CrossRef][Medline].
|
| 7.
|
Derrien, M.,
A. Punjabi,
R. Khanna,
O. Grubisha, and P. Traktman.
1999.
Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase.
J. Virol.
73:7287-7296[Abstract/Free Full Text].
|
| 8.
|
Duncan, S. A., and G. L. Smith.
1992.
Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.
J. Virol.
66:1610-1621[Abstract/Free Full Text].
|
| 9.
|
Earl, P. L., and B. Moss.
1991.
Generation of recombinant vaccinia viruses, p. 16.17.1-16.17.16.
In
F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. Greene Publishing Associates & Wiley Interscience, New York, N.Y.
|
| 10.
|
Engelstad, M.,
S. T. Howard, and G. L. Smith.
1992.
A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope.
Virology
188:801-810[CrossRef][Medline].
|
| 11.
|
Engelstad, M., and G. L. Smith.
1993.
The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence.
Virology
194:627-637[CrossRef][Medline].
|
| 12.
|
Frischknecht, F.,
V. Moreau,
S. Rottger,
S. Gonfloni,
I. Reckmann,
G. Superti-Furga, and M. Way.
1999.
Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.
Nature
401:926-929[CrossRef][Medline].
|
| 13.
|
Grimley, P. M.,
E. N. Rosenblum,
S. J. Mims, and B. Moss.
1970.
Interruption by rifampin of an early stage in vaccinia virus morphogenesis: accumulation of membranes which are precursors of virus envelopes.
J. Virol.
6:519-533[Abstract/Free Full Text].
|
| 14.
|
Hiller, G.,
C. Jungwirth, and K. Weber.
1981.
Fluorescence microscopical analysis of the life cycle of vaccinia virus in chick embryo fibroblasts. Virus-cytoskeleton interactions.
Exp. Cell Res.
132:81-87[CrossRef][Medline].
|
| 15.
|
Hiller, G., and K. Weber.
1985.
Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment.
J. Virol.
55:651-659[Abstract/Free Full Text].
|
| 16.
|
Hiller, G.,
K. Weber,
L. Schneider,
C. Parajsz, and C. Jungwirth.
1979.
Interaction of assembled progeny pox viruses with the cellular cytoskeleton.
Virology
98:142-153[CrossRef][Medline].
|
| 17.
|
Isaacs, S. N.,
E. J. Wolffe,
L. G. Payne, and B. Moss.
1992.
Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.
J. Virol.
66:7217-7224[Abstract/Free Full Text].
|
| 18.
|
Parkinson, J. E., and G. L. Smith.
1994.
Vaccinia virus gene A36R encodes a Mr 43-50 K protein on the surface of extracellular enveloped virus.
Virology
204:376-390[CrossRef][Medline].
|
| 19.
|
Payne, L. G.
1980.
Significance of extracellular virus in the in vitro and in vivo dissemination of vaccinia virus.
J. Gen. Virol.
50:89-100[Abstract/Free Full Text].
|
| 20.
|
Payne, L. G., and E. Norrby.
1976.
Presence of hemagglutinin in the envelope of extracellular vaccinia virus particles.
J. Gen. Virol.
32:63-72[Abstract/Free Full Text].
|
| 21.
|
Roper, R.,
E. J. Wolffe,
A. Weisberg, and B. Moss.
1998.
The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell-to-cell spread of vaccinia virus.
J. Virol.
72:4192-4294[Abstract/Free Full Text].
|
| 22.
|
Roper, R. L.,
L. G. Payne, and B. Moss.
1996.
Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.
J. Virol.
70:3753-3762[Abstract].
|
| 23.
|
Rottger, S.,
F. Frischknecht,
I. Reckmann,
G. L. Smith, and M. Way.
1999.
Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation.
J. Virol.
73:2863-2875[Abstract/Free Full Text].
|
| 24.
|
Sanderson, C. M.,
F. Frischknecht,
M. Way,
M. Hollinshead, and G. L. Smith.
1998.
Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion.
J. Gen. Virol.
79:1415-1425[Abstract].
|
| 25.
|
Schmelz, M.,
B. Sodeik,
M. Ericsson,
E. J. Wolffe,
H. Shida,
G. Hiller, and G. Griffiths.
1994.
Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans-Golgi network.
J. Virol.
68:130-147[Abstract/Free Full Text].
|
| 26.
|
Sefton, B. M.
1997.
Phosphoamino acid analysis, unit 13.3.
In
J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W. Speicher, and P. T. Wingfield (ed.), Current protocols in protein science. John Wiley & Sons, New York, N.Y. [Online.]
|
| 27.
|
Stokes, G. V.
1976.
High-voltage electron microscope study of the release of vaccinia virus from whole cells.
J. Virol.
18:636-643[Abstract/Free Full Text].
|
| 28.
|
Traktman, P.,
K. Liu,
J. DeMasi,
R. Rollins,
S. Jesty, and B. Unger.
2000.
Elucidating the essential role of the A14 phosphoprotein in vaccinia virus morphogenesis: construction and characterization of a tetracycline-inducible recombinant.
J. Virol.
74:3682-3695[Abstract/Free Full Text].
|
| 29.
|
van Eijl, H.,
M. Hollinshead, and G. L. Smith.
2000.
The vaccinia virus A36R protein is a type Ib membrane protein present on intracellular but not extracellular enveloped virus particles.
Virology
271:26-36[CrossRef][Medline].
|
| 30.
|
Wolffe, E. J.,
S. N. Isaacs, and B. Moss.
1993.
Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination.
J. Virol.
67:4732-4741[Abstract/Free Full Text].
|
| 31.
|
Wolffe, E. J.,
E. Katz,
A. Weisberg, and B. Moss.
1997.
The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus.
J. Virol.
71:3904-3915[Abstract].
|
| 32.
|
Wolffe, E. J.,
D. M. Moore,
P. J. Peters, and B. Moss.
1996.
Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.
J. Virol.
70:2797-2808[Abstract].
|
| 33.
|
Wolffe, E. J.,
A. S. Weisberg, and B. Moss.
1998.
Role for the vaccinia virus A36R outer envelope protein in the formation of virus-tipped actin-containing microvilli and cell-to-cell virus spread.
Virology
244:20-26[CrossRef][Medline].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
