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Journal of Virology, May 2001, p. 4453-4458, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4453-4458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of Host Transcription by Vesicular
Stomatitis Virus Involves a Novel Mechanism That Is Independent of
Phosphorylation of TATA-Binding Protein (TBP) or Association of TBP
with TBP-Associated Factor Subunits
Hang
Yuan,
Shelby
Puckett, and
Douglas S.
Lyles*
Department of Microbiology and Immunology,
Wake Forest University School of Medicine, Winston-Salem, North
Carolina 27157
Received 14 August 2000/Accepted 5 February 2001
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ABSTRACT |
The matrix (M) protein of vesicular stomatitis virus (VSV) is a
potent inhibitor in vivo of transcription by all three host RNA
polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the
inhibition is due to lack of activity of the TATA-binding protein
(TBP), which is a subunit of the basal transcription factor TFIID.
Despite the potency of M protein-induced inhibition in vivo,
experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host
factors. The host factors that are known to regulate TBP activity
include phosphorylation by host kinases and association with different
TBP-associated factor (TAF) subunits. However, TBP in VSV-infected
cells was found to be assembled normally with its TAF subunits, as
shown by ion exchange high-pressure liquid chromatography and
sedimentation velocity analysis. A normal pattern of phosphorylation of
TBP in VSV-infected cells was also observed by pH gradient gel
electrophoresis. Collectively, these data indicate that M protein
inactivates TBP activity in RNAPII-dependent transcription by a novel
mechanism, since the known mechanisms for regulating TBP activity
cannot account for the inhibition.
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TEXT |
Many viruses inhibit the expression
of host genes. In most cases, the role of this inhibition of host gene
expression in the viral replicative cycle is to inhibit the expression
of gene products that are involved in the antiviral response of the
host (17). In the case of vesicular stomatitis virus
(VSV), the prototype member of the rhabdovirus family, much of the
inhibition of host gene expression has been attributed to the activity
of the viral matrix (M) protein. M protein plays a role in two very
different aspects of VSV replication. Most of the M protein is present
in the cytoplasm, where it functions in virus assembly by binding the
nucleoprotein core to the cytoplasmic surface of the host plasma
membrane and inducing the budding process that generates the viral
envelope (15). M protein is also present in the nuclei of
infected cells, which is consistent with its major role in the
inhibition of host gene expression (21). This role
includes inhibition of transcription by all three host RNA polymerases (RNAP) (1, 3) and inhibition of nuclear-cytoplasmic
transport of host RNAs and proteins (13, 25, 29). The role
of M protein in the inhibition of host gene expression is genetically
separable from its function in virus assembly, as shown by M protein
mutants that are defective in the inhibition of host gene expression
but function as well as wild-type M protein in virus assembly.
Conversely, other mutants are defective in virus assembly but are as
potent as wild-type M protein in the inhibition of host gene expression (4, 8, 10, 18).
The molecular mechanisms involved in inhibiting host gene expression
are of considerable interest, both because of their implications for
viral pathogenesis and because they may reveal new features of the
regulation of gene expression in host cells. In the case of host RNA
polymerase II (RNAPII), the inhibition by VSV M protein is due to
inactivation of the basal transcription initiation factor TFIID
(31). This inhibition was shown by reconstituting
transcription initiation in vitro using partially purified
transcription initiation factors, in which the TFIID fraction from
VSV-infected cells was not able to reconstitute transcription
initiation in the presence of the other basal transcription factors
from uninfected cells. Conversely, TFIID from uninfected cells was able
to fully restore transcription initiation in nuclear extracts from
infected cells. Thus, TFIID was the only basal transcription factor
whose inhibition could be detected (31).
TFIID is a multisubunit complex consisting of a DNA-binding subunit,
the TATA-binding protein (TBP), and a set of TBP-associated factors
(TAFs) (5). TFIID is the first basal transcription factor
assembled onto RNAPII-dependent promoters through binding of TBP to the
TATA box DNA sequence located upstream of most promoters. TBP is the
only subunit of TFIID required for basal transcription in vitro.
However, activation of transcription by proteins that bind DNA
sequence-specific enhancer elements requires interaction with one or
more TAF subunits, either directly or indirectly through so-called
adapter proteins (32).
M protein does not directly inactivate host TFIID in vitro.
We
have shown previously that purified recombinant TBP is able to fully
restore basal transcription initiation in nuclear extracts from
VSV-infected cells, consistent with the inhibition occurring at the
level of basal transcription initiation (31). This finding
raises the question of how the activity of TBP is inhibited in infected
cells. The inhibition is not due to a reduction in the amount of TBP,
which is present in nuclear extracts from both infected and uninfected
HeLa cells at a level of 0.13 ng of TBP per µg of protein
(31). One possibility is that M protein inhibits TBP
activity by binding directly to TFIID. We examined this possibility by
testing the ability of M protein to inhibit the activity of TFIID from
uninfected cells in vitro.
Figure 1A shows that M protein in nuclear
extracts from VSV-infected cells was not able to inhibit TFIID in
nuclear extracts from uninfected cells in mixing experiments. Nuclear
extracts from infected or uninfected cells were mixed in the
proportions shown in Fig. 1A, and RNAPII-dependent transcription was
assayed using a plasmid DNA template that contained the adenovirus
major late promoter (MLP). The plasmid lacked upstream activating
sequences so that only basal transcription was assayed. Transcripts
were measured by an RNase protection assay with a radiolabeled
riboprobe, as described previously (31). The upper band
and the diffuse background in Fig. 1A result from random initiation on
the plasmid DNA template, whereas the lower band results from
initiation at the MLP (indicated in Fig. 1A). Only the MLP-initiated
product is an assay for the activity of RNAPII transcription initiation factors. The activity of these factors was readily apparent in nuclear
extract from uninfected cells (lane 1), while activity in nuclear
extract from infected cells (lane 5) was markedly reduced. Mixing
nuclear extracts (lanes 2 to 4) resulted in transcription activities
that were proportional to the amounts of uninfected cell nuclear
extract present in the mixture. This result indicated that nuclear
extract from infected cells did not inhibit MLP-initiated transcription
by nuclear extract from uninfected cells. Thus, the inhibitory activity
in infected cell nuclear extracts was either not present in excess or
was not freely diffusible between TFIID complexes from infected and
uninfected cells.
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FIG. 1.
Effect of M protein on transcription by nuclear extracts
in vitro. (A) Nuclear extracts from uninfected or VSV-infected cells
prepared at 6 h postinfection were mixed in the indicated amounts
and incubated in an in vitro transcription reaction with a plasmid DNA
template containing the adenovirus MLP. The transcription products were
analyzed by an RNase protection assay using a radiolabeled riboprobe,
an autoradiograph of which is shown, and quantitated by densitometry as
indicated. The position of the MLP-initiated product is indicated. (B)
Nuclear extracts were prepared from VSV-infected HeLa cells at 3, 6, and 9 h postinfection. The amount of M protein in the nuclear extracts
was determined by Western blot, using the indicated amounts of purified
VSV as a standard. (C) Nuclear extract from uninfected cells was mixed
with M protein purified from VSV virions by ion exchange chromatography
(lane 3). Nuclear extract with no additions (lane 1) or with column
buffer used to elute M protein (lane 2) were used as controls.
Transcription in vitro was assayed as in panel A. (D) Nuclear extract
from uninfected cells was mixed with in vitro translation reactions
containing M protein (lanes 2 to 4) or N protein (lanes 5 to 7) or with
no additions (lane 1). In vitro transcription reactions contained 8 µl of nuclear extract and 1 µl (lanes 2 and 5), 2.5 µl (lanes 3 and 6), or 5.5 µl (lanes 4 and 7) of translation mixture in a total
volume of 25 µl.
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Western blot analysis was used to determine whether M protein was
present in excess over host TFIID in nuclear extracts from
VSV-infected
cells (Fig.
1B). Nuclear extracts were prepared as
described previously
(
31) from VSV-infected HeLa cells at 3,
6, and 9 h
postinfection and analyzed in a Western blot using
a monoclonal M
protein antibody, 23H12 (
21). Serial dilutions
of purified
virions were analyzed in parallel as a standard. The
amount of M
protein in nuclear extracts was nearly constant from
3 to 9 h
postinfection, although in this experiment the amount
of M protein
declined slightly by 9 h postinfection. The amount
of M protein in
the nuclear extracts was calculated to be 2.0
ng of M protein per µg
of extract protein. The concentration of
TBP is 0.13 ng of TBP per µg
of protein (
31). Thus, there is
a 15-fold excess of M
protein compared to TBP. Since TFIID contains
only a fraction of
cellular TBP (
32), the excess of M protein
compared to
TFIID in nuclear extracts was greater than 15-fold.
Despite the excess of M protein over TFIID in nuclear extracts (Fig.
1B), the M protein in nuclear extracts from VSV-infected
cells was not
able to inhibit TFIID in nuclear extracts from uninfected
cells (Fig.
1A). This was confirmed by the addition of purified
M protein (Fig.
1C)
or M protein translated in vitro (Fig.
1D)
to nuclear extracts from
uninfected cells. M protein was purified
from virions by a
phosphocellulose column as described previously
(
23) and
was added to nuclear extracts from uninfected cells
at concentrations
similar to that in nuclear extracts from infected
cells. As a control
for purified M protein, an equal amount of
column buffer was added to
nuclear extracts (Fig.
1C, lane 2).
There was no inhibition of
MLP-initiated transcription when purified
M protein from virions was
added to nuclear extracts prepared
from uninfected cells (Fig.
1C, lane
3). In fact, the reaction
containing M protein actually yielded more
MLP-initiated RNA in
the experiment shown in Fig.
1C, although this was
not consistently
observed in all
experiments.
In the experiment shown in Fig.
1D, M protein synthesized by in vitro
translation was tested for its ability to inhibit RNAPII-dependent
transcription. RNA transcribed from a plasmid containing the M
gene
driven by the bacteriophage T7 promoter (
4) was translated
in a reticulocyte lysate (Promega Corp.). The in vitro translation
mixture containing M protein was added to nuclear extract from
uninfected cells in various amounts (Fig.
1D, lanes 2 to 4). Equal
volumes of in vitro-translated N protein of VSV were added as
a
negative control (lanes 5 to 7), since N protein does not inhibit
host
transcription (
2,
10). The addition of in vitro-translated
M protein did inhibit MLP-initiated transcription slightly (Fig.
1D,
lanes 2 to 4), but this was attributable to other components
in the in
vitro translation mixture, since a similar inhibition
was observed with
the N protein control (lanes 5 to
7).
The data in Fig.
1 indicate that M protein cannot inactivate TFIID by a
direct interaction in vitro. The data leave open the
possibility that M
protein assumes a conformation in vivo that
differs from the M protein
tested in vitro. However, such a conformation
would have to be distinct
from that found in the M protein that
is present in nuclear extracts
from infected cells, as well as
that produced by reticulocyte lysates
or purified from virions.
The M protein in all three of these
preparations was active in
other in vitro assays for M protein
activity, such as the ability
to assemble into viral nucleocapsid-M
protein complexes involved
in virus assembly (
19;
unpublished data). Thus, although this
possibility remains, the fact
that we tested three different types
of M protein preparations made it
unlikely that M protein was
in a nonnative conformation in vitro. It is
also possible that
M protein binds to TFIID in vivo by a mechanism that
is not recreated
in vitro. However, M protein and TBP did not
coimmunoprecipitate
with antibodies to either protein (data not shown).
This suggests
that M protein acts indirectly in vivo through host
mechanisms
that are capable of regulating TFIID activity, in particular
the
activity of TBP, which is the only subunit required for basal
transcription.
Association of TBP with TAF subunits in VSV-infected cells.
There are two ways that the activity of TBP is known to be regulated:
(i) through its subunit interactions and (ii) by phosphorylation. TBP
is a subunit of three different transcription factors that function in
the activation of all three host RNAPs through association with
different TAF subunits (32). The TAF subunits of TFIID are
referred to as TAFIIs. Association of TBP with a set of
TAFIs forms the transcription factor SL1, which functions
in the activation of RNAPI, and association with a set of
TAFIIIs forms the transcription factor TFIIB, which
functions in the activation of RNAPIII. The activity of all three of
these transcription factors can be either enhanced or inhibited by
phosphorylation, depending on which site is phosphorylated (6,
11, 12, 14, 16, 22).
TBP-containing transcription factors in nuclear extracts from
VSV-infected or uninfected cells were analyzed by ion exchange
chromatography to determine whether there was an alteration in
the
association of TBP with TAF subunits in infected cells. The
nature of
the TAF subunits dictates the behavior of these transcription
factors
in ion exchange chromatography, and this technique has
been used
previously to assay association of TBP with TAF subunits
(
33). Nuclear extracts were prepared from uninfected or
VSV-infected
HeLa cells at 6 h postinfection and were
chromatographed on a
1.7-ml preparative cation exchange high-pressure
liquid chromatography
(HPLC) column (POROS HS; Perseptive Biosystems)
eluted with a
17-ml gradient of 50 mM to 1.0 M NaCl in buffer D, as
described
by Dignam et al. (
9), but lacking KCl. TBP in
column fractions
was determined by Western blot analysis (Fig.
2A) as described
previously
(
31), and the results of densitometry analysis of
the
Western blots are shown in Fig.
2B as the percentage of the
total TBP
eluted from the column. TBP in nuclear extracts from
both the infected
cells (Fig.
2B, dark bars) and the uninfected
cells (light bars) eluted
in two peaks. The first peak eluting
at lower salt concentrations was
centered around fraction 18 and
contained TFIIIB (
9). The
second peak was centered around fraction
23 and primarily contained
TFIID and the SL1. The second peak
was broader because TFIID is
heterogeneous in its composition
of TAF subunits (
5). The
elution profiles for TBP-containing
transcription factors from infected
cells were remarkably similar
to those from uninfected cells. In both
cases, approximately 40%
of TBP eluted in the first peak and 60% of
TBP eluted in the second.
These data indicate that there was no
difference in the association
of TBP with TAF subunits in infected
cells versus uninfected cells
that could be detected by ion exchange
HPLC. The same conclusion
was reached when the extracts were analyzed
by conventional chromatography
on a phosphocellulose column, as
described previously (
33),
rather than by HPLC (not
shown). In addition, the TFIID from infected
cells was inactive
following chromatography (
31). Thus, the
mechanism of M
protein inactivation of TFIID does not involve
direct, but labile,
protein-protein or protein-inhibitor interactions
that are dissociated
during chromatography.
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FIG. 2.
Ion exchange HPLC of TBP-containing transcription
factors. Nuclear extracts were prepared from VSV-infected or uninfected
HeLa cells at 6 h postinfection and chromatographed on an ion
exchange HPLC column eluted with a NaCl concentration gradient. (A)
Column fractions were assayed for TBP content by Western blot. (B)
Densitometry of the Western blots in panel A was used to determine the
percent of total TBP in each fraction for nuclear extracts from
infected (dark bars) or uninfected cells (light bars). The
concentration gradient of NaCl used to elute the column is also
shown.
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The conclusion that there was no difference in the association of TBP
with TAF subunits in infected cells versus uninfected
cells was
supported by sedimentation velocity analysis of TBP-containing
transcription factors. TFIID, which has an
s20,w
of 17S (
26,
34) is easily resolved from TFIIIB and SL1,
which have
s20,w of about 5 to 7S
(
28). Nuclear extracts were prepared from uninfected
or
VSV-infected HeLa cells at 6 h postinfection and loaded onto
10 to
30% sucrose gradients. After centrifugation at 40,000 rpm
in a TLS-55
rotor (Beckman Instruments) for 8 h at 4°C, fractions
were
collected and analyzed by Western blotting using antibody
against TBP.
Figure
3 shows the amount of TBP in each
fraction
quantitated by densitometry and represented as the percentage
of the total TBP recovered from the gradient. Two peaks of TBP
were
evident. The peak in the upper half of the gradient (fractions
2 through 6) corresponds to SL1 and TFIIIB, for which the sedimentation
velocity is about 5S. The TBP in fractions 2 through 6 together
amounted to 69% of the total for nuclear extracts from uninfected
cells and 65% for nuclear extracts from infected cells. The peak
in
the lower half of the gradient (fractions 8 through 11) is
TFIID, for
which the sedimentation velocity is about 17S. The
TBP in fractions 8 through 11 amounted to 22% of the total for
nuclear extracts from both
uninfected cells and infected cells.
In repeated experiments, there was
no significant difference in
the level of TFIID (18% for nuclear
extracts from uninfected cells
versus 22% for nuclear extracts from
infected cells in three experiments).
Also, there was no significant
difference in sedimentation velocity
of TFIID, which sedimented
9.33 ± 0.57 fractions for nuclear extracts
from uninfected cells
and 9.66 ± 0.57 fractions for nuclear extracts
from infected
cells. These data support the conclusion that TBP
associates normally
with TAF subunits in transcription factors
from VSV-infected cells.
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FIG. 3.
Sedimentation analysis of TBP-containing transcription
factors. Nuclear extracts were prepared from VSV-infected (dark bars)
or uninfected (light bars) HeLa cells at 6 h postinfection and
sedimented on 10 to 30% sucrose gradients. Gradient fractions and the
pelleted material (P) were assayed for TBP content by Western blots as
in Fig. 2, and the percentage of total TBP in each fraction was
determined by densitometry.
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Phosphorylation of TBP.
In addition to its association with
different subunits, the activity of TBP in each of its three
transcription factors can also be regulated by phosphorylation.
Phosphorylation can either enhance or inhibit the activity of TBP,
depending on which sites are phosphorylated. We considered that TBP
could be inactivated in VSV-infected cells by phosphorylation at an
inhibitory site. An alternative possibility involving dephosphorylation
of an activating site cannot account for the inhibition of
RNAPII-dependent transcription, since TBP is active in basal
RNAPII-dependent transcription in its unphosphorylated form, while TBP
in infected cells is almost completely inactive (31). HPLC
column fractions containing TBP similar to those shown in Fig. 2 were
combined into three pools, and the extent of phosphorylation of TBP was
determined by electrophoresis in a pH gradient gel, followed by Western
blotting with antibody against TBP (Fig.
4). This is a variant of the isoelectric
focusing technique, in which proteins are separated according to their isoelectric points but are not electrophoresed to equilibrium as in the
traditional isoelectric focusing technique (24, 27). This
technique provides better resolution for basic proteins such as TBP and
can detect single charge differences, such as those introduced by
phosphorylation. There are three forms of TBP in nuclear extracts from
HeLa cells (Fig. 4). The fastest migrating form (TBP1) corresponds to
unphosphorylated TBP. TBP2 is probably a singly phosphorylated form,
and TBP3 is a multiply phosphorylated form, as shown by conversion of
TBP2 and TBP3 into the TBP1 form by treatment with alkaline phosphatase
in vitro (not shown). Pool 1 consisted of HPLC fractions corresponding
to fractions 17 through 19 in Fig. 2B, which contained TFIIIB. The TBP
in pool 1 consisted primarily of the phosphorylated forms of TBP. This
is consistent with recent data showing that phosphorylation of TBP is
necessary for TFIIIB activity (11). Pool 2 was the first
half of the TFIID peak, corresponding to fractions 21 through 23, and
pool 3 was the second half of the TFIID peak (fractions 24 through 26).
Both pools contained primarily unphosphorylated TBP and a small amount of the TBP2 form. This is consistent with activity of TBP in
RNAPII-dependent transcription in its unphosphorylated form.
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FIG. 4.
Analysis of TBP phosphorylation by pH gradient gel
electrophoresis. Nuclear extracts were prepared from VSV-infected (I)
or uninfected (U) HeLa cells at 6 h postinfection and
chromatographed on an ion exchange HPLC column eluted with a NaCl
concentration gradient as in Fig. 2. Column fractions were combined
into pool 1 (fractions 17 through 19), pool 2 (fractions 21 through
23), and pool 3 (fractions 24 through 26) and electrophoresed on a pH
gradient gel. TBP was detected by Western blot and migrated as three
forms, with TBP1 being the fastest migrating form and TBP3 the
slowest.
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The most important result of the experiment in Fig.
4 was the finding
that there was no difference in the state of phosphorylation
of TBP
between infected cells and uninfected cells. In particular,
most of the
TBP in TFIID was in the unphosphorylated form in the
nuclear extracts
from both infected and uninfected cells. The
same results were obtained
when the phosphatase inhibitor sodium
vanadate (5 mM) was included in
all the buffers used to prepare
nuclear extracts and perform the HPLC,
as well as when the vanadate
was omitted, as it was in our previous
study demonstrating that
TFIID from infected cells is inactive
(
31). Since the unphosphorylated
form of TBP is fully
active in RNAPII-dependent transcription,
this observation rules out
phosphorylation of TBP as a mechanism
of inactivation of TFIID in
infected cells. Collectively, these
data indicate that M protein
inactivates TBP activity in RNAPII-dependent
transcription by a novel
mechanism, since the known mechanisms
for regulating TBP activity
cannot account for the
inhibition.
These results on the mechanism of inhibition of transcription by VSV
provide an interesting comparison with the inhibition
by poliovirus.
Both viruses inhibit basal RNAPII-dependent transcription
by
inactivation of TBP. In the case of poliovirus, the viral 3C
protease
inactivates TBP directly by cleavage at one or more sites
(
7,
30). Unlike poliovirus 3C protease, M protein does not
have any
known catalytic activity that could be responsible for
the inhibition
of TBP activity. Also, unlike poliovirus 3C protease,
M protein did not
inhibit transcription in nuclear extracts from
uninfected cells when
added in vitro (Fig.
1). M protein is a
very potent inhibitor of
transcription in vivo. We have estimated
that 50% inhibition of host
RNAPII-dependent transcription in
transfection experiments occurs when
the amount of M protein is
approximately 1,000-fold less than the
amount of M protein expressed
in VSV-infected cells (
20).
Thus, it is unlikely that the amounts
of M protein added to nuclear
extract from uninfected cells in
the experiments shown in Fig.
1 were
simply not sufficient, since
they were similar to the amounts present
in nuclear extract from
VSV-infected cells. The conclusion that M
protein does not directly
inactivate TBP was supported by the inability
to coimmunoprecipitate
M protein and TBP with antibodies to either
protein. The coimmunoprecipitation
assays were sufficiently sensitive
that if 10 to 25% of TFIID
coprecipitated with M protein, the
interaction would have been
readily detected (data not shown). These
results support the idea
that M protein acts indirectly in vivo through
host mechanisms
that are capable of inhibiting TBP
activity.
It has been proposed that the inhibition of transcription in
VSV-infected cells is an indirect effect of the inhibition of
nuclear-cytoplasmic transport by M protein, leading to a reduction
in
the levels of the critical transcription factors (
13,
29).
However, in the case of TFIID, we found that there was no reduction
in
the amount of TFIID in nuclear extracts from VSV-infected cells,
but
instead the TFIID was in an inactive form (
31; Fig.
2 and
3). Our observation that the inactivation of TFIID was an indirect
effect caused by M protein leaves open the possibility that the
M
protein-induced block in nuclear-cytoplasmic transport affects
the
activity of a host factor involved in regulating TFIID
activity.
Phosphorylation of TBP by a cellular kinase was a likely candidate for
the mechanism of inhibition of transcription initiation,
since
phosphorylation of TBP can either inhibit or enhance the
activity of
all three TBP-containing transcription initiation
factors, depending on
which sites are phosphorylated. For example,
phosphorylation of TBP by
cdc2/cyclin B kinase has been implicated
in the silencing of
transcription by all three host RNAPs during
mitosis (
12,
14,
16). Analysis of TBP phosphorylation in
VSV-infected cells
showed that there were dramatic differences
among the different
transcription factors, but there was little
if any difference between
infected and uninfected cells (Fig.
4). In particular, TFIIIB contained
primarily phosphorylated TBP,
while TFIID contained primarily
unphosphorylated TBP. These results
extend to HeLa cells the results
obtained in the yeast
Saccharomyces cerevisiae, in which
phosphorylation of TBP in TFIIIB was originally
demonstrated
(
11). In yeasts, phosphorylation of TBP by casein
kinase
II is required for TFIIIB activity. In contrast to TFIIIB,
TFIID is
active when TBP is in the unphosphorylated form, although
its activity
can be enhanced by phosphorylation by DNA-dependent
protein kinase
(
6). Since most of the TBP in TFIID was in the
unphosphorylated form in nuclear extracts from both infected and
uninfected cells, differences in phosphorylation of TBP cannot
account
for the inactivation of
TFIID.
The normal phosphorylation and assembly of TBP with TAF subunits in
VSV-infected cells implies that there is a novel inhibitory
factor
responsible for the inactivation of TFIID. Such an inhibitory
factor
might act by associating with TFIID in a manner similar
to the TAF
subunits. If so, such a factor did not affect the behavior
of TFIID in
ion exchange HPLC nor affect its sedimentation velocity.
Alternatively,
TFIID could be inhibited by a posttranslational
modification other than
phosphorylation of TBP. If so, then this
modification would not alter
the charge on TBP, since there was
no change in its isoelectric point.
Both of these possibilities
are currently being
investigated.
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ACKNOWLEDGMENTS |
We thank Barbara Yoza, Griffith Parks, and David Ornelles for
helpful advice and comments on the manuscript.
This work was supported by Public Health Service grant AI 32983 from
the National Institute of Allergy and Infectious Diseases.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1064. Phone: (336) 716-4237. Fax: (336) 716-9928. E-mail: dlyles{at}wfubmc.edu.
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Journal of Virology, May 2001, p. 4453-4458, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4453-4458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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