Department of Oncology, University of
Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada
During lytic infection, herpes simplex virus type 1 (HSV-1)
represses host transcription, recruits RNA polymerase II (RNAP II) to
viral replication compartments, and alters the phosphorylation state of
the RNAP II large subunit. Host transcription repression and RNAP II
modifications require expression of viral immediate-early (IE) genes.
Efficient modification of the RNAP II large subunit to the
intermediately phosphorylated (IIi) form requires expression of ICP22
and the UL13 kinase. We have further investigated the mechanisms by
which HSV-1 effects global changes in RNAP II transcription by
analyzing the RNAP II holoenzyme. We find that the RNAP II general
transcription factors (GTFs) remain abundant after infection and are
recruited into viral replication compartments, suggesting that they
continue to be involved in viral gene transcription. However, virus
infection modifies the composition of the RNAP II holoenzyme, in
particular triggering the loss of the essential GTF, TFIIE. Loss of
TFIIE from the RNAP II holoenzyme requires viral IE gene expression,
and viral IE proteins may be redundant in mediating this effect.
Although viral IE proteins do not associate with the RNAP II
holoenzyme, they interact with RNAP II in complexes of lower molecular
mass. As the RNAP II holoenzyme containing TFIIE is necessary for
activated transcription initiation and RNAP II large subunit
phosphorylation in uninfected cells, virus-induced modifications to the
holoenzyme may affect both of these processes, leading to aberrant
phosphorylation of the RNAP II large subunit and repression of host
gene transcription.
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INTRODUCTION |
Herpes simplex virus type 1 (HSV-1)
is a 152-kb double-stranded DNA virus, the genome of which is
transcribed and replicated within the host cell's nucleus (reviewed in
reference 54). During lytic infection, the HSV-1 genes are
transcribed by the host's RNA polymerase II (RNAP II) transcription
machinery (20, 71). The HSV-1 genes are expressed in a
regulated cascade and are classified into three groups based on their
order of expression: immediate-early (IE), delayed-early (DE), and late
(L). The five IE genes (encoding ICP4, ICP0, ICP27, ICP22, and ICP47)
are expressed immediately after infection, and all IE gene products
except ICP47 are regulatory proteins involved in controlling expression
of the DE and L genes. Their synthesis reaches a peak between 2 and
4 h postinfection, but IE proteins persist throughout infection.
Infection with HSV-1 results in dramatic alterations to host gene
transcription. Within 6 h postinfection, RNAP II transcription of
many, if not most, cellular genes is repressed to less than 40% of
uninfected levels (36, 61, 64, 66), and transcription levels decline for at least another 6 h. At the same time, RNAP II
transcription of HSV-1 genes is induced to high levels (20, 64). We have shown that repression of host gene transcription does not require DE or L gene transcription or viral DNA replication. Also, virion components do not trigger host transcription repression in
the absence of viral IE gene expression (64). The IE
proteins may be redundant in their effects on host gene transcription, as transcription is repressed after infection with viruses bearing null
mutations in individual IE genes (64).
The preferential transcription of viral DE and L genes over host cell
genes cannot be explained by sequence differences between host and
viral gene promoters (reviewed in reference 61). Each of
the viral gene promoters displays features of typical RNAP II
promoters. IE gene promoters contain TATA boxes, start sites, and
TAATGARAT elements that bind cellular complexes containing Oct-1. The
virion transactivator VP16, in association with Oct-1 and HCF,
binds to these elements and stimulates transcription of each IE gene
(31, 41). The viral DE gene promoters are simple RNAP II
promoters, containing TATA boxes, start sites, and promoter-proximal
cis-acting sequences that bind basal cellular transcription
factors such as Sp1. L gene promoters are even simpler than those of DE
genes, comprising only TATA boxes and sequences surrounding
transcription start sites. Neither DE nor L gene promoters contain
specific binding sites for viral regulatory proteins that are necessary
for DE or L gene transcription during infection (60). The
sequence independence of host and viral gene transcription regulation
is demonstrated by studies of cellular genes expressed within the viral
genome. For example, the 
-globin gene, with 1.2 kb of upstream
promoter DNA, is expressed as a viral DE gene after infection of MEL
cells with the recombinant -globin-HSV; however, the endogenous
cellular -globin gene is transcriptionally repressed (59,
61). These and similar data have led to the hypothesis that the
global shift in RNAP II promoter recognition that occurs after HSV-1
infection may be due to sequence-independent mechanisms, such as
recruitment of transcription proteins into viral compartments,
differences between host and viral chromatin, and modification of the
host's RNAP II transcription machinery (61).
We have reported previously that RNAP II is recruited into viral
replication compartments following HSV-1 infection (51). However, considering that repression of host transcription occurs after
infection with ICP4 mutant viruses, and that replication compartments
do not form during these infections, relocation of RNAP II to viral
replication compartments is unlikely to be a requirement for host
transcription repression (64). Nonetheless, RNAP II
recruitment to viral replication compartments may be a requirement for
efficient activation of viral DE and L gene transcription.
We have also reported that HSV-1 infection alters the phosphorylation
state of the RNAP II large subunit (51). RNAP II consists of at least 10 subunits, the largest of which contains sites of catalysis and of DNA and nascent RNA binding and the
carboxy-terminal domain (CTD). The CTD is a highly conserved structure
of 52 repeats of the sequence YSPTSPS and is essential for cell
viability (reviewed in reference 10). Due to cyclic
phosphorylation of serine, threonine, and tyrosine residues on the CTD,
RNAP II is present in two forms in vivo
RNAP IIO
(hyperphosphorylated) and RNAP IIA (hypophosphorylated). RNAP IIA is
recruited to preinitiation complexes, and RNAP IIO is the form of the
enzyme involved in transcription elongation. The transition from IIA to
IIO occurs during transcription initiation and is due to the activities
of CTD kinases including cyclin-dependent kinase 7 (cdk7, a subunit of
TFIIH) and cdk9 (a subunit of P-TEFb) (9, 10, 35, 69). The
CTD has several functions in vivo, including mediating responses to
transcription activators and stimulating transcription elongation. The
CTD may also play a role in pre-mRNA processing by recruiting mRNA
processing factors to nascent transcripts (26).
We have reported that within 5 h postinfection, RNAP IIO, the
hyperphosphorylated form of RNAP II, is replaced by an intermediately phosphorylated, transcriptionally active form, RNAP III. The viral IE
protein ICP22 and the viral kinase UL13 are required for efficient production of RNAP III, but the hyperphosphorylated RNAP IIO form is
lost after infection with both wild-type and individual IE null mutant
viruses (33, 50, 51). Given the importance of the CTD and
its phosphorylation cycle to transcription regulation, we have
hypothesized that HSV-1-induced alterations to CTD phosphorylation may
contribute to the shift in transcription from host to viral genes.
In order to initiate transcription, RNAP II requires a number of
accessory factorsthe general transcription factors (GTFs). These
include TFIID, TFIIB, TFIIF, TFIIH, and TFIIE. It is now thought that
most or all of the GTFs participate in stable, preassembled RNAP II
complexes (the ~2-MDa holoenzymes) that are thought to be the
physiologically relevant forms of RNAP II that enter most (but not all)
preinitiation complexes in vivo (7, 22, 24, 38, 67).
Genetic studies in yeast show that artificial recruitment of a
holoenzyme subunit to promoter DNA is sufficient to activate transcription and remodel chromatin (17, 19). Although the composition of the RNAP II holoenzyme varies somewhat depending on the
method of isolation, holoenzymes from human cells contain at least RNAP
II, TFIIE, TFIIF, and TFIIH, and some contain TFIIB and TFIID. Besides
the GTFs, RNAP II holoenzymes contain a number of factors that mediate
transcription activation, modify chromatin structure, and interact with
mRNA processing factors (reviewed in reference 24).
In this study, we examine virus-induced modifications to the RNAP II
holoenzyme. We find that HSV-1 infection leads to depletion of TFIIE
from the RNAP II holoenzyme and a reduction in holoenzyme activity in
vitro. TFIIE depletion requires IE gene expression but occurs in the
absence of individual IE proteins, suggesting that two or more IE
proteins may be redundant in their effects on RNAP II holoenzyme
composition. Since TFIIE is an essential holoenzyme GTF and stimulates
RNAP II large subunit phosphorylation mediated by TFIIH, its absence
from the holoenzyme may contribute to aberrant phosphorylation of the
RNAP II large subunit and altered transcription patterns during HSV-1 infection.
| |
MATERIALS AND METHODS |
Cells, viruses, and infections.
HeLa S3 cells (American Type
Culture Collection, Manassas, Va.) were used for infections. Cells were
propagated in Dulbecco modified Eagle medium containing 10% fetal
bovine serum. HeLa S3 cells were grown either as monolayers or as
suspension cells in spinner flasks.
Infections were performed at a multiplicity of infection (MOI) of 10 PFU per cell. Infections of monolayer cells were performed as described
elsewhere (50). Suspension cells were infected by addition
of virus at 10 PFU/cell and incubated for 12 h.
The following HSV-1 strains were used: the wild-type strain F22, the
ICP4 mutant strain d120 (13), the ICP0 mutant
strain n212 (5), the ICP27 mutant strain
d27-1 (49), and the ICP22 mutant
d22-lacZ (33). The F22 virus
contains a FLAG epitope-tagged ICP22 gene and is a derivative of the
wild-type virus KOS1.1. The 12-codon FLAG epitope was inserted between
codons 6 and 7 of the ICP22 gene in KOS1.1. F22 is phenotypically wild
type by viral transcription and growth in ICP22 permissive and
restrictive cell lines. Details of the construction and
characterization of F22 will be described elsewhere (C. A. Spencer
and S. A. Rice, unpublished data). Growth and titering of virus
strains have been described previously (33, 50).
UV-inactivated virus stock was prepared as described previously
(51) from a stock of the wild-type HSV-1 strain KOS1.1. The titer of UV-inactivated virus was 4 to 5 orders of magnitude reduced from that of the parent stock. Infections with UV-inactivated virus were carried out using the stock's preirradiation titer to
obtain an MOI of 10.
Western blotting and immunofluorescence.
Preparation of
whole-cell extracts and immunoblotting were performed as described
previously (51). Blots were probed with the following
primary antibodies: the anti-RNAP II large subunit antibodies ARNA-3
(29) (Research Diagnostics) at a 1:2,000 dilution and
8WG16 (68) at a 1:7,500 dilution, the anti-p56E antibody TFIIE-
C-17 (Santa Cruz) at a 1:1,000 dilution, an anti-TATA-binding protein (anti-TBP) antibody (Promega) at a 1:1,000 dilution, the anti-MO15 antibody MO1.1 (NeoMarkers) at a 1:400 dilution, the anti-RAP74 antibody 7B3 (Austral Biologicals) at a 1:300 dilution, the
anti-MLH antibody hMLH1 C-20 (Santa Cruz) at a 1:100 dilution, the anti-FLAG antibody M2 (Eastman Kodak) at 1:1,000, the anti-ICP4 antibody H1101 at 1:2,000, the anti-ICP27 antibody H1119 at 1:8,000, and the anti-ICP0 antibody H1112 at 1:2,500 (Goodwin Institute). Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse (or anti-rabbit) immunoglobulin G (IgG) (Jackson
ImmunoResearch). Blots were visualized using the ECL Western blotting
detection system (Amersham).
Immunostaining was performed as described previously (65).
Primary antibodies were the anti-ICP4 antibody H1101 conjugated to
either Texas Red or Oregon Green fluorochrome (Molecular Probes) and
used at a 1:200 dilution, 8WG16 at 1:200, anti-TBP at 1:200, TFIIE-
C-17 at 1:50, and anti-p62H (Upstate Biotechnology) at 1:100. Secondary
antibodies were goat anti-mouse Alexa 488 at a 1:250 dilution, goat
anti-rabbit Alexa 488 at a 1:250 dilution (Molecular Probes), or
lissamine rhodamine-conjugated goat anti-mouse IgG at a
1:200 dilution (Jackson ImmunoResearch). DNA was visualized by staining
cells with 4',6'-diamidino-2-phenylindole (DAPI) at 5 µg/ml.
Coverslips were mounted in glycerol, and cells were visualized with a
Zeiss LSM510 confocal microscope using sequential laser scans for each
fluorochrome. Images for Fig. 8 were converted to tagged-image format
files (TIFFs) and were assembled and labeled using Adobe Photoshop
software. Control stains included secondary antibodies only, ICP4
stains only, and primary or secondary stains only in the absence of
ICP4 staining. Bleedthrough staining was undetectable using confocal
microscopy and sequential laser scans.
Nuclear extract preparation.
Nuclear extracts were prepared
from 109 uninfected or infected HeLa S3 cells by
following the methods of Shapiro et al. (58). HeLa S3
cells were grown to a density of approximately
106/ml in spinner flasks and were infected with
wild-type or mutant HSV-1 strains at an MOI of 10. At 12 h
postinfection, cells were harvested and nuclei were isolated as
described previously (58). Nuclear extracts were dialyzed
into nuclear dialysis buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 2 mM dithiothreitol [DTT], and 20% [vol/vol]
glycerol) and typically contained protein concentrations between 8 and
15 mg per ml. Extracts were frozen in liquid nitrogen until use.
Sepharose CL-2B gel filtration chromatography.
Approximately
2 mg of nuclear extracts was fractionated on 25-ml Sepharose CL-2B
columns as described previously (45). The CL-2B columns
were equilibrated and run with CL-2B buffer (20 mM HEPES [pH 7.9],
100 mM KCl, 20% glycerol) at 4°C. Fractions of 500 µl were
collected and precipitated with acetone. Precipitated proteins were
resuspended in sodium dodecyl sulfate (SDS) loading buffer and analyzed
by immunoblotting. Bands on Western blots were quantitated by scanning
autoradiographs with an LKB Ultroscan XL Enhanced Laser Densitometer.
Densitometer readings were normalized against identical standards (20 µg of nuclear extract) on each gel. Normalized readings were plotted
using DeltaGraph 4.0 software, and plots were assembled and labeled
using Quark Xpress software (Fig. 3, 5, and 9).
Sepharose CL-2B columns were calibrated with Blue Dextran 2000 (2 MDa;
Pharmacia) and thyroglobulin (670 kDa; Bio-Rad). To control for protein
binding to RNA and DNA, an infected and an uninfected nuclear extract
were incubated at 4°C for 20 min in 20 µg of ethidium bromide/ml,
100 µg of RNase A/ml, and 500 U of DNase I/ml prior to CL-2B chromatography.
Affinity purification of the RNAP II holoenzyme.
TFIIS
affinity chromatography was carried out as described by Pan et al.
(45). Affinity columns were prepared by binding glutathione S-transferase (GST) or GST-TFIIS fusion protein
to glutathione-Sepharose 4B beads as described by the
manufacturer (Amersham Pharmacia). Affinity matrices of 50 µl
were dispensed into 250-µl columns (Promega) and washed with 20 bed
volumes of ACB (10 mM HEPES [pH 7.9], 50 mM NaCl, 0.1 mM EDTA, 0.1 mM
DTT, 10% glycerol). Columns were blocked with ACB plus 0.1% bovine serum albumin (BSA). Affinity matrices were incubated with either pooled 2-MDa fractions (fractions surrounding the 2-MDa peak) or pooled
670-kDa fractions (fractions at and below 670 kDa) from Sepharose CL-2B
chromatography (fractions indicated in Fig. 2 and 3). The flowthrough
was reapplied once. Columns were washed with 20 bed volumes of ACB.
Bound proteins were then eluted successively with 4 bed volumes of ACB
plus 0.3 M NaCl and ACB plus 0.5 M NaCl. Proteins in the eluates were
concentrated by acetone precipitation and analyzed by immunoblotting.
In vitro transcriptions.
The DNA template for in vitro
transcriptions consisted of the 2.4-kb SmaI fragment
of adenovirus (Ad), containing the Ad major late promoter (MLP)
(14), cloned into vector pSG5 (Stratagene) to create
plasmid pSG5Ad. pSG5Ad was digested with BamHI, 540 bp
downstream of the Ad MLP transcription start site. Reaction mixtures
contained either crude nuclear extracts (46 µg) or concentrates from
the ~2-MDa or ~670-kDa peaks of Sepharose CL-2B columns. Fractions
surrounding the 2-MDa or 670-kDa peaks (as indicated in Fig. 2 and 3)
were pooled and concentrated 50- or 10-fold in UltraFree-0.5
concentrators (Millipore Corp.). Ten microliters of concentrate was
used in each reaction. An aliquot of each nuclear extract and
concentrate was analyzed by immunoblotting, to estimate the relative
quantities of RNAP II in each reaction. Transcription reactions were
carried out as follows.
DNA template (600 ng) was preincubated at 4°C for 15 min in a
solution containing nuclear extract (or column concentrate), 10 mM
HEPES (pH 7.6), 50 mM KCl, 8% glycerol, 0.04 mM EDTA, and 0.1 mM DTT.
After preincubation, magnesium chloride was added to 8 mM, plus ATP,
GTP, and CTP to 0.4 mM, and 10 µCi of
5'-[-32P]UTP. Reactions were terminated
after 45 min at 30°C by addition of 300 mM Tris HCl (pH 7.4)-300 mM
sodium acetate-0.5% SDS-2 mM EDTA. Reaction products were treated
with 50 µg of proteinase K for 20 min at 42°C, then phenol
extracted and precipitated. RNA was separated on 5% acrylamide-urea
gels, and radioactivity was visualized by autoradiography. Signals were
quantitated by phosphorimaging (Fujix Bas100 analyzer).
Artwork for autoradiographs.
Autoradiographs (Fig. 1, 2, 4,
6, 7, and 10) were scanned on a Hewlett-Packard desk scanner, and scans
were cropped and saved as TIFFs using Adobe Photoshop software. Images
were assembled and labeled using Quark Xpress software.
| |
RESULTS |
HSV-1 infection alters the composition of the RNAP II
holoenzyme.
We have previously reported that HSV-1 infection
brings about rapid modifications in RNAP II large subunit
phosphorylation and repression of host transcription (51,
64). Since transcription initiation and large subunit
phosphorylation are carried out by the RNAP II holoenzyme in uninfected
cells, we reasoned that these changes may reflect virus-induced
modifications to the composition of the RNAP II holoenzyme.
To assess the abundance of RNAP II GTFs during infection, we prepared
whole-cell lysates from mock-infected cells or cells infected with the
wild-type virus KOS1.1 and analyzed their GTF contents by
immunoblotting. None of the GTFs examined displayed significant
alterations in abundance or migration on SDS-polyacrylamide gel
electrophoresis (PAGE) gels, up to 9 h postinfection (Fig. 1). We also examined the abundance of
p62H (a subunit of TFIIH), cdk8, cdk9, and cyclin T1 after infection
and found no significant changes (data not shown). As seen previously,
the RNAP II large subunit was aberrantly phosphorylated after
infection, leading to the loss of the hyperphosphorylated IIo form and
the appearance of the intermediately migrating IIi form (Fig. 1).
Treatment of lysates with calf intestinal phosphatase collapsed
hyperphosphorylated forms of the RNAP II large subunit to the
hypophosphorylated (IIa) form and eliminated the apparent abundance
differences, which are due to the polydispersed migration of
phosphorylated forms (reference 51; also data not shown).
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FIG. 1.
Abundance of RNAP II and GTFs in mock-infected and
HSV-1-infected cells. Whole-cell lysates were prepared from
mock-infected cells or cells infected with wild-type virus for the
times indicated. Samples representing equal numbers of cells were
analyzed by immunoblotting, and blots were probed with antibodies that
recognize subunits of the factors indicated. The RNAP II large subunit
shows three phosphorylation variants: IIo, IIi, and IIa.
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We next determined the GTF composition of RNAP II holoenzymes from
uninfected cells. In order to analyze RNAP II holoenzymes, we used a
combination of Sepharose CL-2B gel filtration chromatography and TFIIS
affinity chromatography. These methods have been used by others to
purify and characterize RNAP II holoenzymes from mammalian cells
(37, 45, 72). We first prepared nuclear extracts from
uninfected HeLa cells and then separated proteins using Sepharose CL-2B
gel filtration chromatography. Column fractions were concentrated and
analyzed by immunoblotting, using antibodies that recognize the large
subunit of RNAP II, the p56 subunit of TFIIE, the TBP subunit of TFIID,
and the cdk7 subunit of TFIIH. Gel filtration chromatography showed
that RNAP II, TFIID (TBP), TFIIE (p56E), and TFIIH (cdk7) from
uninfected nuclear extracts eluted in fractions surrounding 2 MDa (Fig.
2A). These factors also eluted in
fractions below 670 kDa, similar to patterns seen previously (43,
45). Sepharose CL-2B efficiently separates proteins from
approximately 1 to 10 MDa; proteins below approximately 1 MDa elute
together over a broad peak. Hence, our molecular mass estimates for
macromolecules and complexes below ~1 MDa are based on the elution
peak of the 670-kDa thyroglobulin marker. Treatment of uninfected
nuclear extracts with ethidium bromide, DNase I, and RNase A prior to
CL-2B chromatography did not alter the elution profiles of RNAP II and
the GTFs (data not shown), indicating that their presence in
high-molecular-mass fractions was not due to interactions with nucleic
acids in the nuclear extracts. In addition, the DNA repair protein MLH1
did not elute in the 2-MDa peak (Fig. 2A), demonstrating that the
presence of RNAP II GTFs in high-molecular-mass complexes is not a
general feature of proteins that interact with DNA.
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FIG. 2.
Gel filtration chromatography of RNAP II and GTFs.
Nuclear extracts from uninfected cells (A) or cells infected with
wild-type HSV-1 (B) were chromatographed on Sepharose CL-2B columns.
Fractions were concentrated and analyzed by immunoblotting. Blots were
probed with antibodies that recognize the RNAP II large subunit, the
p56E subunit of TFIIE, the TBP subunit of TFIID, and the cdk7 subunit
of TFIIH. Blots were also probed with an antibody that recognizes the
DNA repair protein MLH1. Lane 1 contains 20 µg of uninfected (A) or
infected (B) nuclear extract. Fractions 21 to 31 surrounded ~2 MDa;
fractions 42 to 52 were at and below ~670 kDa. Ten fractions between
approximately 1 MDa and 670 kDa are not shown in this figure. Fractions
24 through 28 (2 MDa Pool) were pooled for GST-TFIIS affinity
chromatography and in vitro transcription assays (Fig. 4, 6, and 10A),
as were fractions 43 through 52 (670 kDa Pool) (Fig. 7 and 10B).
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In order to assess the relative quantities of GTFs in the 2-MDa peak
from uninfected nuclear extracts, we scanned Western blots by
densitometry, normalized bands to identical standards (nuclear
extracts) on each gel, and plotted these as relative densitometry units
(Fig. 3, top panels). The relative
densitometry units in eight fractions surrounding the 2-MDa peak were
added and expressed as a percentage of total densitometry units over the entire chromatography run (Table 1).
Approximately 14% of RNAP II, 15% of TFIIE (p56), and 29% of TFIIH
(cdk7) were present in the ~2-MDa fractions. In contrast,
approximately 73% of total TFIID (TBP) was present in these fractions.
Since TBP participates in a number of high-molecular-mass complexes,
including RNAP I and RNAP III holoenzymes (7, 56, 70), the
presence of TBP in the ~2-MDa fractions likely represents a mixture
of TBP-containing holoenzyme complexes of all three nuclear RNA
polymerases.
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FIG. 3.
Elution profiles of RNAP II and GTFs from Sepharose
CL-2B gel filtration chromatography. Nuclear extracts from uninfected
cells or cells infected with HSV-1 were chromatographed and analyzed by
immunoblotting, as described in the legend to Fig. 2. The intensity of
each band was quantitated by densitometry. Densitometer readings were
normalized against identical standards on each gel (20 µg of infected
or uninfected nuclear extract) and expressed as relative densitometry
units. Relative densitometry units were plotted against fraction
number. Positions of 2-MDa and 670-kDa molecular standards are
indicated. Horizontal lines below graphs indicate fractions at ~2 MDa
and ~670 kDa that were pooled for GST-TFIIS affinity chromatography
and in vitro transcriptions (Fig. 4, 6, 7, and 10).
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To determine whether GTF components of the ~2-MDa peak from
uninfected extracts copurify with RNAP II, we pooled fractions surrounding the 2-MDa peak from CL-2B columns (fractions indicated in
Fig. 2 and 3) and subjected these to GST-TFIIS affinity chromatography. The RNAP II transcription elongation factor TFIIS is used as an immobilized ligand for RNAP II holoenzyme affinity purification (45). TFIIS binds directly to RNAP II, although it may
also interact with other members of the RNAP II holoenzyme during
affinity chromatography (1, 45, 63). Holoenzymes purified
by GST-TFIIS chromatography are ~2 MDa in mass and contain RNAP II
and all GTFs including TFIID. Pan et al. (45) have shown
that RNAP II holoenzymes elute in the 0.3 M elution. Fractions
surrounding the 2-MDa peak were pooled and passed over GST-TFIIS
affinity columns. The columns were washed, and bound proteins were
eluted successively with buffers containing 0.3 and 0.5 M NaCl.
Eluates were concentrated and analyzed by immunoblotting.
GST-TFIIS chromatography of pooled 2-MDa fractions showed that TFIIE
(p56E), TFIID (TBP), and TFIIH (cdk7) coeluted with RNAP II in
holoenzymes from uninfected cells (Fig.
4A). Holoenzyme subunits did not elute
from control GST-Sepharose columns.
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FIG. 4.
Affinity chromatography of pooled ~2-MDa fractions
from gel filtration chromatography. Nuclear extracts from uninfected or
infected cells were fractionated by Sepharose CL-2B gel filtration
chromatography. Fractions surrounding the 2-MDa peak were pooled
(fractions indicated in Fig. 2 and 3) and chromatographed on GST and
GST-TFIIS columns. Bound proteins were eluted sequentially with buffers
containing 0.3 and 0.5 M NaCl. Eluates were concentrated and analyzed
by immunoblotting with antibodies that recognize the large
subunit of RNAP II, the p56E subunit of TFIIE, the TBP subunit of
TFIID, and the cdk7 subunit of TFIIH. Each blot contained a lane of
uninfected (A) or infected (B) nuclear extract (20 µg each) to
indicate positions of GTFs, as well as a lane containing one-quarter of
the flowthrough (Fl-Thru) and a lane containing one-half of the wash.
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In summary, RNAP II holoenzymes from uninfected cells contain RNAP II,
TFIIE, TFIIH, and TFIID, as seen previously (43-45, 72).
We next wanted to analyze the GTF contents of RNAP II holoenzymes
isolated from cells infected with HSV-1. We infected HeLa cells with
wild-type HSV-1, prepared nuclear extracts, and fractionated the
extracts using Sepharose CL-2B gel filtration chromatography. Fractions
from CL-2B columns were concentrated and analyzed by immunoblotting,
using the antibodies that recognize the RNAP II large subunit, p56E,
TBP, and cdk7. A typical elution profile is shown in Fig. 2B. Although
RNAP II, TBP, and cdk7 continued to elute in the ~2-MDa fractions
after infection, p56E appeared to be depleted in these
high-molecular-mass fractions from HSV-1-infected nuclear extracts.
In order to quantitate the relative abundance of each of these factors
in the ~2-MDa fractions, we scanned Western blots by densitometry,
normalized band intensities to identical standards on each gel, and
plotted values as relative densitometry units (Fig. 3, bottom panels).
The relative densitometry units in eight fractions surrounding the
2-MDa peak were added and expressed as a percentage of total relative
densitometry units over the entire chromatography run (Table 1). As
seen in Fig. 3, the Sepharose CL-2B elution profile of RNAP II from
infected extracts was similar to that from uninfected extracts.
Approximately 14% of the total RNAP II eluted in the 2-MDa peak in
both infected and uninfected nuclear extracts (Table 1). The CL-2B
elution profile of TFIIH (cdk7) from infected extracts was similar to
that for uninfected extracts (Fig. 2B and Fig. 3, lower panels) but
showed a shift towards lower-molecular-mass fractions. Approximately
19% of total cdk7 from infected extracts eluted in the 2-MDa range,
compared to approximately 29% from uninfected extracts. A more
pronounced change was apparent for TFIID (TBP). After infection,
approximately 32% of the total TBP eluted in the ~2-MDa fractions,
compared to 73% eluting in these fractions prior to infection. As
noted above, TBP participates in a number of high-molecular-mass
complexes including RNAP I, II, and III holoenzymes. Depletion of TBP
in high-molecular-mass fractions after HSV-1 infection may indicate disruption of any of these complexes. The greatest change was apparent
for TFIIE (p56E). Although p56E was abundant in the 2-MDa peak from
uninfected nuclear extracts (15% of total p56E), p56E eluting in the
2-MDa peak was reduced to <1% of total p56E in infected
nuclear extracts (Fig. 2B and 3; Table 1).
In order to determine whether GTF components of the ~2-MDa fractions
from HSV-1-infected extracts copurified with RNAP, we pooled fractions
surrounding the ~2-MDa peak from CL-2B columns (pooled fractions
indicated in Fig. 2 and 3) and performed GST-TFIIS affinity
chromatography. GST-TFIIS chromatography showed that TFIID (TBP) and
TFIIH (cdk7) copurified with RNAP II in holoenzymes from infected cells
(Fig. 4B). TFIIE (p56E), although present at low levels in the pooled
fractions, did not elute in either the 0.3 or the 0.5 M salt elution
from GST-TFIIS columns. RNAP II and GTF subunits did not elute from
control GST-Sepharose columns. In addition, the GST-TFIIS affinity
elution patterns of RNAP II holoenzymes were altered after infection,
with a portion of complexes eluting in the 0.5 M fraction. In summary,
RNAP II holoenzymes from HSV-1-infected cells contain RNAP II, TFIIH,
and TFIID but are depleted of the GTF TFIIE.
Loss of TFIIE from the RNAP II holoenzyme requires expression of
viral IE genes.
The observation that TFIIE is depleted in the
postinfection RNAP II holoenzyme was surprising, since TFIIE is a
consistent component of mammalian RNAP II holoenzymes and is an
essential GTF, required for TFIIH-mediated CTD phosphorylation and
transcription activation. This raised the possibility that loss of
TFIIE from the holoenzyme may contribute to some of the alterations in
RNAP II and transcription patterns that occur after HSV-1 infection, including aberrant phosphorylation of the RNAP II CTD and selective repression of host gene transcription. We previously showed that CTD
modification and host transcription repression require expression of
viral IE proteins and that these proteins may be redundant in their
capacity to modify RNAP II and host transcription. In order to
determine whether loss of TFIIE from the holoenzyme has similar
requirements for IE gene expression, we first analyzed the TFIIE
content of RNAP II holoenzymes isolated from cells infected with the
ICP4-null mutant virus d120 (13). Because ICP4
is required for transcription of DE and L genes and hence for viral DNA
replication, infections with ICP4-null viruses are arrested at the IE
stage (20). We prepared nuclear extracts from cells
infected with the d120 virus, fractionated the extracts by
Sepharose CL-2B gel filtration chromatography, and analyzed the
fractions by immunoblotting (Fig. 5). The
levels of p56E were low in the 2-MDa peak, similar to patterns seen in
nuclear extracts prepared from cells infected with wild-type HSV-1
(Fig. 3). These data indicate that expression of ICP4 is not required
for loss of TFIIE from the 2-MDa RNAP II holoenzyme. In addition, DE
and L gene expression, as well as viral DNA replication, are not
required for this effect.
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FIG. 5.
Elution profiles of TFIIE (p56E subunit) from Sepharose
CL-2B gel filtration chromatography. Nuclear extracts were prepared
from cells infected with viruses bearing null mutations in one of the
IE genes ICP4, ICP0, ICP22, and ICP27, as well as with UV-inactivated
virus, as indicated. Nuclear extracts were fractionated on Sepharose
CL-2B columns, and fractions were analyzed by immunoblotting, as
described for Fig. 2. Relative densitometry units were determined and
plotted as described for Fig. 3. Positions of 2-MDa and 670-kDa
molecular standards are indicated.
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These results suggested that loss of TFIIE from the RNAP II holoenzyme
may require expression of other viral IE proteins and/or the presence
of virion components. In order to test the requirements for other IE
gene products, we analyzed TFIIE elution patterns in extracts prepared
from cells infected with the following null mutants: n212,
which bears a nonsense mutation in the ICP0 gene (5);
d22-lacZ, which bears a deletion of the ICP22
gene (33); and d27-1, which bears a deletion of
the ICP27 gene (49). We infected cells with each of these
mutant viruses, prepared nuclear extracts, fractionated extracts by
Sepharose CL-2B gel filtration chromatography, and analyzed fractions
by immunoblotting (Fig. 5). The elution profiles of TFIIE were similar
in extracts prepared from cells infected with each of the mutant
viruses in that all showed low levels of TFIIE in fractions surrounding
2 MDa.
To assess the relative quantities of TFIIE in the ~2-MDa fractions
from cells infected with each of the viral mutants, we added the
relative densitometry units in eight fractions surrounding the 2-MDa
peak and expressed these as a percentage of total densitometry units
over the entire chromatography run. This analysis showed that the TFIIE
content in the ~2-MDa peak was 0.2% for d120-infected cells, 1.0% for n212 infected cells, 0.6% for
d22-lacZ-infected cells, and 2.0% for
d27-1-infected cells. These values are similar to those seen
in wild-type virus-infected cells (0.6%) and are below those seen in
uninfected cells (15%).
We next examined the contribution of virion components to depletion of
TFIIE from the RNAP II holoenzyme fractions by analyzing TFIIE elution
profiles in cells infected with UV-inactivated virus. UV-inactivated
virus was prepared as described previously (51) and showed
a 4- to 5-log reduction in virus titer. UV-inactivated HSV-1 is used to
distinguish the relative contributions of virion proteins and viral
gene expression. Treatment of HSV-1 with 254-nm UV light cross-links
viral DNA and prevents viral gene transcription but does not damage
virion proteins (18, 25, 51). We infected cells with
UV-inactivated virus, prepared nuclear extracts, fractionated the
extracts by Sepharose CL-2B gel filtration chromatography, and analyzed
the fractions by immunoblotting (Fig. 5). UV-inactivated virus was
unable to deplete TFIIE from the ~2-MDa fractions. Approximately 19%
of total TFIIE was present in the ~2-MDa peak, compared with results
for uninfected cells (15%) and cells infected with wild-type virus
(0.6%) (Table 1). These data suggest that virion components are not
sufficient to cause loss of TFIIE from the RNAP II holoenzyme. They do
not eliminate the possibility, though, that virion components contribute to TFIIE depletion, in conjunction with IE gene expression.
In summary, HSV-1 infection results in loss of TFIIE from the RNAP II
holoenzyme, and this loss requires viral IE gene expression but not
viral DE or L gene expression or viral DNA replication.
RNAP II holoenzymes from infected cells are transcriptionally
inactive.
One possible consequence of the loss of TFIIE from the
RNAP II holoenzyme is that RNAP II holoenzymes may be inactive after HSV-1 infection. As a first step in analyzing the transcriptional consequences of RNAP II holoenzyme modification, we performed in vitro
transcription assays on nuclear extracts and RNAP II holoenzymes from
infected and uninfected cells. We prepared nuclear extracts from
infected or uninfected cells as described in Materials and Methods.
Nuclear extracts were fractionated by Sepharose CL-2B gel filtration
chromatography, and fractions surrounding the 2-MDa peak (shown in Fig.
2 and 3) were pooled and concentrated 50-fold. Crude nuclear extracts
or holoenzyme concentrates containing comparable amounts of RNAP II (as
assessed by immunoblotting) were used for in vitro transcription
assays, along with a linear DNA template bearing the Ad MLP 540 bp
upstream of the linear DNA end. Radioactive RNAs were analyzed by
acrylamide-urea gel electrophoresis and autoradiography (Fig.
6). Infected nuclear extracts were
consistently less active than uninfected nuclear extracts during in
vitro transcription assays (Fig. 6, lanes 1 and 3). Phosphorimaging
revealed that infected nuclear extracts yielded 10 to 40% less
promoter-specific transcription than uninfected nuclear extracts
(depending on the extract). We do not know the basis for this lower
activity. DNA templates were not detectably degraded during the
transcription assays with either infected or uninfected nuclear
extracts (data not shown). Interestingly, nuclear extracts prepared
from cells infected with the ICP4 mutant virus, d120, were
as transcriptionally active as uninfected nuclear extracts (M. L. Long and C. A. Spencer, unpublished data), suggesting that one or
more DE or L gene products may inhibit RNAP II transcription in vitro.
However, extracts prepared from cells infected with the d120
virus also contain high levels of the viral IE proteins ICP0, ICP22,
and ICP27; therefore, their high transcriptional activity may be due to
the presence of these proteins.
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FIG. 6.
In vitro transcription activities of nuclear extracts
(NE), RNAP II holoenzymes (Holo), and pooled low-molecular-mass
fractions (Low). In vitro transcription reactions were carried out
using a linear DNA template containing the Ad MLP. Reactions were
performed with nuclear extracts (46 µg) or 10 µl of concentrated
fractions from gel filtration chromatography, adjusted to contain
similar quantities of RNAP II. Transcription was template dependent and
sensitive to 2 µg of -amanitin/ml, indicating transcription by
RNAP II. Arrow indicates runoff transcripts originating at the Ad MLP.
Lanes 1 and 3, nuclear extracts prepared from infected and uninfected
cells. Lanes 2 and 4, concentrated fractions from the ~2-MDa peak of
Sepharose CL-2B gel filtration chromatography (fractions indicated in
Fig. 2 and 3). Transcription signals were at background levels in lane
4. Lanes 5 and 6, crude uninfected nuclear extracts and pooled
concentrates from the ~670-kDa peak of Sepharose CL-2B chromatography
(fractions shown in Fig. 2 and 3). Lanes 7 and 8, crude infected
nuclear extracts and pooled concentrates from the ~670-kDa peak of
Sepharose CL-2B chromatography (fractions indicated in Fig. 2 and 3).
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RNAP II holoenzymes from uninfected cells supported in vitro
transcription (Fig. 6, lane 2), albeit at lower levels than the parent
crude nuclear extract (~30% of nuclear extract activity). Although
we do not know the reason for this lower activity, it may be due to a
loss of activity during the 2 to 3 h required to concentrate the
pooled column fractions. We were unable to detect transcriptional
activity above background levels in RNAP II holoenzyme concentrates
prepared from infected nuclear extracts (Fig. 6, lane 4), even though
the amounts of RNAP II were comparable in the infected and uninfected
holoenzyme concentrates. Studies are in progress to determine the
biochemical basis for low transcriptional activities in infected
nuclear extracts and RNAP II holoenzymes, and whether loss of TFIIE
from the RNAP II holoenzyme contributes to this loss of activity.
Low-molecular-mass RNAP II-containing fractions are
transcriptionally active and exhibit RNAP II-GTF interactions.
Our
in vitro transcription assays suggested that RNAP II holoenzymes may be
inactive after HSV-1 infection. If holoenzymes are also inactive in
vivo after HSV-1 infection, it is possible that free RNAP II and GTFs,
or low-molecular-mass complexes containing these factors, may be the
transcriptionally relevant species after infection with HSV-1. To test
the transcription activity of low-molecular-mass material, we performed
in vitro transcription assays on concentrated fractions from Sepharose
CL-2B columns. Nuclear extracts were prepared from uninfected or
wild-type virus-infected cells, extracts were fractionated on Sepharose
CL-2B columns, fractions at and below ~670 kDa were pooled (as
indicated in Fig. 2 and 3), and pooled fractions were concentrated
10-fold. Crude nuclear extracts or ~670-kDa concentrates containing
comparable amounts of RNAP II (as assayed by immunoblotting) were used
in in vitro transcription assays, as described above for RNAP II
holoenzymes. The transcription activities of ~670-kDa concentrates
from both infected and uninfected cells were greater (~3- to 4-fold)
than those of the nuclear extracts from which they were derived (Fig.
6, lanes 5 to 8). In addition, the ~670-kDa concentrates from
infected nuclear extracts directed synthesis of multiple bands,
suggesting inaccurate transcription initiation and/or premature
transcription termination. These data suggest that RNAP II and GTFs in
infected nuclear extracts are transcriptionally active; however, this
activity may be masked or repressed within the context of a crude
nuclear extract. The biochemical basis for these different activities
in crude versus fractionated material will be addressed in future studies.
As low-molecular-mass material derived from nuclear extracts supports
high levels of transcription activity, we wanted to determine whether
the RNAP II and GTFs in these fractions exist as free factors or
whether they interact in low-molecular-mass complexes. In order to
examine the interactions of GTFs with RNAP II in low-molecular-mass
fractions, we pooled fractions from CL-2B gel filtration chromatography
(fractions indicated in Fig. 2 and 3), representing material from
~670 kDa and lower peaks, and subjected these to GST-TFIIS
affinity chromatography. GST-TFIIS affinity chromatography of pooled
low-molecular-mass fractions from uninfected extracts showed that TFIID
(TBP), TFIIH (cdk7), and TFIIE (p56E), coeluted with RNAP II (Fig.
7A). Similarly, TFIID, TFIIH, and TFIIE
coeluted in GST-TFIIS affinity chromatography of pooled low-molecular-mass fractions from extracts prepared from cells infected
with HSV-1 (Fig. 7B). None of these factors eluted from control
GST-Sepharose columns (Fig. 7, lower panels). In summary, although
TFIIE is absent from RNAP II holoenzymes after HSV-1 infection, it is
capable of interacting with RNAP II in complexes of lower molecular
mass. As these complexes are in the pooled fractions of ~670 kDa, it
is likely that RNAP II interacts with the GTFs in a number of different
complexes. This is consistent with observations that free RNAP II is
capable of binding individual GTFs and GTF subunits in vitro (4,
32).
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FIG. 7.
TFIIS affinity chromatography of pooled
low-molecular-mass fractions from gel filtration chromatography.
Nuclear extracts prepared from uninfected or infected cells were
fractionated by Sepharose CL-2B gel filtration chromatography, as
described for Fig. 2. Fractions at and below ~670 kDa (indicated in
Fig. 2 and 3) were pooled and chromatographed on GST and GST-TFIIS
columns. Bound proteins were eluted sequentially with buffers
containing 0.3 and 0.5 M NaCl. Eluates were concentrated and analyzed
by immunoblotting with antibodies that recognize the large subunit of
RNAP II, the p56E subunit of TFIIE, the TBP subunit of TFIID, and the
cdk7 subunit of TFIIH. Each blot contained a lane of uninfected (A) or
infected (B) nuclear extract (20 µg each) to indicate positions of
GTFs, as well as a lane containing one-quarter of the flowthrough
(Fl-Thru) and a lane containing one-half of the wash.
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If TFIIE continues to be involved in transcription of viral genes after
HSV-1 infection, despite its absence from the RNAP II holoenzyme, it
may be visibly recruited to viral replication compartments. We
previously reported that RNAP II is recruited to viral replication
compartments after HSV-1 infection and that this recruitment coincides
with repression of host gene transcription and induction of viral gene
transcription, at approximately 4 to 6 h postinfection (51,
64). To examine the intracellular localization of GTFs after
infection, we infected HeLa cells for 6 h, fixed them, and
immunostained them with antibodies recognizing RNAP II and GTFs. Cells
were also stained with an antibody against the viral IE protein ICP4 to
visualize viral replication compartments (Fig.
8). Infected cells displayed typical
intranuclear viral replication compartments, which are sites of viral
DNA synthesis and transcription (11, 12, 47, 51, 65).
Immunostains showed that the GTFs TFIID (TBP), TFIIH (p62H), and TFIIE
(p56E) were present in viral replication compartments, along with RNAP II. In summary, these data show that TFIIE and other GTFs interact with
RNAP II in complexes of low molecular mass, both before and after
infection with HSV-1. In addition, TFIIE localizes to viral replication
compartments along with RNAP II and other GTFs, consistent with its
potential role in transcription of viral genes.
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FIG. 8.
Intracellular distribution of RNAP II and GTFs in
HSV-1-infected cells. HeLa S3 cells were infected with wild-type virus
for 6 h, fixed, and stained with anti-ICP4 antibody conjugated to
either Texas Red or Oregon Green in order to visualize viral
replication compartments. Cells were costained with DAPI to visualize
DNA and with antibodies recognizing the RNAP II large subunit, TBP,
p56E, or p62H (a subunit of TFIIH) and secondary antibodies conjugated
to either rhodamine (TBP, red stain) or Alexa 488 (green stain).
Staining patterns were visualized by confocal microscopy.
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Viral IE proteins ICP4, ICP0, and ICP27 interact with RNAP II in
low-molecular-mass complexes.
Our data suggest that RNAP II
holoenzymes may not be involved in viral or host gene transcription
after infection; however, free RNAP II and GTFs (or various
low-molecular-mass complexes containing RNAP II and GTF subunits) may
be the transcriptionally relevant forms. One way in which RNAP II and
GTFs could be assembled on viral DNA in the absence of the RNAP II
holoenzyme is for viral regulatory proteins to interact with RNAP II
and GTFs and to recruit these individual components (or small
complexes) to viral promoters. One candidate for such a regulatory
protein is ICP4, which is able to interact with TFIID and TFIIB in
vitro and to stimulate the assembly of transcription preinitiation
complexes (6, 23, 62).
In order to detect the association of viral IE proteins with RNAP II
and GTFs, we first analyzed the IE protein content in fractions from
CL-2B gel filtration chromatography. Nuclear extracts from cells
infected with wild-type HSV-1 were fractionated on CL-2B columns, and
fractions were analyzed by immunoblotting. Western blots were scanned
by densitometry, values were normalized to identical standards on each
gel, and values were plotted as relative densitometry units. Each of
the viral IE proteins was present in the ~2-MDa fractions from CL-2B
chromatography (Fig. 9). The CL-2B
elution profiles of the IE proteins were not affected by prior
treatment of nuclear extracts with ethidium bromide, DNase I, and RNase
A, indicating that their presence in high-molecular-mass fractions was
not due to interactions with nucleic acids in the nuclear extracts
(data not shown). The densitometry units in fractions surrounding the
2-MDa peak were expressed as a percentage of the total densitometry
units over the entire run. These calculations indicated that
approximately 2% of total ICP4, 1% of total ICP0, 64% of total
ICP22, and 40% of total ICP27 eluted in high-molecular-mass fractions
from CL-2B columns.
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FIG. 9.
Elution profiles of viral IE proteins from Sepharose
CL-2B gel filtration chromatography. Nuclear extracts prepared from
cells infected with wild-type HSV-1 were chromatographed on Sepharose
CL-2B columns, and fractions were analyzed by immunoblotting. Blots
were probed with antibodies that recognize ICP4, ICP0, ICP22, and ICP27
as indicated. Relative densitometry units were determined and plotted
as described for Fig. 3. Positions of 2-MDa and 670-kDa molecular
standards are indicated.
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To determine whether the IE proteins in high-molecular-mass fractions
interact with RNAP II holoenzyme complexes, we pooled fractions
surrounding the 2-MDa peak from CL-2B columns (indicated in Fig. 9) and
subjected these to GST-TFIIS affinity chromatography. None of the IE
proteins eluted in the 0.3 or 0.5 M elutions or from GST columns (Fig.
10A). These data indicate that viral IE proteins, although present in high-molecular-mass complexes, do not
interact with the RNAP II holoenzyme after HSV-1 infection.
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FIG. 10.
TFIIS affinity chromatography showing viral IE
proteins in low-molecular-mass fractions containing RNAPII. Nuclear
extracts, prepared from cells infected with wild-type HSV-1, were
fractionated by Sepharose CL-2B gel filtration chromatography, as
described for Fig. 2. (A) Fractions surrounding the 2-MDa peak
(indicated in Fig. 2, 3, and 9) were pooled and subjected to GST and
GST-TFIIS affinity chromatography. Bound proteins were eluted
sequentially with buffers containing 0.3 and 0.5 M NaCl. Eluates were
concentrated and analyzed by immunoblotting with antibodies that
recognize the IE proteins ICP4, ICP0, ICP22, and ICP27. Each blot
contained a lane of nuclear extract (20 µg) to indicate the position
of each IE protein. The Fl-Thru lane contained one-quarter of the
flowthrough, and the wash lane contained one-half of the wash. (B)
Fractions at and below ~670 kDa (indicated in Fig. 2, 3, and 9) were
pooled and subjected to GST and GST-TFIIS affinity chromatography.
Bound proteins were eluted and analyzed as described for panel A. The
black dot indicates an ICP22 band above the ICP27 band, visible from a
previous probing of the Western blot.
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Although IE proteins appeared not to interact with RNAP II holoenzyme
complexes, we wanted to examine their interactions with RNAP II in
complexes of lower molecular mass. To do this, we pooled fractions of
~670 kDa and lower from CL-2B columns (fractions indicated in Fig. 9)
and subjected these to GST-TFIIS affinity chromatography. Small but
detectable amounts of ICP4, ICP27, and ICP0 coeluted with RNAP II from
pooled low-molecular-mass fractions but not from GST columns (Fig.
10B). ICP22 was not detectable in GST-TFIIS eluates. In addition, ICP22
consistently displayed a mobility shift and altered immunostaining
after TFIIS or GST chromatography, perhaps due to phosphatase activity
during the chromatography run. Since RNAP II and IE proteins interact
in fractions of approximately 670 kDa, it is likely that these
associations represent a mixture of individual IE proteins with free
RNAP II or RNAP II-GTF complexes of low molecular mass. These data do
not distinguish between direct and indirect interactions of RNAP II
with ICP4, ICP0, or ICP27. It is possible that IE proteins may interact
directly with TFIIS; however, no IE proteins were detected in TFIIS
affinity chromatography of high-molecular-mass fractions which also
contained viral IE proteins (Fig. 10A). Further biochemical analysis
will be required to precisely define the composition of each of the
RNAP II-containing low-molecular-mass complexes and to determine
whether the associations of GTF subunits or viral IE proteins with free
RNAP II occur in vivo.
| |
DISCUSSION |
In this study, we show that HSV-1 infection modifies the
composition of the RNAP II holoenzyme. A number of biochemical methods have been used to isolate RNAP II holoenzymes from mammalian cells. Although the composition of these large multisubunit complexes varies
depending on the method of isolation, they contain at least RNAP II and
the GTFs TFIIH, TFIIF, and TFIIE (8, 34, 39, 40, 43, 45, 46,
55). TFIIB and TFIID are also components of RNAP II holoenzymes
isolated using one- or two-step purification procedures; hence, their
association with RNAP II complexes is thought to be less stable than
that of other GTFs. RNAP II holoenzymes, unlike free RNAP II and GTFs,
are responsive to transcription activators and are thought to be
responsible for most transcription initiation in yeast and mammalian
cells (2, 17, 30, 43, 67).
Using a two-step purification procedure (gel filtration chromatography
and TFIIS affinity purification), we show that TFIIE (p56E), TFIID
(TBP), and TFIIH (cdk7) copurify with RNAP II in high-molecular-mass
holoenzyme complexes prior to infection. TBP and cdk7 also copurify
with RNAP II holoenzyme complexes after infection; however, p56E levels
are low in the ~2-MDa peak from CL-2B columns and undetectable in
eluates from TFIIS chromatography.
The observation that the interaction of the RNAP II holoenzyme with
TFIIE is disrupted after infection is intriguing, given the biochemical
functions ascribed to TFIIE (21, 42, 53, 57). TFIIE
consists of a 180-kDa heterotetramer of 56- and 34-kDa subunits and is
essential for most RNAP II transcription in vivo and in vitro. TFIIE
interacts physically with TFIIH and stimulates the CTD kinase and
ATPase activities of TFIIH. Interestingly, certain mutant recombinant
TFIIEs, when added to in vitro kinase assays containing RNAP II, TFIIH,
and other purified GTFs, lead to partial CTD phosphorylation, yielding
forms that resemble the HSV-1 modified form, RNAP
III (42). These mutant TFIIE
proteins are also defective for RNAP II transcription in vitro. In
addition to stimulating CTD phosphorylation, TFIIE plays roles in open complex formation and promoter clearance (15, 27). These
roles may involve the ability of TFIIE to stimulate TFIIH helicase
activity, as well as binding to single-stranded and double-stranded DNA immediately upstream of the transcription start site (52).
The requirement for TFIIE can be circumvented by premelting the
promoter DNA (27, 28).
It is conceivable that disruption of TFIIE from the postinfection
holoenzyme could contribute to the changes in CTD phosphorylation and
RNAP II transcription that occur after infection with HSV-1. In the
absence of TFIIE, the CTD kinase activity of holoenzyme-associated TFIIH may be impaired, leading to aberrant phosphorylation of RNAP II.
Aberrant phosphorylation of RNAP II could in turn contribute to
repression of host gene transcription by interfering with the ability
of RNAP II transcription initiation complexes to make the transition to
productive elongation (10, 15, 28, 57). It is possible
that viral proteins such as ICP22 and the UL13 kinase may partially
compensate for TFIIE's stimulatory function in CTD phosphorylation,
thereby facilitating viral gene transcription. In addition, disruption
of TFIIE from the holoenzyme could interfere with the ability of RNAP
II transcription initiation complexes to open DNA during transcription
elongation on cellular genes, thereby contributing to host
transcription repression after infection. Specific structural features
of the viral genome or the presence of viral regulatory proteins could
compensate for an RNAP II holoenzyme that is impaired in promoter
opening. Finally, loss of TFIIE from the RNAP II holoenzyme could lead
to defects in host RNA processing, since CTD hyperphosphorylation is
required for interactions of the CTD with mRNA processing factors
(3).
Another possibility is that the RNAP II holoenzyme may not be involved
in HSV-1 gene transcription after infection. In this scenario, free
RNAP II and GTFs, or small complexes containing these factors, may be
the transcriptionally relevant forms of these factors on viral DNA
templates in vivo. This hypothesis would be consistent with the known
role of the RNAP II holoenzyme as the form of the enzyme responsible
for activator-responsive transcription initiation. Since viral DE and L
gene promoters contain no known class-specific enhancer elements that
are responsible for transcription activation by IE proteins
(61), it is possible that DE and L gene transcription
represents a kind of basal transcription, resembling in vitro
transcription on linear or supercoiled templates. In the absence of a
requirement for activator-driven transcription of viral genes, free
RNAP II and GTFs (including TFIIE) may be recruited to these basal
viral promoters in a stepwise manner, perhaps assisted by their
individual interactions with viral regulatory proteins. In this
scenario, the postinfection RNAP II holoenzyme would be incompetent for
transcription initiation on cellular genes due to its lack of TFIIE and
its inability to hyperphosphorylate the RNAP II CTD. However, viral
genes would circumvent the requirement for the RNAP II holoenzyme by
recruiting free RNAP II and GTFs to promoters that do not utilize
classical transcription activators and are maintained in a
nucleosome-free conformation. This hypothesis is consistent with our
immunofluorescence staining data showing that RNAP II and GTFs
including TFIIE appear to concentrate within replication compartments.
Data from our laboratory (C. A. Spencer, unpublished data) and
from others (12, 48, 65) show that ICP4 and ICP27 also
localize to viral replication compartments, consistent with their
possible roles as recruiters of RNAP II and GTFs to viral DNA. ICP0
also localizes to viral replication compartments, although this
association is less defined than that of ICP4 and ICP27
(16).
Our data show that loss of TFIIE from the RNAP II holoenzyme requires
viral IE gene expression and that two or more viral IE proteins may be
redundant in bringing about this modification. We previously showed
that repression of host transcription and loss of the
hyperphosphorylated form of the RNAP II large subunit also require
viral IE gene expression and that viral IE genes may be redundant in
mediating these changes. Hence, loss of TFIIE from the RNAP II
holoenzyme correlates with loss of RNAP IIO and repression of host
transcription, suggesting a functional link between these virus-induced events.
In this study, we report that the RNAP II holoenzyme is depleted of
TFIIE and is transcriptionally inactive after HSV-1 infection. In
addition, free RNAP II or RNAP II in low-molecular-mass complexes may
interact with the viral IE proteins ICP4, ICP0, and ICP27. It will be
of interest to determine the biochemical basis for the
transcriptional-activity differences between infected and uninfected
RNAP II holoenzymes, as well as their intrinsic CTD kinase activities.
In addition, analysis of RNAP II complexes in uninfected cells
expressing one or more IE genes may provide clues as to the mechanisms
by which HSV-1 infection modifies the RNAP II holoenzyme and redirects
the host's transcription machinery from host to viral genes.
This work was supported by grants from the Canadian Institutes of
Health Research and the Alberta Cancer Board. C.A.S. is a Senior
Scholar of the Alberta Heritage Foundation for Medical Research.
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Abstract
Introduction
