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Journal of Virology, April 1999, p. 3404-3409, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activation of Baculovirus Very Late Promoters
by Interaction with Very Late Factor 1
Song
Yang1 and
Lois K.
Miller1,2,*
Departments of
Genetics1 and
Entomology,2 University of Georgia,
Athens, Georgia 30602
Received 22 September 1998/Accepted 5 January 1999
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ABSTRACT |
Very late factor 1 (VLF-1) of Autographa californica
multicapsid nuclear polyhedrosis virus (AcMNPV) activates
the transcription of two genes, polyhedrin (polh) and
p10, during the final, occlusion-specific phase of
infection. Using transient expression assays responsive to VLF-1, we
identified linker scan mutations in the polh and p10 promoters which abolished or weakened the ability of
the promoters to respond to stimulation by VLF-1. These mutations were
located between the transcriptional and translational initiation sites, a region previously shown to be essential for the burst of expression during the very late phase. Addition of partially purified,
epitope-tagged VLF-1 to DNA encompassing this "burst sequence"
resulted in a shift in the gel electrophoretic mobility of the DNA,
indicating that VLF-1 forms a complex with DNA. Addition of an antibody
specific for the epitope tag of VLF-1 decreased the mobility of the DNA further, confirming the presence of VLF-1 in the complex. DNase I
footprint assays revealed that VLF-1 partially purified from either
insect cells or bacterial cells interacted with the burst sequences of
both the polh and p10 very-late promoters.
Linker scan mutations within the burst sequences severely impaired
interaction between VLF-1 and the promoters. We propose that VLF-1
transactivates the polh and p10 promoters by
interacting with the burst sequences.
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INTRODUCTION |
The VLF-1 (very late factor 1) gene
(vlf-1) of Autographa californica multicapsid
nuclear polyhedrosis virus is required for expression of very late
genes, e.g., polyhedrin (polh) and p10, during
the final phase of infection involving the formation of occlusion
bodies. vlf-1 was originally identified by characterization of an occlusion-defective mutant virus, tsB837, which
produces only low levels of polh and p10
transcripts during the very late phase (11). In
transient-expression assays, vlf-1 also stimulates expression from very late promoters but has no effect on expression from late promoters (22). Construction of recombinant
viruses with altered vlf-1 expression revealed that
polh expression is regulated by the timing of
vlf-1 expression and/or the concentration of VLF-1 in the
cell (27). vlf-1 itself is expressed as a late gene, and its product is an essential and limiting factor in
polh expression (28).
Transcription of late and very-late baculovirus genes initiates
from a TAAG sequence which is an essential element for both classes of
promoters. The strength of expression from these promoters during the
late phase depends on the context of the TAAG. The 18 bp encompassing
the TAAG of the late vp39 promoter are the primary, if not
the sole, determinants of expression levels from this promoter
(12). In contrast, the strength of expression from the
polh promoter during the very late phase of infection depends not only on the context of the TAAG but also on the nature of
the sequence located between the TAAG and the translational initiation
site, i.e., the sequence specifying the untranslated leader of very
late mRNAs (10, 14, 17, 19, 25). This sequence is required
for the burst of expression during the very late phase and is therefore
referred to as the burst sequence. Mutations within the burst sequence
reduce expression during the very late phase (e.g., 48 h
postinfection) by 10- to 20-fold and lower both the steady-state levels
of polh RNA and the rate of transcriptional initiation from
the polh promoter (14). In contrast, mutations in
sequences upstream of the TAAG sequence of the polh promoter
have comparatively mild effects on the level of very late gene
expression (14, 17, 19). Progressive deletions of the
p10 promoter also suggest the presence of a burst sequence that is essential for strong expression during the very late phase (18, 24, 25).
The important roles of both VLF-1 and the burst sequence in
hyperexpression of very late promoters suggest that they are two components of the same transactivation mechanism. We have examined the
possibility that VLF-1 exerts its effect through interaction with very
late promoters. We show that the burst sequence is important for a very
late promoter to respond to stimulation by VLF-1 in transient-expression assays. Our data also suggest that VLF-1 interacts
with the burst sequence in vitro.
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MATERIALS AND METHODS |
Cell line, plasmids, and recombinant viruses.
The
Spodoptera frugiperda (fall armyworm) IPLB-SF-21 (SF-21)
cell line (23) was grown at 27°C in TC-100 medium
(GIBCO/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine
serum (Intergen, Purchase, N.Y.) and 0.26% tryptose broth
(15).
phcwt, phcLSXVII, phcLSVI, and phcLSVII contain a chloramphenicol
acetyltransferase (CAT)-encoding gene driven by a wild-type (wt) or
linker scan mutant polh promoter (14, 19).
pCAPCAT contains a vp39 promoter-driven cat gene
(21). pXA76.9 (28) and pXA76.9d (27)
contain wt vlf-1 or frameshift mutant vlf-1 under
the control of the p6.9 promoter.
p10hcBS, p10hc-81, p10hc-47, and p10hc-17 contain a
cat gene
driven by a wt or linker scan mutant
p10 promoter (Fig.
1). p10hcBS
was constructed by moving the
KpnI-
HindIII fragment of plasmid
p10hc
(
22) containing the
p10-promoted
cat
gene into pBluescriptII
KS(+) (Stratagene, La Jolla, Calif.) between
the
SmaI and
HindIII
sites. Plasmids
p10hc-81, p10hc-47, and p10hc-17 were constructed
by site-directed
mutagenesis (
2) using p10hcBS as the template
plasmid and
oligomers P10hc-81 (5'-CTTATTTAACTATCCGGATCCGTGTTGGGTTG-3'),
P10hc-47 (5'-GTATTTTAATTAATATGGCTCGAGATTGATAATAATTC-3'),
and P10hc-17
(5'-GTAAATAAAATGTGCGGCCGCGTATAGTATTTTAA-3'),
respectively, as
mutagenic primers. pETvlf1 was constructed by
inserting the intact
vlf-1 open reading frame (ORF) between
the
ClaI and
BamHI sites
of pET-15b (Novagen,
Madison, Wis.) so that a 6×His tag was fused
to the N terminus of
vlf-1. pETvlf1
SstIII is identical to pETvlf1,
except that
it lacks the sequence between the
SstI and
SstII
sites
in the
vlf-1 ORF. Thus, pETvlf1
SstIII contains a
6×His-tagged
vlf-1 truncation that encodes the first 151 residues of VLF-1.
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FIG. 1.
Sequences of the polh and p10
promoter regions within reporter plasmids. Plasmids phcwt and p10hcBC
contain the cat gene driven by wt polh (A) and
p10 (B) promoter sequences, respectively. Sequences modified
by linker scan mutations are indicated by single underlining, and the
mutant sequences and names of the corresponding reporter plasmids are
shown below the mutated sequences. TAAG sequences are boxed.
Restriction sites are doubly underlined. The numbering above the
sequences is relative to the original translational initiation sites of
the polh and p10 genes. In both cases, the ATG
has been modified to a BglII site so that only the A (+1)
remains. The translation initiation codon of the cat gene is
marked by asterisks. Thick arrows indicate the positions of primers
used to generate probes by PCR for DNase I footprint assays. The
radioactively labeled 5' end of the CAT3 primer is indicated by a
star.
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vcFgvlf1 is a recombinant of
A. californica multicapsid
nuclear polyhedrosis virus with a FLAG epitope tag inserted at the
C
terminus of the
vlf-1 ORF (
28).
Transient-expression assays.
Transient-expression
assays used to assess the ability of mutant polh and
p10 promoters to respond to stimulation by VLF-1 were
similar to those described previously (22). In each
transfection, 2 µg of the reporter plasmid and 0.5 µg of any
additional plasmid were introduced into 2 × 106 SF-21
cells using Lipofectin (GIBCO/BRL) (15). CAT assays were performed 72 h posttransfection as described previously
(4). A PhosphorImager 4000 (Molecular Dynamics, Sunnyvale,
Calif.) was used for quantification.
Protein expression and purification.
SF-21 cells (40 × 106) were infected with recombinant virus vcFgvlf1 at a
multiplicity of infection of 20 PFU per cell (15) and lysed
24 h postinfection with 5 ml of 50 mM Tris-HCl (pH 8.0)-50 mM
NaCl-1% Nonidet P-40. Cleared cell lysates were incubated with 200 µl of anti-FLAG M2 monoclonal antibody affinity gel (Kodak, New
Haven, Conn.) with gentle agitation at 4°C for 4 h. The gel pellets were washed three times with 50 mM Tris-HCl (pH 8.0)-50 mM
NaCl. Bound proteins were eluted with 100 µl of 200-µg/ml FLAG peptide (Kodak) in 10 mM Tris-HCl (pH 7.4)-150 mM NaCl. Glycerol was
added to the eluates to a final concentration of 20%. Purified FLAG-VLF-1 was diluted with 10 mM Tris-HCl (pH 7.4)-150 mM NaCl-20% glycerol when necessary.
To express 6×His-tagged
vlf-1 in
Escherichia
coli, strain BL2(DE3)pLysS (Novagen) was transformed with pETvlf1
or pETvlf1
SstIII.
A single colony was picked to inoculate 2 ml of
Luria-Bertani
growth medium containing 50-µg/ml ampicillin and
34-µg/ml chloramphenicol
and grown at 30°C overnight. The overnight
cell culture was used
to inoculate, at a 1:100 dilution, 50 ml of
Luria-Bertani medium
containing ampicillin and chloramphenicol. Cells
were grown at
30°C until the optical density at 600 nm reached 0.5 to
0.6 and
induced by adding
isopropyl-
-
D-thiogalactopyranoside to a final
concentration of 1 mM. Cells were harvested 3 h later and lysed
by
freezing and thawing in 5 ml of 50 mM Tris-HCl (pH 8.0)-50
mM
NaCl-1% Nonidet P-40. The supernatant of the cell lysate was
incubated with 100 µl of Ni-nitrilotriacetic acid (NTA) resin
(QIAGEN, Chatsworth, Calif.) at 4°C with gentle shaking for 4
h.
The resin was washed three times with 50 mM Tris-HCl (pH 8.0)-50
mM
NaCl before elution with 120 µl of 250 mM imidazole-10 mM Tris-HCl
(pH 7.4)-150 mM NaCl. Glycerol was added to the eluate to 20%.
Immunoblot analysis.
Proteins of the FLAG-VLF-1 preparation
were separated by sodium dodecyl sulfate-10% polyacrylamide gel
electrophoresis and transferred to nylon membranes (Millipore). Blots
were blocked in TBST buffer (10 mM Tris-HCl [pH 7.6], 150 mM NaCl,
0.1% Tween 20) in the presence of 5% nonfat dried milk, probed first
with a 1:5,000 dilution anti-FLAG M2 monoclonal antibody and then a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G in TBST buffer, and visualized with an
enhanced-chemiluminescence Western blot kit (Amersham).
Gel mobility shift assays.
To prepare radioactively labeled
promoters, plasmids phcwt and p10hcBC were digested with
EcoRV and BglII (Fig. 1). The BglII site was filled in with [
-32P]dATP by using T4 DNA
nucleotide polymerase, and the DNA fragments containing the
polh and p10 promoters were gel purified. A
1-µl sample of purified FLAG-VLF-1 was mixed with 10 µl of binding buffer (10 mM HEPES-NaOH [pH 7.9], 50 mM KCl, 0.1 mM EDTA, 10% glycerol) containing 50 ng of poly(dI-dC) and 20,000 cpm of the promoter-containing fragment (0.56 and 0.35 ng of the polh
and p10 promoters, respectively). The mixtures were kept at
room temperature for 20 min before being analyzed by electrophoresis in
0.5× Tris-borate-EDTA-6% polyacrylamide gels. For the supershift
reactions, 1 µl of anti-FLAG M2 monoclonal antibody was added and the
mixture was incubated for an additional 5 min before electrophoresis.
For competition experiments, EcoRI-linearized pBluescript
was used as a nonspecific competitor, while specific competitors were
the same DNA fragments used as probes but lacked the radiolabel.
Competition experiments were performed under the same conditions as
described above with various amounts of competitors added
simultaneously with probes.
DNase I protection assays.
Radioactively labeled
polh, p10, and vp39 promoter fragments
were made by PCR using plasmids phcwt, p10hcBC, and pCAPCAT as templates and primers polhEV, p10EV, and CAP104, respectively, plus a
-32P-labeled CAT3 primer (Fig. 1). A footprint assay was
initiated by adding 15 µl of the purified VLF-1 fusion (9.45 µg of
FLAG-VLF-1 or 11.24 µg of 6×His-VLF-1) to 35 µl of 15 mM
HEPES-NaOH (pH 7.9)-75 mM KCl-0.15 mM EDTA-15% glycerol containing
30,000 cpm of promoter fragment and allowing the binding reaction to
proceed at room temperature for 20 min. A 50-µl volume of 10 mM
MgCl2-5 mM CaCl2 was added to each reaction
mixture, which was then cooled on ice for 5 min. The probe was digested
with 1 µl of RQ1 RNase-free DNase I (Promega, Madison, Wis.) at 4°C
for 5 min. The digestion was terminated by adding 90 µl of 200 mM
NaCl-20 mM EDTA-1% sodium dodecyl sulfate-20-µg/ml
single-stranded salmon sperm DNA. The DNA was purified by
phenol-chloroform extraction and analyzed on a sequencing gel. A
sequence ladder of the corresponding template plasmid was generated in
parallel with the same radioactively labeled CAT3 primer for use as a marker.
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RESULTS |
Involvement of the burst sequence in the stimulation of expression
from the polh and p10 promoters by
vlf-1 in transient-expression assays.
To determine the
region(s) of very late promoters involved in VLF-1 stimulation, we
examined the effects of linker scan mutations on the ability of the
polh and p10 promoters to respond to
transcriptional stimulation by VLF-1 in transient-expression assays
(22). Reporter plasmids containing a cat gene
driven by a wt or mutant promoter were cotransfected into SF-21 cells
with a set of genomic clones collectively containing all of the late
expression factor genes (lef) required for late gene
expression in transient-expression assays (9, 20) but
lacking vlf-1. Either a truncated version of
vlf-1 or wt vlf-1 was supplied in the
transfections on a separate plasmid. The difference between CAT
expression levels in the presence of wt or mutant vlf-1 was
a reflection of the sensitivity of the promoter to VLF-1 stimulation.
In these assays, both wt vlf-1 and mutant vlf-1
were under the control of the p6.9 promoter, which provided
elevated expression of vlf-1 and greater stimulation of
expression from very late reporter plasmids (27).
Mutant
polh and
p10 promoters with 10-bp linker
mutations introduced at selected positions (Fig.
1) were examined and
compared
to wt promoters. While the late
vp39 promoter
(pCAPCAT) was not
affected by addition of functional
vlf-1
as expected, the wt
polh promoter (phcwt) was stimulated
approximately 20-fold in the presence
of wt
vlf-1 (Fig.
2 and Table
1). A linker scan mutation 13 nucleotides
(nt) upstream of the
polh TAAG (phcLSXVII) was also
stimulated
approximately 20-fold by VLF-1. Mutations 12 nt
(phcLSVI) or 25
nt (phcLSVII) downstream of the TAAG reduced the level
of expression
approximately eightfold in the absence of wt
vlf-1 but either
abolished the response of the mutant
polh promoter (phcLSVI) to
VLF-1 stimulation or reduced the
response to only twofold (phcLSVII)
(Table
1). The
p10
promoter series showed a similar pattern.
The wt
p10
promoter (p10hcBS) and the mutant
p10 promoter with
a linker
10 nt upstream of the TAAG (p10hc-81) were stimulated
10- and 22-fold
by VLF-1. Mutations 13 nt (p10hc-47) or 43 nt
(p10hc-17) downstream of
the
p10 TAAG were stimulated only three-
to fourfold or not
affected at all by VLF-1, respectively. The
fact that the response of
the
polh and
p10 promoters to stimulation
by
VLF-1 is severely impaired by mutations in their burst sequences
suggests that VLF-1 exerts its effects through these sequences.
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FIG. 2.
Response of mutated very-late promoters to stimulation
by VLF-1 in transient-expression assays. All transfections included
genomic clones BC5, HL8, HL5, ETL7, PstH4, PstH1, HC10, XmaB, HK5,
IE15, and pSDEM2, which collectively supplied all of the lef
genes required for late gene expression in transient-expression assays
(22). A plasmid carrying p6.9 promoter-driven
frameshift mutant vlf-1 (pXA76.9d) or wt vlf-1
(pXA76.9) was also transfected into the cells. The reporter plasmid
used in each pair of transfections is shown at the bottom. The
positions of their mutated sequences are shown in Fig. 1. pCAPCAT
carries a late vp39 promoter-driven cat gene and
served as a negative control. Cells were harvested 72 h after
transfection, and levels of CAT activity were determined. The CAT
activity of the transfection with pCAPCAT and frameshift mutant
vlf-1 was arbitrarily set as 100%. Relative CAT activities
of other transfections were calculated based on this standard. Standard
errors were determined from results of triplicate experiments. (A)
polh promoters. (B) p10 promoters.
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Interaction between VLF-1 and the burst sequences of the
polh and p10 promoters.
Based on these
results, we explored the possibility that VLF-1 physically interacts
with very late promoters. Gel electrophoresis mobility shift assays
provided the first evidence of such interaction (Fig.
3). DNA fragments containing the
polh promoter (from 
1 to 92) and the p10
promoter (from 1 to 107) were radioactively labeled for use as
probes. VLF-1 tagged with a FLAG epitope at the C terminus was purified
from SF-21 cells infected with vcFgvlf1, a recombinant virus expressing
the FLAG-vlf-1 fusion (28). As increasing
amounts of purified FLAG-VLF-1 were added to the probes, two
FLAG-VLF-1-polh promoter complexes and three
FLAG-VLF-1-p10 promoter complexes were formed. The
presence of FLAG-VLF-1 in these complexes was verified by the
observations that they could be supershifted by the addition of a
monoclonal antibody against the FLAG epitope (Fig. 3A, lanes 5) and
that FLAG-VLF-1 was the only protein recognized by the anti-FLAG
antibody in the preparation (Fig. 3B).
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FIG. 3.
Gel mobility shift assays showing interactions between
very late promoters and FLAG-VLF-1. (A) Complex formation of the
polh and p10 promoters with FLAG-VLF-1. The
polh and p10 promoters were cut from plasmids
phcwt and p10hcBC, respectively, with EcoRV and
BglII (Fig. 1), end labeled, and gel purified. Each probe
was incubated with increasing amounts of partially purified
FLAG-VLF-1: 0, 0.16, 0.32, 0.64, and 0.64 µg of total protein were
used in lanes 1, 2, 3, 4, and 5 with the polh promoter,
respectively, and 0, 0.04, 0.08, 0.16, and 0.16 µg of FLAG-VLF-1
were used in lanes 1, 2, 3, 4, and 5 with the p10 promoter,
respectively. The resulting complexes were resolved by a
Tris-borate-EDTA-6% polyacrylamide gel. Lanes 5 in each panel also
included a monoclonal antibody (Ab) against the FLAG epitope to form
antibody-FLAG-VLF-1 complexes and further altered the mobility of the
DNA probes (supershifted DNA). (B) Western blot analysis showing
cross-reactivity of the FLAG-VLF-1 preparation with the anti-FLAG
monoclonal antibody. A 1.28-µg sample of total protein was analyzed.
Molecular masses (in kilodaltons) of standard proteins are indicated on
the right.
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The specificity of interaction between FLAG-VLF-1 and very-late
promoters was investigated by competition gel shift assays.
Interaction
of FLAG-VLF-1 with the probes was efficiently inhibited
by addition of
a 20- to 80-fold excess of the same DNA fragments
as the probes (Fig.
4, lanes 3, 4, and 5). The same amounts
of
linearized pBluescript DNA had no significant effect on complex
formation between FLAG-VLF-1 and the
polh or
p10
promoter (Fig.
4, lanes 6, 7, and 8), suggesting that interaction of
FLAG-VLF-1
with very late promoters is specific.
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FIG. 4.
Competition gel shift assays showing the specificity of
interactions between very late promoters and FLAG-VLF-1. Lanes 3, 4, and 5 contained 20×, 40×, and 80× specific competitors (the
polh or p10 promoter). Lanes 6, 7, and 8 contained 20×, 40×, and 80× nonspecific competitors (linearized
pBluescript). Competitors were mixed with probes before addition to the
reactions. Other conditions were as described in the legend to Fig.
3.
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To confirm the interaction of FLAG-VLF-1 with very late promoters and
to locate the regions within the
polh and
p10
promoters
which were involved in complex formation, we performed DNase
I
protection assays. The
polh and
p10 promoters,
labeled on the
template strands, were incubated with partially purified
C-terminally
tagged VLF-1 and then subjected to DNase I digestion. As
shown
in Fig.
5A, the wt
polh
promoter was protected from
1 to
40
and the wt
p10
promoter was protected from
5 to
56 in the presence
of FLAG-VLF-1,
which was produced in insect cells and purified
with anti-FLAG M2
affinity gels. Neither the
polh nor the
p10 promoter was protected by equivalent extracts from wt virus-infected
cell lysates lacking FLAG-VLF-1 (Fig.
5A, lanes with asterisks),
indicating that the observed footprints were not due to proteins
binding nonspecifically to the column. The
vp39 promoter, a
late
promoter, was not protected by FLAG-VLF-1 (Fig.
5A).
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FIG. 5.
Mapping of the binding site of VLF-1 by DNase I
footprint assays. polh promoters, p10 promoters,
and the vp39 promoter were amplified by PCR using primer
polhEV (5'-GATATCATGGAGATAATTAAAATG-3'), p10EV
(5'-GATATCCTTTAATTCAACCC-3'), or CAP104
(5'-GAATTTAAAATTTTATACAAC-3') plus radioactively labeled
primer CAT3 (5'-CAACGGTGGTATATCCAGTG-3'), respectively (Fig.
1). The wt, LSVI, and LSVII polh promoters were amplified
from plasmids phcwt, phcLSVI, and phcLSVII (Fig. 1), respectively. The
wt, 47, and 17 p10 promoters were amplified from
plasmids p10hc, p10hc-47, and p10hc-17 (Fig. 1), respectively. Labeled
promoters were digested with DNase I after incubation with purified
FLAG-VLF-1 and analyzed on a sequencing gel. The markers on the left
were determined by alignment with a sequence ladder generated with
primer CAT3. Footprints are marked by brackets. Positions of mutations
are indicated by black bars between lanes. (A) Protection of late and
very late promoters by FLAG-VLF-1. vcFgvlf1, a recombinant virus
expressing a FLAG-vlf-1 fusion, was used to infect SF-21
cells for production of FLAG-VLF-1, which was partially purified with
an anti-FLAG M2 affinity gel. As a negative control, lysate from wt
virus-infected cells was processed in parallel and used to protect the
DNA probes (lanes marked with asterisks). (B) Protection of wt
polh and p10 promoters by 6×His-VLF-1.
Full-length 6×His-tagged VLF-1 and truncated 6×His-VLF-1 (lanes with
asterisks) were partially purified from bacteria expressing
plasmid-borne genes by using Ni-NTA resin and used to protect probes.
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Similar footprints on very-late promoters were also revealed by
protection with 6×His-VLF-1 that was produced in
E. coli
cells
and purified with Ni-NTA resin (Fig.
5B). The footprint on the
polh promoter is indistinguishable from that resulting from
interaction
with insect cell-derived FLAG-VLF-1 (Fig.
5A). The
p10 promoter
was protected by 6×His-VLF-1 from
5 to
45
(Fig.
5B), a region
slightly shorter than that protected by
FLAG-VLF-1. A 6×His-VLF-1
truncation containing the N-terminal
two-fifths of VLF-1 was generated
in parallel with 6×His-VLF-1 as a
control and did not produce
these footprints, although a small region
(from
40 to
56) appeared
to be protected. The basis for this other
footprint is not clear.
The fact that two preparations of full-length
VLF-1 produced in
completely different expression systems and purified
by different
affinity methods resulted in footprints at the same
locations
strongly suggests that VLF-1 is involved in the formation of
these
footprints.
Since the protected sites constitute the majority of the burst
sequences, we examined interactions between FLAG-VLF-1 and
mutant
polh or
p10 promoters containing linker scan
mutations
in their burst sequences. Three of the four examined
promoters
with mutated burst sequences were no longer protected by
FLAG-VLF-1
under these conditions (Fig.
5A). Only a weaker and shorter
footprint
(from
10 to
44) was detected for the fourth one, the
p10 (
47)
promoter with a 10-bp mutation 13 nt downstream
of the TAAG (Fig.
5A). These data indicate that the ability of VLF-1 to
interact
with burst sequences of very late promoters depends on the
sequence
of the burst sequence and correlates with VLF-1's ability to
transactivate
expression from the
promoters.
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DISCUSSION |
We have examined the ability of mutant very late promoters to
respond to VLF-1. While the wt polh promoter and a mutant
promoter with a linker mutation upstream of the TAAG sequence were
stimulated approximately 20-fold by VLF-1 in transient-expression
assays, the two mutant promoters with linker mutations in their burst sequences either failed to respond or responded only slightly to VLF-1
stimulation. Since VLF-1 has previously been shown to influence the
level and timing of polh expression (11, 27), this observation indicates that the effect of VLF-1 is exerted through
the burst sequence. Although the p10 promoter had not been
mutationally analyzed as extensively as the polh promoter previously, our analysis of linker mutations in the p10
promoter confirmed the presence of a burst sequence between the TAAG
and the translational initiation codon and further showed that VLF-1 stimulates expression from the p10 promoter through the
burst sequence.
The connection between the burst sequence and VLF-1 transactivation is
further supported by our observation that VLF-1 may physically interact
with the polh and p10 promoters through their burst sequences. DNase I protection assays detected similar footprints within the burst sequences of both polh and p10
promoters when different VLF-1 fusion preparations generated in insect
and E. coli cells were used. Thus, it appears that VLF-1 is
able to bind to very-late promoters, although the possibility cannot be
excluded that this interaction is facilitated by factors in infected
insect cells. The observation that VLF-1 partially purified from insect cells resulted in a larger footprint on the p10 promoter
than the VLF-1 partially purified from E. coli cells (Fig.
5) may be an indication that other factors copurifying with VLF-1
participated in the protection. Multiple VLF-1-containing complexes
were detected in gel shift assays, and they may represent involvement
of other factors or multimerization of VLF-1. The large size of the
protected region (40 to 45 bp) suggests that VLF-1 binds as a multimer. Since VLF-1 is a relative of the 
phage integrase family (11, 13, 28), the members of which usually form tetramers upon association with DNA, it would not be surprising to find that VLF-1
binds to the burst sequence as a tetramer. Binding of VLF-1 to the
burst sequence may be synergistic, since VLF-1 may form multimers and
activation of the polh promoter requires a threshold level
of VLF-1 (27). Although the burst sequences of both the polh and p10 promoters are capable of interacting
with VLF-1, there is no obvious similarity between them, nor are there
short consensus sequences reminiscent of the phage integrase
binding sites. Both promoters are AT rich, which may facilitate VLF-1 complex formation. Interaction of VLF-1 with the p10
promoter is more readily detected than that with the polh
promoter in both gel shift assays and DNase I protection assays,
suggesting that VLF-1 has higher affinity to the burst sequence of the
p10 promoter.
Mutational analyses show that complex formation of VLF-1 with the burst
sequences of the polh and p10 promoters is
closely correlated with transactivation of these promoters by VLF-1. In most cases, mutations in the burst sequences disrupted VLF-1 complex formation and also abrogated the responses of the corresponding very
late promoters to stimulation by VLF-1. One of the p10
promoter mutants (47) bound partially to VLF-1 and responded
partially to VLF-1 stimulation. These data provide strong support for
the view that the interaction of VLF-1 with the burst sequences of these promoters is essential for VLF-1 transactivation.
Both late and very late genes are transcribed by a novel virus-induced
RNA polymerase (1, 3, 5, 7) whose major components are four
viral gene products, LEF-8, LEF-9, LEF-4, and p47 (6). LEF-8
and LEF-9 have sequence motifs found in subunits of cellular RNA
polymerases (8, 16). In vitro transcription studies suggest
that the polymerase responsible for late gene transcription is
biochemically distinguishable from the polymerase responsible for very
late gene transcription, although the two activities may differ by only
a subunit (26). Both late and very late promoters require a
polymerase which can recognize and initiate at a TAAG sequence, while
very late promoters apparently require an additional factor which can
recognize the burst sequence. It is possible that interaction of VLF-1
with the burst sequence facilitates the recruitment of RNA polymerase
to the promoter or stabilizes the transcription complex and thereby
enhances transcription initiation. The interaction of VLF-1 with
components of the very-late transcription complexes remains to be demonstrated.
Our current model is that very-late promoters have TAAG sequences which
are in relatively poor contexts for polymerase initiation during the
late phase, when VLF-1 levels are low. When VLF-1 accumulates above a
threshold level (27), binding of VLF-1 to the burst sequence
becomes sufficiently potent and facilitates polymerase interaction with
and initiation from the TAAG sequence. Other very late factors may be
involved in the interaction of VLF-1 with the burst sequence or with
the RNA polymerase. However, no such additional factors have been
identified in transient-expression assays (22), and
regulation of polh expression is governed by the level of
VLF-1 (27). Thus, VLF-1 seems to be the primary factor in
the regulation of very late gene expression, and it appears to act by
interaction with the burst sequence.
| |
ACKNOWLEDGMENTS |
We thank Yonghong Li, Jeanne McLachlin, Janet Hatt, Alex Harvey,
Jeff Rapp, Domagoj Vucic, and Joyce Wilson for help and advice.
This work was supported in part by Public Health Service grant AI23719
from the National Institute of Allergy and Infectious Diseases.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Entomology, 413 Biological Sciences Bldg., The University of Georgia, Athens, GA 30602. Phone: (706) 542-2294. Fax: (706) 542-2279. E-mail:
miller{at}arches.uga.edu.
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Journal of Virology, April 1999, p. 3404-3409, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
