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INTRODUCTION |
Vaccinia virus, the prototypic poxvirus, possesses a
double-stranded DNA genome of 191,686 base pairs (1) capable of
encoding ~200 proteins. Poxviruses, which replicate within the
cytoplasm of the infected cell, evolved to encode the enzymes required
to carry out viral DNA replication, transcription, and mRNA
processing (2). Studies with vaccinia virus revealed three temporal
stages of gene expression: early, intermediate, and late. Capped and polyadenylated early mRNAs are synthesized within the virus
particle immediately after infection (3-5). Following early gene
expression and the onset of DNA replication, intermediate genes are
transcribed on a replicating template. Late gene transcription follows
that of intermediate genes and employs a similar template.
Transcription of each gene class requires class-specific transcription
initiation factors and employs class-specific promoters. Host factors
are also required for both intermediate (6) and late (7, 8) mRNA synthesis.
Initiation of early vaccinia virus transcription requires the
heterodimeric early transcription factor
VETF1 (9) and the virion RNA
polymerase possessing the RNA polymerase-associated protein RAP94, the
product of H4L gene (10, 11). Initiation is coupled to ATP hydrolysis
by VETF (12), which binds to the promoter sequence and recruits RNA
polymerase to the template (13). Only the virion RNA polymerase
molecules containing RAP94 exhibit VETF-dependent
transcription of a double-stranded DNA template possessing a viral
early promoter (14). Unlike the other subunits of vaccinia virus RNA
polymerase, RAP94 is present in submolar amounts and is synthesized
exclusively late in infection, whereupon it is packaged into nascent
virions (10). Free H4L protein was not detected in virion extracts (10)
and was not dissociable from purified viral polymerase even under
denaturing conditions (14).
Early viral genes are unique in that transcription terminates in a
signal- and factor- dependent manner (15-17). Elongation proceeds
through the sequence TTTTTNT in the nontemplate strand, yielding
UUUUUNU in the nascent mRNA, which serves as a signal required for
the termination event (18). Termination requires both the vaccinia
termination factor (VTF; also serves as viral mRNA capping enzyme)
(16) and nucleoside triphosphate phosphohydrolase I (NPH I), the
product of gene D11L, as the ATPase employed in transcription
termination (19, 20). During infection, transcription termination is
restricted to early genes. In vitro, only RNA polymerase capable of recognizing early promoters is subject to
signal-dependent termination, demonstrating that this form
of RNA polymerase is uniquely termination-competent (21).
Prior work demonstrated a physical interaction between the
COOH-terminal end of NPH I and the NH2-terminal end of the
H4L subunit of the virion RNA polymerase (22). Furthermore,
COOH-terminal NPH I mutants failed both to bind to H4L (22) and to
support transcription termination (20) and transcript release (22), indicating that the interaction between NPH I and H4L is needed for
these processes. The requirement for NPH I/H4L binding would explain
the known restriction of transcription termination to early genes,
since the H4L subunit is only found on the form of RNA polymerase that
transcribes early genes.
In order to test this model by a different approach, we
evaluated the effect of region-specific H4L antibodies on early gene transcription termination, in vitro. We show that antibodies
directed against the NH2-terminal region of H4L
(representing amino acids 1-256) inhibit early gene transcription
termination in an extract prepared from cells infected at the
nonpermissive temperature with C50, a temperature-sensitive mutant
virus. This extract lacks NPH I activity (20). Preincubation of the
antibodies with H4L fragments representing amino acids 1-256 or 1-195
prevented the antibody inhibition. Interestingly, antibody inhibition
of transcription termination is reduced in a wild type virus-infected
cell extract containing NPH I. Moreover, addition of NPH I to a C50
mutant virus-infected cell extract, lacking NPH I, or to a ternary
complex isolated from a C50 mutant virus-infected cell extract,
prevented antibody inhibition of termination. These results demonstrate that the amino-terminal end of H4L is required for early gene transcription termination and confirm the importance of the H4L/NPH I
interaction in this process.
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EXPERIMENTAL PROCEDURES |
Cells and Viruses--
Wild type (WT) vaccinia virus
strain WR and the temperature-sensitive (ts) mutant virus, C50 (23,
24), were propagated in BSC40 African green monkey cells at 37 °C,
or the permissive temperature for ts mutants, 31 °C, respectively,
as described (23). Crude extracts of virus-infected cells were prepared
by freeze/thaw, and infectious virus titer was determined by plaque assay on BSC40 cells at the permissive temperature, 31 °C, and the
nonpermissive temperature, 40 °C.
Purification of Anti-H4L Antibodies--
Polyclonal rabbit
antisera were prepared against segments of the H4L subunit of the
virion RNA polymerase, representing amino acids 1-256, 258-556, and
568-795. The IgG fraction of each antiserum was isolated using
Affi-gel Protein A MAPS II kit (Bio-Rad) following the manufacturer's
instructions. Protein A-agarose was packed in a column (10-ml Poly-Prep
column, Bio-Rad) and washed with the binding buffer provided with the
kit. One ml of antiserum was diluted 1:1 with the binding buffer and
loaded onto a Protein A-agarose column. The unbound fraction of the
serum was washed off the column, and the bound fraction (IgG)
subsequently eluted with the elution buffer, pH 2.8. The eluates were
collected in 1-ml fractions in tubes containing 200 µl of 0.1 M Tris-HCl, pH 8.0, to neutralize the eluates. The column
effluent was monitored spectrophotometrically at 280 nm in order to
detect the protein peaks. Fractions were pooled, aliquoted, and stored
at
80 °C.
Western Analysis--
Proteins were resolved in a 12.5%
polyacrylamide SDS gel and transferred to a nitrocellulose membrane.
The blot was blocked with 3% gelatin and probed with the IgG fractions
(diluted 1:500 in 1% gelatin) raised against MalE fusion proteins
containing amino acids 1-256 or 568-795 of the H4L subunit of virion
RNA polymerase. Following several washes, the membranes were incubated with goat anti-rabbit IgG (diluted 1:3000 in 1% gelatin) conjugated to
alkaline phosphatase. The alkaline phosphatase conjugate substrate (Bio-Rad) was then used for the development of immunoblots.
Immunoprecipitation--
For each immunoprecipitation, 5 µl of
the IgG fraction (0.7 mg/ml) was employed with 1 µl of the
35S-labeled transcription translation product in a total
incubation of 200 µl. The reaction was incubated at 4 °C overnight
with constant rocking. The antibodies were collected by the addition of
20 µl of Protein A-agarose, and the precipitate was washed four times as described (25). The final pellet was resuspended in sample buffer,
boiled, and applied to an SDS-polyacrylamide gel. The gel was then
soaked in 1 M salicylic acid for 30 min and dried, and
fluorography was then performed at
80 °C.
Transcription Extracts--
Extracts of virus-infected cells
were prepared by lysolecithin treatment, as described (21). A549 cells
were infected with either wild type or ts mutant viruses at a
multiplicity of infection of 15, at 37 °C or 31 °C, respectively.
In the case of the ts mutant virus, after 24 h, the medium was
removed and replaced with 40 °C medium containing 100 µg/ml
cycloheximide. After an additional 24 h at 40 °C, cells were
washed and treated with 250 µg/ml lysolecithin and extracts prepared.
This procedure permits the initial synthesis of active NPH I, which is
required for intermediate and late gene expression. After switching to
40 °C, the endogenous NPH I is inactivated and cycloheximide
prevents the synthesis of new protein (20).
Transcription Termination Assay--
The plasmid template,
pSBterm (21), possesses tandem termination signals within the G-less
cassette. Transcription reactions were carried out in a 20-µl total
volume containing 6 µl of extract, 1 mM ATP, 0.1 mM UTP, 20 µM CTP, 4 µCi of
[
-32P]CTP (800 Ci/mmol), 0.1 mM 3'OMeGTP,
0.2 µg of supercoiled plasmid DNA, 20 mM Tris-HCl buffer,
pH 8.0, 6 mM MgCl2, 2 mM
dithiothreitol, and 8% glycerol for 30 min at 30 °C. When
indicated, the virus-infected cell extracts were preincubated with the
IgG fractions for 30 min on ice. After proteinase K treatment, RNA was
isolated by extraction with phenol/chloroform, precipitated with
isopropanol, and resuspended in formamide dye solution. Samples were
heated at 90 °C and separated by electrophoresis in 5% acrylamide,
8 M urea gels, and the RNA was visualized by
autoradiography. Termination efficiency was calculated as the molar
ratio of terminated RNA to the sum of read-through and terminated RNA.
Construction of the G21(TER29)A78 plasmid containing a vaccinia early
promoter was described by Deng et al. (26); this plasmid was
generously provided by Dr. Stewart Shuman. The prototype G21(TER29)A78 transcription unit consists of a synthetic early promoter fused to a
20-nucleotide G-less cassette, which is flanked by a run of three G
residues at positions +21 to +23. A 57-nucleotide A-less cassette lies
downstream of the G-less cassette and flanked at its 3' end by four A
residues at positions +78 to +81. A termination signal, TTTTTTTTT, lies
within the A-less cassette, spanning position +29 to +37. The
biotinylated 324-base pair DNA template was amplified by polymerase
chain reaction employing a 5' biotin tag on the upstream primer and
isolated by preparative agarose gel electrophoresis. The purified DNA
fragment was then immobilized to streptavidin-coated magnetic beads
(Dynabeads M280; Dynal) as described (27). The bead-bound (B) template
(typically, 100 fmol) was first incubated with 6 µl of C50 or WT
virus-infected cell extracts, in the presence of 1 mM
ATP, 10 µCi of [
-32P]CTP (800 Ci/mmol), 0.1 mM UTP, and 0.625 mM 3'OMeGTP to synthesize the G21 transcript. The ternary complex was isolated, washed, and
incubated in the presence or absence of the IgG fractions, or NPH I. When indicated, the IgG fractions were preincubated with H4L fragments
for 20 min on ice, prior to incubation with the ternary complexes. The
ternary complexes were collected by centrifugation and resuspended, and
termination was then assessed after elongation in the presence of 1 mM UTP, 1 mM GTP, 1 mM CTP, and 1 mM ATP, and the presence or absence of VTF and NPH I. Elongation of the nascent RNA chains beyond the arrest site at
G21 depended on removal of the blocking 3'OMeGMP moiety by the
hydrolytic activity intrinsic to the vaccinia RNA polymerase elongation
complex (28). Elongation of the transcript beyond the G21 position, in
the presence of all four NTPs, would yield a transcript of about 177 bases in length. Greater than 90% of the isolated ternary complexes were routinely elongated in the second RNA synthesis reaction. Signal-dependent termination would be expected to produce a
family of RNA products about 70 bases in length. Termination efficiency was calculated as the molar ratio of terminated RNA to the sum of
read-through and terminated RNA.
Plasmids--
pCITE-4a-H4L plasmid containing full-length H4L
was constructed by inserting an NcoI-SalI DNA
fragment derived from pET-14a-H4L (obtained from Dr. Stewart Shuman),
containing the coding sequence of H4L, into the
NcoI-SalI digested pCITE-4a. H4L retains a 5' His6 tag encoded in pET-14a. pCITE-4a-H4L1-195
was constructed by digesting the pCITE-4a-H4L plasmid with
AccI restriction enzyme and religation of the digested
construct. pET-30a-H4L1-195 was constructed by excising
the DNA fragment corresponding to amino acids 1-195 from
pCITE-4a-H4L1-195 construct, using NcoI and
XhoI restriction enzymes, and inserting it into pET-30a (22).
In Vitro Transcription/Translation--
Novagen Single Tube
Protein system 3 (STP3) was used for the in vitro synthesis
of 35S-labeled proteins directly from DNA templates
containing T7 RNA polymerase promoter. The DNA template (typically 0.5 µg) was transcribed in 10 µl at 30 °C for 15 min, followed by
the addition of 40 µl of translation mix, and incubated for another
60-90 min. pCITE-4a-derived recombinant plasmid was used.
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RESULTS |
Characterization of Anti-H4L Antibodies--
A recent report by
Mohamed and Niles (22) showed that the NH2-terminal end of
the H4L subunit of the virion RNA polymerase can interact with the
COOH-terminal end of NPH I, a single-stranded DNA-dependent
ATPase. In addition, mutations in the COOH-terminal end of NPH I, which
failed to bind H4L, also failed to mediate both transcription
termination (20) and transcript release (22). These observations
indicate that this interaction is required for early gene transcription
termination and transcript release. In order to further investigate the
involvement of the H4L subunit of the virion RNA polymerase in early
gene transcription termination, we evaluated the effect of
region-specific H4L antibodies on early gene transcription termination,
in vitro. Polyclonal antibodies were raised in rabbits
against MalE fusion proteins containing segments of H4L, representing
amino acids 1-256, 258-556 and 568-795 (Fig.
1A). The IgG fraction of each
antiserum was isolated by adsorption to Protein A-agarose and
subsequently eluted using a low pH elution buffer. The relative
affinity of each of these IgG fractions to the H4L protein was examined
in both immunoprecipitation and Western blot analyses. Antibodies
H4L1-256 and
H4L568-795 precipitated
35S-H4L to a similar extent (Fig. 1B). In
addition, each antibody exhibited similar reactivity to H4L in Western
blot analyses (Fig. 1C). These results demonstrate the
specificity of the antibody fractions and show that both fractions,
H4L1-256 and
H4L568-795, have
comparable affinity to H4L protein. In contrast, antibodies directed
against H4L 258-556 exhibited low binding affinity toward H4L in both
immunoprecipitation and Western blot analyses (data not shown).
Therefore, only
H4L1-256 and
H4L568-795 were employed throughout the rest of the study.

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Fig. 1.
Characterization of anti-H4L antibodies.
A, a diagram showing the different regions of H4L to which
antibodies were raised. B, immunoprecipitation of
35S-H4L with H4L1-256 and
H4L568-795. For each immunoprecipitation, 5 µl of the
IgG fraction (0.7 mg/ml) was employed with 1 µl of the
35S-labeled transcription translation product in a total
incubation of 200 µl. Input, 100% of the input
radioactivity; I, IgG from immunized rabbit serum;
PI, preimmune rabbit serum. C, Western blot
analysis of wild type vaccinia virus and C50 mutant virus-infected cell
extracts using H4L1-256 and H4L568-795.
Proteins were resolved on a 12.5% polyacrylamide SDS gel, transferred
to a nitrocellulose membrane, and probed with the IgG fractions raised
against MalE fusion proteins containing amino acids 1-256 or 568-795
of the H4L subunit of the virion RNA polymerase. H4L,
migration position of full-length H4L protein.
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Antibodies Directed against the NH2-terminal Region of
H4L Inhibit Early Gene RNA Synthesis--
Prior work by Deng and
Shuman (11), using an anti-H4L antibodies directed against the
NH2-terminal 364 amino acids of H4L, showed that the
NH2-terminal region of H4L is required for early gene
transcription initiation. In confirmation of these results and in an
attempt to evaluate our antibody reagents, the following in
vitro analyses were conducted. Transcription-competent extracts were prepared from cells infected with either wild type virus or C50
mutant (lacking NPH I) virus. To test the ability of the virus-infected
cell extracts to mediate early gene transcription termination, the
pSB24-term plasmid template (21) was used (Fig. 2A). The plasmid template was
designed to contain a G-less cassette possessing a tandem early gene
transcription termination signal downstream from a strong synthetic
vaccinia virus early promoter (21). Synthesis of a transcript that
extends from the initiation site to the end of the G-less cassette
yields a product of about 540 bases in length.
Signal-dependent termination would be expected to produce a
family of RNA products about 450 bases in length. Using the WT
virus-infected cell extract, either control or pretreated with anti-H4L
antibodies, transcription of pSB24-term was carried out in the presence
or absence of exogenously added VTF, and the RNA products were analyzed
by gel electrophoresis (Fig. 2B). In the absence of any
added factor, a 55% termination efficiency was achieved. Addition of
VTF enhanced the level of termination to about 78% without affecting
total RNA synthesis. However, preincubation of the extract with
antibodies directed against the NH2-terminal region of H4L
(1) inhibited total RNA synthesis, whereas preincubation with
antibodies directed against other regions of H4L did not diminish total
RNA synthesis and had no effect on termination (Fig. 2B). A
similar pattern was obtained using C50 mutant virus-infected cell
extract, which lacks NPH I (Fig. 2C). As reported
previously, synthesis of only the read-through product was seen in the
C50 extract (20). Addition of VTF alone did not significantly stimulate termination since NPH I was missing. However, addition of both VTF and
NPH I enhanced the level of termination to about 66% (Fig. 2C). Preincubation of this extract with antibodies directed
against the NH2-terminal region of H4L (1) inhibited
total RNA synthesis, whereas antibodies directed against other regions
of H4L did not alter total RNA synthesis. These results are in
agreement with that obtained by Deng and Shuman (11), showing that
antibodies raised against the NH2-terminal region of H4L
inhibited viral RNA synthesis, further limiting the essential region to
the first 256 amino acids. However, one important difference can
be observed. Although
H4L1-256 inhibits RNA synthesis
in both wild type virus and C50 mutant virus-infected cell extracts, in
the C50 extract, termination is inhibited as well (Fig. 2, B
and C).

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Fig. 2.
Antibodies directed against the
NH2-terminal region of H4L inhibit early gene RNA
synthesis. A, a diagram of the pSB24-term plasmid
template (21) is shown. Synthesis of a transcript that extends from the
initiation site to the end of the G-less cassette would yield a product
of about 540 bases in length. Signal-dependent termination
would be expected to produce a family of RNA products about 450 bases
in length. FL, full-length transcript; Term,
terminated transcript; P, promoter; T,
terminator. B, transcription was carried out employing a
wild type virus-infected cell extract, without or with preincubation
with anti-H4L antibodies H4L1-256,
H4L258-556, and H4L568-795 (in the
presence or absence of the indicated amounts of VTF). C,
transcription was carried out employing a C50 mutant virus-infected
cell extract, which lacks NPH I activity, without or with preincubation
with anti-H4L antibodies H4L1-256,
H4L258-556, and H4L568-795 (in the
presence or absence of the indicated amounts of VTF and wild type NPH
I. The percentage of RNA synthesis and transcription termination
(indicated below the autoradiograph) was quantified by scanning the
autoradiogram with a PhosphorImager. FL, 540-base full
length transcript; Term, 450-base terminated
transcript.
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To further evaluate the affect of
H4L1-256 on
transcription termination, it was necessary to separate transcription initiation from elongation and termination. To do this, a bead-bound template was employed in the following studies. The prototype G21(TER29)A78 transcription unit (26) consists of a synthetic early
promoter fused to a 20-nucleotide G-less cassette, which is flanked by
a run of three G residues at positions +21 to +23. A 57-nucleotide
A-less cassette was inserted downstream of the G-less cassette and
flanked at its 3' end by a run of four A residues at positions +78 to
+81. A termination signal, TTTTTTTTT, was placed within the A-less
cassette, spanning positions +29 to +37 (Fig.
3A). Synthesis of a transcript
that extends from the initiation site to the end of the G-less cassette
would yield a product of 21 bases in length (G21). Elongation of the
transcript beyond the G21 position, in the presence of all 4 NTPs,
would yield a transcript of about 177 bases in length.
Signal-dependent termination would be expected to produce a
family of RNA products about 70 bases long. RNA synthesis was initiated
in a C50 mutant virus-infected cell extract, lacking NPH I. The
template-engaged 32P-labeled (bead-bound) G21 RNA product
was then separated and washed, and transcription termination was
measured after a second incubation in all four NTPs, in the presence or
absence of VTF and NPH I. In the absence of any added factor, synthesis
of a major read-through product was seen. Minor and variable amounts of
premature release products can also be seen. Addition of VTF alone did
not stimulate termination. However, addition of both VTF and NPH I
enhanced the level of termination to about 56% (Fig. 3B).

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Fig. 3.
Transcription termination assay using the
bead-bound template. A, a map of the bead-bound
G21(TER29) A78 DNA template (26) is shown. The transcription unit
consists of a synthetic early promoter fused to a 20-nucleotide G-less
cassette, which is flanked by a run of three G residues at positions
+21 to +23. A 57-nucleotide A-less cassette was inserted downstream of
the G-less cassette and flanked at its 3' end by four A residues at
positions +78 to +81. A termination signal, TTTTTTTTT, was placed
within the A-less cassette, spanning position +29 to +37.
Arrows represent the products produced by the various
reaction conditions. FL, full-length; P,
promoter; Term, termination product. B, ternary
complexes containing the G21 transcript were synthesized in a C50
virus-infected cell extract, lacking NPH I. The ternary complexes were
then isolated and the nascent transcript was elongated with all four
NTPs, in the presence or absence of the indicated amounts of VTF and
NPH I. The percentage of transcription termination (indicated below the
autoradiograph) was quantified by scanning the autoradiogram with a
PhosphorImager. P denotes the migration position of paused
transcripts.
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Antibodies Directed against the NH2-terminal Region of
H4L Inhibit Early Gene Transcription Termination in a C50 Mutant
Virus-infected Cell Extract--
In order to evaluate the role of H4L
in early gene transcription termination, the effect of the purified
antibodies,
H4L1-256 and
H4L568-795, on
transcription termination was assessed using the bead-bound template.
The template-engaged 32P-labeled G21 RNA product was
synthesized employing a C50 mutant virus-infected cell extract, lacking
NPH I. The ternary complex containing the 32P-labeled G21
product was washed and preincubated for 30 min on ice in the presence
or absence of increasing amounts of IgG antibodies, directed against
either H4L 1-256 or H4L 568-795. The ability of the antibody-treated
ternary complexes to terminate transcription was then evaluated by an
additional incubation in all four NTPs, in the presence or absence of
VTF and NPH I. In the absence of any added factor, synthesis of the
major read-through product and the minor pause products was seen (Fig.
4, lane 1).
Addition of VTF alone did not stimulate termination; however, addition of both VTF and NPH I enhanced the level of termination to about 43%
(Fig. 4, lanes 2 and 3, respectively).
Preincubation of the G21 ternary complex with the IgG antibodies
directed against the NH2-terminal region of H4L (1)
inhibited transcription termination in a
concentration-dependent fashion (Fig. 4, lanes
4-6). Antibodies directed against the COOH-terminal region
of H4L had no effect on transcription termination (Fig. 4,
lane 7). These results indicate the requirement
for the NH2-terminal region of H4L for early gene transcription termination.

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Fig. 4.
Antibody inhibition of transcription
termination. Ternary complexes containing the G21 transcript were
synthesized in a C50 virus-infected cell extract, lacking NPH I. The
ternary complexes were isolated and preincubated for 30 min on ice in
the presence or absence of increasing amounts of anti-H4L antibodies,
H4L1-256 (lanes 4-6) and
H4L568-795 (lane 7). The G21
ternary complexes were then elongated with all four NTPs, in the
presence or absence of the indicated amounts of VTF and NPH I. The
percentage of transcription termination (indicated below the
autoradiograph) was quantified by scanning the autoradiogram with a
PhosphorImager. FL, full-length; Term,
termination product.
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Antibody Inhibition of Transcription Termination Is Prevented by
Preincubation of
H4L1-256 with H4L Fragments--
In
order to evaluate the specificity of the antibody inhibition of
termination, antibodies were preincubated with a H4L fragment, containing amino acids 1-256, that was originally used to raise the
antibodies. The template-engaged 32P-labeled G21 RNA
product was synthesized employing the C50 mutant virus-infected cell
extract, lacking NPH I. The G21 ternary complexes were then incubated
for 30 min on ice in the presence or absence of 250 ng of IgG
antibodies, which either were or were not preincubated with H4L
fragment 1-256. The preincubated G21 ternary complexes were then
elongated in the presence of all four NTPs, in the presence or absence
of VTF and NPH I. In the absence of any added factor, synthesis of only
the major read-through product was seen (Fig. 5A, lane
1). Addition of VTF alone did not stimulate termination; however, addition of both VTF and NPH I enhanced the level of termination to about 41% (Fig. 5A, lanes
2 and 3, respectively). Preincubation of the G21
ternary complex with
H4L1-256 inhibited transcription
termination (Fig. 5A, lane 4).
Preincubation of
H4L1-256 with H4L 1-256 prevented the
antibody inhibition of transcription termination (Fig. 5A,
lanes 5-8).

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Fig. 5.
Antibody inhibition of transcription
termination is prevented by preincubation of
H4L1-256 with H4L fragments.
Ternary complexes containing the G21 transcript were synthesized in a
C50 virus-infected cell extract, lacking NPH I. The G21 ternary
complexes were then incubated, for 30 min on ice, in the presence or
absence of 250 ng of H4L1-256 antibodies that were
(lanes 5-8) or were not (lane
4) preincubated with increasing amounts of H4L fragments
1-256 or 1-195. The preincubated G21 ternary complexes were then
elongated with all four NTPs, in the presence or absence of the
indicated amounts of VTF and NPH I. A,
H4L1-256 antibodies were preincubated in the presence
(lanes 5-8) or absence (lane
4) of H4L 1-256. B, H4L1-256
antibodies were preincubated in the presence (lanes
5-8) or absence (lane 4) of H4L
1-195. The percentage of transcription termination (indicated below
the autoradiograph) was quantified by scanning the autoradiogram with a
PhosphorImager. FL, full-length; Term,
termination product.
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Recently, the interaction between the H4L subunit of the virion RNA
polymerase and NPH I, a single-stranded DNA-dependent ATPase, was shown to be required for early gene transcription termination and transcript release (22). This interaction was mapped to
the NH2-terminal 195 amino acids of H4L. The ability of the
H4L 1-195 to prevent the antibody inhibition of transcription termination was tested. Preincubation
H4L1-256 with H4L 1-195 also prevented the antibody inhibition of transcription termination (Fig. 5B, lanes 5-8).
These results demonstrate that the inhibition of termination requires
antibody binding to one or more epitopes in the
NH2-terminal end of H4L between amino acids 1 and 195. Furthermore, these results also confirm that the
NH2-terminal region of H4L, containing amino acids 1-195, is required for transcription termination.
H4L1-256 Antibodies Exhibit Reduced Inhibition of
Early Gene Transcription Termination in a Wild Type Virus-infected Cell
Extract--
The template-engaged 32P-labeled G21 RNA
product was synthesized employing a wild type virus-infected cell
extract. The G21 ternary complexes were then incubated for 30 min on
ice, in the presence or absence of 250 ng of
H4L1-256.
Preincubation with the
H4L568-795, directed against the
COOH-terminal region of H4L, was used as a negative control. The
preincubated G21 ternary complexes were then elongated with all four
NTPs, in the presence or absence of VTF and NPH I. In the absence of any added factor, synthesis of the major read-through product was seen
(Fig. 6, lane 1).
Addition of VTF enhanced the level of termination to about 61% (Fig.
6, lane 2). Addition of extra NPH I to the
isolated ternary complexes had little effect on the level of
transcription termination observed (Fig. 6, lane
3). In contrast to the C50 mutant virus-infected cell
extract, preincubation of the G21 ternary complexes synthesized in wild
type virus-infected cell extract with
H4L1-256
exhibited minor inhibition of transcription termination (Fig. 6,
lane 4), which was prevented by addition of NPH I
to the ternary complexes (Fig. 6, lane 5). Preincubation with
H4L568-795 had no effect on
termination (Fig. 6, lane 6). These results
indicate that the presence of NPH I in the ternary complex prevents
antibody inhibition of transcription termination. Prior work from this
laboratory showed that NPH I is not an integral component of the
ternary complex, but rather that a reversible interaction between NPH I
and the ternary complex occurs (22). Addition of NPH I to ternary
complexes synthesized in wild type-infected cell extract had little
effect on termination (Fig. 6, lane 3), but NPH I
addition prior to the antibody exhibited an additional protection from
antibody inhibition of transcription termination (Fig. 6,
lane 5). These data indicate that both NPH I and
H4L1-256 compete for the same site(s) on H4L.

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Fig. 6.
H4L1-256
antibodies exhibit reduced inhibition of early gene transcription
termination in a wild type virus-infected cell extract. The
template-engaged 32P-labeled G21 RNA product was
synthesized in a wild type virus-infected cell extract. The isolated
G21 ternary complexes were preincubated in the presence or absence of
NPH I, prior to the antibody addition (lane 5).
The G21 ternary complexes were then incubated for 30 min on ice, in the
presence or absence of 250 ng of H4L1-256
(lanes 4 and 5) or
H4L568-795 (lane 6). The
preincubated G21 ternary complexes were then elongated with all four
NTPs, in the presence or absence of the indicated amounts of VTF and
NPH I. The percentage of transcription termination (indicated
below the autoradiograph) was quantified by scanning the
autoradiogram with a PhosphorImager. FL, full-length;
Term, termination product.
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|
Addition of NPH I to a C50 Mutant Virus-infected Cell Extract,
Lacking NPH I, Reduces Antibody Inhibition of Transcription
Termination--
If NPH I and
H4L1-256 compete for the
same site(s) on H4L, addition of NPH I to a C50 mutant virus-infected
cell extract should prevent antibody inhibition of transcription
termination. To test this hypothesis, the template-engaged
32P-labeled G21 RNA product was synthesized in a C50 mutant
virus-infected cell extract preincubated with NPH I (C50 extract/NPH
I). The G21 ternary complexes were isolated and then incubated in the presence or absence of additional NPH I, prior to the antibody addition. Subsequently, the ternary complexes were incubated in the
presence or absence of the
H4L1-256. Preincubation of
the G21 ternary complexes with the
H4L568-795 was used as a negative control (Fig. 7,
lane 6). The preincubated G21 ternary complexes
were then elongated with all four NTPs, with or without added VTF and
NPH I. In the absence of any added factor, synthesis of the major
read-through product was seen (Fig. 7, lane 1).
Addition of VTF enhanced the level of termination to about 75% (Fig.
7, lane 2). Addition of extra NPH I to the
ternary complexes did not enhance the level of transcription
termination any further (Fig. 7, lane 3).
Incubation of the G21 ternary complexes, prepared in a C50 mutant
virus-infected cell extract complemented with added NPH I, with
H4L1-256 exhibited reduced inhibition of transcription
termination (Fig. 7, lane 4). Moreover,
preincubation of the isolated ternary complexes with additional NPH I,
prior to the antibody addition, further prevented the antibody
inhibition of transcription termination (Fig. 7, lane
5). These results support the hypothesis that NPH I binds to
the site(s) on H4L containing the epitopes that bind to the inhibitory
antibodies.

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Fig. 7.
Addition of NPH I to a C50 mutant
virus-infected cell extract, lacking NPH I, reduces antibody inhibition
of transcription termination. The template-engaged
32P-labeled G21 RNA product was synthesized in a C50 mutant
virus-infected cell extract preincubated in the presence of NPH I (C50
extract/NPH I). The G21 ternary complexes were isolated and then
incubated in the presence or absence of additional NPH I, prior to the
antibody addition (lane 5). Subsequently, the
ternary complexes were incubated in the presence or absence of
H4L1-256 (lanes 4 and
5) or H4L568-795 (lane
6). The preincubated G21 ternary complexes were then
elongated with all four NTPs, in the presence or absence of the
indicated amounts of VTF and NPH I. The percentage of transcription
termination (indicated below the autoradiograph) was
quantified by scanning the autoradiogram with a PhosphorImager.
FL, full-length; Term, termination product.
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NPH I Addition to Ternary Complexes Isolated from a C50 Mutant
Virus-infected Cell Extract Prevents the Antibody Inhibition of
Transcription Termination--
The template-engaged
32P-labeled G21 RNA product was synthesized employing a C50
mutant virus-infected cell extract, lacking NPH I. The isolated G21
ternary complexes were incubated in the presence or absence of NPH I. The preincubated G21 ternary complexes were then incubated in the
presence or absence of
H4L1-256. The preincubated G21
ternary complexes were then elongated with all four NTPs, with or
without added VTF and NPH I. Preincubation of the G21 ternary complexes
with
H4L568-795 was used as a negative control (Fig.
8, lane 6). In the
absence of any added factor, synthesis of the major read-through
product was seen (Fig. 8, lane 1). Addition of
VTF alone did not stimulate termination; however, addition of both VTF
and NPH I enhanced the level of termination to about 46% (Fig. 8,
lanes 2 and 3, respectively). Incubation of the G21 ternary complexes without added NPH I, with
H4L1-256 inhibited transcription termination (Fig. 8,
lane 4). However, preincubation of the isolated
ternary complexes with NPH I, prior to the antibody addition, prevented
the antibody inhibition of transcription termination (Fig. 8,
lane 5). These results demonstrate that NPH I can
block the antibody access to the NH2-terminal region of
H4L.

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Fig. 8.
NPH I addition to ternary complexes isolated
from a C50 mutant virus-infected cell extract prevents the antibody
inhibition of transcription termination. The template-engaged
32P-labeled G21 RNA product was synthesized in a C50 mutant
virus-infected cell extract, lacking NPH I. The G21 ternary complexes
were incubated further in the presence or absence of NPH I, prior to
the antibody addition (lane 5). The preincubated
G21 ternary complexes were then incubated in the presence or absence of
H4L1-256 (lanes 4 and
5). Preincubation of the G21 ternary complexes with
H4L568-795 was used as a negative control
(lane 6). The preincubated G21 ternary complexes
were then elongated with all four NTPs, in the presence or absence of
the indicated amounts of VTF and NPH I. The percentage of transcription
termination (indicated below the autoradiograph) was
quantified by scanning the autoradiogram with a PhosphorImager.
FL, full-length; Term, termination product.
|
|
 |
DISCUSSION |
Signal-dependent transcription termination is
restricted to early poxvirus genes (29), whose transcription is
catalyzed by the virion form of RNA polymerase (10). Effective
termination of early gene transcription requires the productive
interplay of at least four factors: the virion RNA polymerase (21), the signal UUUUUNU in the nascent mRNA (15, 18, 30), VTF, a multifunctional transcription factor and mRNA processing enzyme (16), and the ATP-hydrolyzing enzyme NPH I (19, 20). It is clear that
only the form of RNA polymerase that recognizes an early promoter is
sensitive to signal-dependent termination (21). The H4L
protein, RAP94, is an integral RNA polymerase subunit found only in the
virion form of RNA polymerase that recognizes and initiates at early
gene promoters (10, 11, 31).
Prior work from this laboratory demonstrated a physical interaction
between the COOH-terminal end of NPH I and the NH2-terminal end of the H4L subunit of the virion RNA polymerase (22). This observation correlated with NPH I mutants that failed both to bind to
H4L (22) and to support transcription termination (20) and transcript
release (22), indicating that the interaction between NPH I and H4L may
be required for these processes. The essential interaction of NPH I and
H4L provided an explanation for the observed restriction of
UUUUUNU-dependent transcription termination to early genes,
where only the H4L-containing RNA polymerase would be able to
terminate. This interaction also defined H4L as a termination cofactor,
recruiting NPH I to the ternary complex. However, this observation
conflicted with a prior report (32) indicating that H4L was not
required for NPH I mediated transcription termination in
vitro. Therefore, further experiments were needed to resolve this
discrepancy and to evaluate the requirement of H4L for early gene
transcription termination.
Antibodies directed against H4L were tested for their ability to
inhibit transcription termination in vitro. Preincubation of
a ternary complex prepared in the absence of NPH I with the antibodies
directed against the NH2-terminal region of H4L (1) specifically inhibited transcription termination. Inhibition was blocked by preincubation of
H4L1-256 antibodies with
H4L 1-195, demonstrating that the inhibition was mediated by antibody binding to one or more epitopes in the NH2-terminal end of
H4L. These data demonstrate that the NH2-terminal domain of
H4L is required for early gene transcription termination.
Prior results from this laboratory mapped the site of interaction
between NPH I and H4L to the NH2-terminal 195 amino acids of H4L (22). Therefore, we investigated whether NPH I and
H4L1-256 compete for the same site(s) on H4L.
Preincubation of the G21 ternary complexes synthesized in a wild
type-infected cell extract with
H4L1-256 exhibited
reduced inhibition of transcription termination. Furthermore,
preincubation of a NPH I minus virus-infected cell extract with NPH I
prior to antibody addition, or readdition of NPH I to isolated ternary
complexes prepared in the absence of NPH I, prevented antibody
inhibition of transcription termination, indicating that NPH I binds to
the site on H4L containing the epitopes that bind to the inhibitory antibodies.
One can propose three models to explain the mechanism of antibody
inhibition of NPH I binding to H4L. In the simplest and least likely
model, the antibody binding site and the NPH I interaction site are the
same. Binding is mutually exclusive. In other models, NPH I and the
antibody bind to different sites on H4L. In one case, binding of either
antibody or NPH I blocks binding of the other protein. Alternatively,
binding of one protein to H4L could change the conformation of H4L,
weakening the binding of the other protein. In the absence of
additional data, direct competition is the favored model.
These results demonstrate that inhibition of termination requires
antibody binding to one or more epitopes in the
NH2-terminal end of H4L between amino acids 1 and 195. These data also confirm the prior conclusion (22) that the
NH2-terminal region of H4L, containing amino acids 1-195,
is required for transcription termination. The finding that the
NH2-terminal domain of H4L, an integral RNA polymerase
subunit found only in the virion form of RNA polymerase that recognizes
and initiates at early gene promoters (10, 11, 31), is required for
termination provides an explanation for the known restriction of
transcription termination to early genes (29).
It is known that NPH I must bind single-stranded DNA to reveal a
cryptic ATPase activity (33, 34) and that ATP hydrolysis is required
for termination (19, 20, 27). Therefore, in the termination complex,
NPH I must have access to single-stranded DNA. Since much of the
template strand is annealed to nascent RNA in the transcription bubble,
the most likely source for single-stranded DNA is the free non-template
strand in the paused ternary complex. Therefore, the role of H4L in
early gene transcription termination could be simply a docking site for
NPH I, which would permit NPH I association with the ternary complex,
and provide access to the non-template strand when termination occurs.
Alternatively, H4L may play an active role in termination yet to be
described, in addition to its role in recruiting NPH I. Further genetic
and biochemical studies are under way to evaluate the exact role of H4L
in termination.