(Received for publication, September 15, 1994; and in revised form, November 7, 1994)
From the
The predicted amino acid sequence of the vaccinia virus gene
A18R shows significant homology to the human ERCC3 gene product, which
is a member of the DEXH subfamily of the DNA and RNA helicase
superfamily II and which plays a role in both RNA polymerase II
transcription and nucleotide excision repair of DNA. The vaccinia virus
A18R gene product is expressed throughout infection and is encapsidated
in virions. Vaccinia virions containing mutant A18R gene product are
defective in early viral transcription in vitro, and infection
with A18R mutant virus results in aberrant viral transcription late
during infection. Thus we hypothesize that the vaccinia virus A18R gene
product is a helicase that plays a role in viral transcription and
possibly DNA repair. As a first test of this hypothesis, we have
affinity purified an amino-terminal polyhistidine-tagged A18R protein
and shown that it has DNA-dependent ATPase activity. The A18R ATPase
activity is stimulated by both single-stranded and double-stranded DNA
and by RNADNA hybrids, but not by either single-stranded or
double-stranded RNA.
Vaccinia virus, the prototype poxvirus, is a large
double-stranded (ds) ()DNA-containing virus that replicates
in the cytoplasm of eukaryotic cells(1) . Vaccinia virus
encodes most of the enzymes required for synthesis and modification of
viral mRNA, and therefore the virus provides a valuable model system
for genetic and biochemical investigation of eukaryotic transcription
and its regulation(2, 3) . During infection, vaccinia
genes are expressed in a cascade that can be divided into three
distinct temporal classes: early, intermediate, and late(4) .
Early viral transcription is catalyzed by enzymes contained within the
infecting virions. Early mRNA encodes factors required for synthesis of
intermediate mRNA, intermediate mRNA encodes factors required for
synthesis of late mRNA, and late mRNA encodes early transcription
factors which, along with the viral RNA polymerase, are packaged into
virions in preparation for the next round of infection. Biochemical
experiments have defined many of the viral gene products required for
the proper expression of vaccinia genes, including the multisubunit RNA
polymerase and several of the protein factors specific for each of the
three stages of viral transcription (see Refs. 3, 5-11 and
references therein). Genetic analysis of vaccinia has identified
several additional genes that, when mutated, result in disruption of
the normal orderly program of viral gene
expression(12, 13, 14, 15, 16) .
Mutants in one such gene, A18R (13), have a complex phenotype,
which suggests that the gene plays an important role in transcription
throughout vaccinia infection.
The A18R gene is transcribed
and translated at both early and late times during a vaccinia
infection, and the A18R gene product is packaged in the virion core
particle(17, 18) . Virions isolated from A18R temperature-sensitive mutant infections done at the permissive
temperature contain normal amounts of A18R and other viral proteins but
are diminished in their ability to synthesize early mRNAs in an in
vitro assay, suggesting that the A18R protein is involved in early
viral transcription by virus particles(17) . In vivo,
however, the effect of the A18R mutation on early
transcription is somehow masked, and abnormalities become apparent only
after late viral gene expression has begun, when several seemingly
related phenomena are observed simultaneously. First, viral
transcription loses its usual specificity, and regions of the viral
genome which are normally not transcribed late during infection become
transcriptionally active(19) . This promiscuous late
transcription is accompanied by synthesis of abnormally high levels of
dsRNA(19) , elevation of the intracellular concentration
ppp(A2`p)A (2-5A)(20) , degradation
of ribosomal and viral messenger RNA(13, 19) , and
premature cessation of late viral protein synthesis(13) . A
straightforward hypothesis that explains these events is that the
primary effect of A18R mutations is to induce promiscuous
transcription, which includes transcription of complementary strands of
viral DNA, which in turn results in overproduction of dsRNA. The dsRNA
then triggers the cellular 2-5A pathway(21) , a cascade
of events which includes activation of the dsRNA-dependent 2-5A
synthetase, synthesis of 2-5A, activation of the
2-5A-dependent RNase L, RNase L-mediated RNA degradation, and
cessation of protein synthesis. Other hypotheses, described below, are
inspired by the existence of helicase structural motifs within the
amino acid sequence of A18R.
Computer-assisted analysis of the amino acid sequence of the A18R protein shows that it contains an NTP binding site and that it is homologous to the DEXH subfamily of the DNA and RNA helicase superfamily II(22, 23) . The most significant homology between A18R and any protein in the DEXH family is to the human ERCC3 gene product(23) . ERCC3 (or XPB) is one of several genes associated with xeroderma pigmentosum and Cockayne's syndrome, hereditary disorders characterized by defective nucleotide excision repair of DNA(24) . Interestingly, several recent reports show that ERCC3 functions not only in nucleotide excision repair but also that it is a component of the basal transcription initiation factor TFIIH and that recombinant ERCC3 has DNA helicase activity(25, 26, 27, 28) . Likewise, the yeast homolog of ERCC3, RAD25, complexes with the yeast RNA polymerase II transcription factor b (the yeast homolog of TFIIH), RAD25 is essential for viability, and mutants in RAD25 are defective in both transcription and nucleotide excision repair of DNA(29, 30, 31) . In fact, a growing body of evidence demonstrates that in both mammalian cells and in yeast, several proteins originally associated with DNA repair are also components of the basal transcription machinery, suggesting a tight coupling between transcription and DNA repair(32, 33) . The homology between A18R and the DEXH family in general or ERCC3 specifically, combined with the observations on A18R mutants described above, suggests four alternative hypotheses that describe the role of A18R in vaccinia infection. Central to these hypotheses is the notion that A18R is a DNA or RNA helicase or part of a helicase complex. First, it has been suggested that A18R may be an RNA helicase that unwinds the dsRNA that accumulates during a normal infection and therefore prevents activation of the 2-5A pathway(23) . Second, it is possible that A18R provides an unwinding function associated specifically with transcription. Third, it is possible that A18R is a DNA repair protein and that DNA damage that accumulates in the absence of A18R function indirectly results in promiscuous late transcription. Lastly, it is possible that A18R functions in both transcription and DNA repair in a fashion analogous to subunits of the human transcription factor TFIIH and the yeast transcription factor b. A biochemical analysis of the A18R protein should help to clarify its role in vaccinia infection.
The vaccinia recombinant VVTMHisA18 was constructed by transfecting wild type vaccinia virus-infected cells with the plasmid pTMHisA18 (see below) and selecting for thymidine kinase negative recombinants(38) . The structure of the recombinant was confirmed by polymerase chain reaction analysis using appropriate primers. This virus contains a polyhistidine-A18R fusion, driven by a T7 RNA polymerase promoter and an EMC internal ribosome entry site element, embedded in the vaccinia virus thymidine kinase gene.
pTMHisA18 contains the polyhistidine-A18R fusion protein driven by a T7 RNA polymerase promoter and an EMC internal ribosome entry site element, flanked by the 5` and 3` halves of the vaccinia virus thymidine kinase gene. pTMHisA18 was made by cloning an NcoI-BamHI fragment from p16A18, which contains the entire polyhistidine-A18R fusion coding region, into pTM1 (38) that had been digested with NcoI and BamHI.
For purification of histidine-tagged
A18R protein overexpressed in E. coli, a 2.5-ml column of
His-Bind resin was used. Supernatants were loaded at a rate of 10 ml/h,
the column was washed sequentially with 30-40 ml of binding
buffer, 40-60 ml of wash buffer 1, and 40-60 ml of wash
buffer 2. Bound proteins were eluted with strip buffer containing 5%
glycerol. Peak fractions were identified by electrophoresis of samples
on polyacrylamide gel and silver staining. Fractions were pooled and
dialyzed first into buffer A (20 mM Hepes, pH 7.6, 0.25 M KCl, 0.1 mM EDTA, 1 mM MgCl, 5%
glycerol) and finally into buffer B (buffer A plus 1 mM dithiothreitol).
Figure 2: Analysis of His-A18R by glycerol gradient centrifugation. His-A18R protein (130 µg in 400 µl) purified on a His-Bind column (Fig. 1, lanes 3) was centrifuged on a 10-ml, 15-30% glycerol gradient in buffer B (see ``Experimental Procedures'') in a Beckman SW 41 rotor at 40,000 rpm for 70.5 h at 4 °C. Forty-seven 200-µl fractions were collected. Three µl of each fraction was assayed for ATPase activity (panel A), and 10 µl of each fraction was assayed by polyacrylamide gel electrophoresis and silver staining (panel B).
Figure 4:
The effects of salt, MgCl,
DNA, and substrate on His-A18R ATPase activity. Reactions were done
under standard conditions except as indicated. Panel A, the
KCl concentration was varied, or KCl was substituted with various
concentrations of NaCl or NH
Cl as indicated. Panel
B, the final concentrations of ATP and MgCl
were
varied as indicated. Panel C, m13 ssDNA was substituted with
various concentrations dsDNA or ssDNA. dsDNA is linear, blunt-ended (SmaI-cleaved) pGEM3ZF(+) plasmid DNA; ssDNA is dsDNA
heated to 100 °C for 3 min and quick cooled immediately before
addition to reactions. Panel D, ATP and MgCl
were
added in a 1:1 ratio at various concentrations as
indicated.
Figure 5: The effects of incubation time and enzyme concentration on ATPase activity. Reactions were done under standard conditions except that the final volume was increased to 400 µl, and the concentration of enzyme and DNA contained in the reactions was varied as indicated. At various times after the addition of enzyme, 50-µl samples were removed, quenched by the addition of EDTA, and assayed for ATP hydrolysis as described under ``Experimental Procedures.'' Panel A, time course of reactions containing varying amounts of enzyme or DNA. Panel B, graphical analysis of the 15-min time points from panel A.
Figure 6: Hydrolysis of alternate nucleoside triphosphates by the His-A18R ATPase. Reactions were done under standard conditions except that ATP was replaced by alternate nucleoside triphosphates at a final concentration of 4 mM as indicated. The results are the average of two experiments.
Figure 7: The effects of different nucleic acid cofactors on His-A18R ATPase activity. Reactions were done under standard conditions except that m13 ssDNA was replaced by alternate nucleic acid cofactors at final concentrations of either 10 or 100 µg/ml as indicated.
Figure 1: Polypeptide composition of purified His-A18R. His-A18R was purified from recombinant vaccinia virus-infected cells or from His-A18R-expressing E. coli as described under ``Experimental Procedures.'' Samples were analyzed by electrophoresis on polyacrylamide gels followed by silver staining (panel A), staining with Coomassie Blue (panel B), or Western blotting with anti-A18R antibody (panel C). Lanes 1, molecular weight markers; lanes 2, eluate from a His-Bind column loaded with extract from recombinant vaccinia virus infected cells; lanes 3, eluate from a His-Bind column loaded with extract from His-A18R expressing E. coli; lanes 4, glycerol gradient purified 55 kDa protein from His-A18R expressing E. coli (see text and Fig. 2). Sizes of the molecular weight markers, in kDa, are shown at the left of each panel.
For chromatographic analysis of reaction products
(see Fig. 2), reactions contained 3 µCi of
[-
P]ATP. One µl of the reaction mix was
spotted on polyethyleneimine-cellulose thin layer plates and developed
by ascending chromatography in 0.8 M acetic acid, 0.9 M LiCl(41) . Nucleotide standards were visualized by
illumination with a UV light, and radioactive products were visualized
by autoradiography.
Extracts were prepared from induced E. coli or from vaccinia virus-infected cells and chromatographed on the His-Bind resin as described under ``Experimental Procedures.'' In both cases, the eluate from the His-Bind resin contained two major protein bands of 55 and 30 kDa (Fig. 1, A and B, lanes 2 and 3). The predicted molecular mass of the A18R gene product is 56 kDa. The 55-kDa protein in the affinity-purified material was recognized by an antiserum specific for the carboxyl-terminal 10 amino acids of the A18R protein (Fig. 1C, lanes 2-4), demonstrating that this 55-kDa protein is the A18R gene product. Silver staining shows that although the vaccinia virus-expressed protein contains essentially no additional contaminating proteins (Fig. 1A, lane 2), the protein purified from E. coli contains two minor protein bands of 60 and 85 kDa (Fig. 1A, lane 3). The Coomassie-stained gel shows that compared with protein expressed in E. coli, the protein expressed from the vaccinia virus recombinant contains a lower quantity of the 30-kDa protein relative to the 55-kDa protein (Fig. 1B, lanes 2 and 3). Both preparations of affinity-purified protein, from E. coli or vaccinia virus-infected cells, contained DNA-dependent ATPase activity with virtually identical characteristics (data not shown).
To distinguish which of the two major proteins contained ATPase activity, the proteins were separated by centrifugation on 15-35% glycerol gradients (Fig. 2). Fractions from the gradient were analyzed by polyacrylamide gel electrophoresis (Fig. 2B) and by ATPase assay (Fig. 2A). Almost complete separation of the 55- and 30-kDa protein was achieved (Fig. 2B). The peak of ATPase activity cosedimented with the peak of 55-kDa protein, and fractions containing essentially pure 30-kDa protein contained no ATPase activity (Fig. 2A). The glycerol gradient-purified 55-kDa protein (Fig. 1, A and B, lane 4) possessed DNA-dependent ATPase activity virtually identical to the eluate from the His-Bind column (Fig. 1, A and B, lanes 2 and 3, and data not shown) and was recognized by the A18R antibody (Fig. 1C, lane 4). We conclude that the 55-kDa protein is the vaccinia virus A18R gene product and that it has DNA-dependent ATPase activity.
For the reasons described below, we believe that the 30-kDa polypeptide is the polyhistidine-tagged amino-terminal 53% of His-A18R, produced by premature termination of transcription by T7 RNA polymerase. The presence of the 30-kDa protein in both the vaccinia and E. coli preparations of affinity-purified A18R suggests that this protein is encoded by the A18R gene and that it contains a polyhistidine tag. This protein would not be recognized by the A18R antiserum since the antiserum is specific for the carboxyl terminus of the A18R protein. The A18R mRNA contains an AT-rich stem-loop structure near the middle of the coding sequence which strongly resembles a T7 RNA polymerase transcription terminator (42, 43, 44) . Termination of transcription at this stem-loop structure would produce an mRNA that, when translated, would yield a polypeptide of 30 kDa. Since the 30-kDa protein is inert in ATPase reactions (Fig. 2), the remaining characterization of the ATPase activity of His-A18R was done with material eluted from the His-Bind column, without further purification. Since the material produced from the vaccinia recombinant had higher purity and a lower amount of the 30-kDa protein relative to protein purified from E. coli (Fig. 1), the characterization was done with protein purified from the vaccinia recombinant.
Figure 3:
Analysis of His-A18R ATPase reaction
products. ATPase reactions were done under standard conditions except
that [-
P]ATP was included at a
concentration of 60 µCi/ml, and the ATP and MgCl
concentrations were lowered to 0.5 mM each to maximize
the percent conversion of ATP to ADP. At various times after addition
of enzyme, samples were removed from reactions, quenched by addition of
EDTA, and 1 µl was analyzed by ascending chromatography on
polyethyleneimine-cellulose plates as described under
``Experimental Procedures.'' An autoradiogram of the
polyethyleneimine plate is shown. Arrows at the left of the autoradiogram indicate the origin (Ori) and the
migration of ATP, ADP, and AMP standards. The three lanes on
the left are from a reaction that contained His-A18R, and the three lanes on the right are from a reaction that did
not contain His-A18R, as indicated at the bottom (+ and
-). Time of incubation is also indicated at the bottom of the figure.
ATPase reaction optima for
several salts were determined. The enzyme was maximally stimulated by
KCl, NaCl showed only slight stimulation, and NHCl was
inhibitory (Fig. 4A). Stimulation by KCl exhibited a
broad optimum between 50 and 150 mM. Based on these results,
we chose to use 100 mM KCl in ATPase reactions.
The
dependence of ATPase activity on magnesium was determined. Magnesium
titrations were done at several different ATP concentrations in an
attempt to distinguish whether any requirement for magnesium
represented utilization of the Mg ion as an enzyme
cofactor or whether magnesium was required simply to complex ATP. The
results show that the optimum magnesium concentration was always
equivalent to the ATP concentration in a given reaction (Fig. 4B). When reactions were done with
ATP:Mg
ratios of greater than 1, the activity
observed correlated with the magnesium concentration. The simplest
interpretation of these results is that the primary function of
magnesium in these reactions is to complex ATP.
The effect of DNA cofactor concentration on ATPase activity was determined. The His-A18R ATPase activity showed a strict dependence on the presence of a DNA cofactor (Fig. 4C). Although both single-stranded DNA (ssDNA) and dsDNA stimulate ATPase activity, the His-A18R ATPase exhibits a slight preference for ssDNA. Based on these results, we have chosen to use ssDNA at a concentration of 10 µg/ml as an optimum in ATPase reactions.
The effect of substrate concentration on ATPase
activity was determined. Since the ATPase reaction is optimal only at
Mg concentrations which are equal to the ATP
concentration (Fig. 4B), ATP titrations were done by
adding to reactions a mixture of ATP and MgCl
in a 1:1
molar ratio. The ATPase reaction velocity reached a plateau at
approximately 4 mM ATP (Fig. 4D). A
double-reciprocal plot (1/Vversus 1/[S])
of this data yields a K
for the His-A18R ATPase of
2.4 mM and a V
of 37.5 nmol ATP
hydrolyzed/15 min (data not shown).
A specific activity for His-A18R was determined by measuring the time dependence of ATP hydrolysis at varying enzyme concentrations. The reaction displays linear kinetics for at least 20 min at enzyme concentrations of up to 1.35 µg/ml (Fig. 5A). When the 15-min time points from this experiment are compared, the reaction shows a linear dependence on enzyme concentration up to 1.35 µg/ml of enzyme (Fig. 5B). Using these data, we calculate a specific activity of 18,000 nmol of ATP hydrolyzed/min/µg of enzyme, or approximately 1,000 molecules of ATP hydrolyzed/min/molecule of His-A18R.These values are a minimum estimate since the His-A18R protein used in these reactions is contaminated with the 30-kDa polypeptide (Fig. 1).
An apparent inconsistency in these results is the
failure of some homopolymeric ssDNAs to stimulate ATPase activity. It
seems likely that the homopolymeric ssDNAs assume unusual and
irrelevant structures that are not found in naturally occurring ssDNAs
and therefore do not behave as genuine ssDNAs in this assay. It is
noteworthy in this regard that a wide variety of heteropolymeric ssDNAs
stimulate ATPase activity and also that homopolymeric DNAs that are
inactive as single-stranded molecules are active as dsDNAs. We conclude
from these data that the His-A18R ATPase is stimulated by ssDNA as
short as 17 nucleotides, and by dsDNA or RNADNA hybrids but not
by ssRNA or dsRNA.
Investigation of the biochemical activity of the vaccinia
virus A18R gene product was inspired by computer analysis of the A18R
amino acid sequence, which revealed motifs in A18R which are found in
the DEXH subfamily of the DNA and RNA helicase superfamily
II(22, 23) . In this study we have shown that the
vaccinia virus A18R gene product has ATPase activity that is stimulated
by either ssDNA, dsDNA, or an RNADNA duplex but not by ssRNA or
dsRNA. These results strongly suggest that A18R is not an RNA helicase,
but may be a DNA helicase. In fact, preliminary results indicate that
the A18R protein has ATP-dependent DNA helicase activity. (
)
Based on the homology between A18R and the DEXH family of helicases, in particular ERCC3 and RAD25, four hypotheses have been proposed to explain the phenotype of A18R temperature-sensitive mutants of vaccinia virus (see Introduction). (i) A18R could be an RNA helicase that unwinds the dsRNA that accumulates during a normal infection, thereby preventing activation of the 2-5A pathway(23) . (ii) A18R could play a direct role in viral transcription, perhaps as a DNA helicase that provides an unwinding function associated with transcription. (iii) A18R could be a DNA repair protein, and DNA damage that accumulates in the absence of A18R function could indirectly result in effects on transcription. (iv) A18R could function in both transcription and DNA repair. The finding that the A18R protein is not an RNA-dependent ATPase and thus cannot be an RNA helicase disproves the first hypothesis. The finding that A18R is a DNA-dependent ATPase (and probably a helicase) is consistent with a role in transcription, DNA repair, or both. Although no gross defects in DNA synthesis have been noted during A18R mutant infections, no information is available concerning the effects of A18R mutations on DNA repair in vivo or in vitro, and therefore this topic merits further investigation. The experiments reported here provide new perspective on the hypothesis that A18R plays a direct role in viral transcription.
If A18R is directly involved in viral transcription,
then one might expect that purification of virus-specific transcription
factors would identify A18R and that any step in virus transcription
which required A18R would be inhibited by ATP analogs containing a
nonhydrolyzable -
-phosphate bond. Virtually all of the viral
factors required for transcription of both early and intermediate
promoters in vitro have been identified (see Refs. 3,
5-11, and references therein), and A18R is not among them. Both
early and intermediate gene transcription require ATP hydrolysis, but
this requirement can be accounted for by the ATPase activity of
transcription factors other than
A18R(45, 46, 47) . Not all of the viral
factors required for late transcription in vitro have yet been
identified; however, transcription of late vaccinia promoters in
vitro does not require ATP hydrolysis(48) . Therefore most
of the available evidence argues against a requirement for A18R in the in vitro vaccinia virus transcription systems that have been
described to date.
It is possible that A18R is directly involved in virus transcription in vivo but that the design of the in vitro transcription systems currently in use circumvents or masks the requirement for A18R. Three lines of reasoning support this hypothesis. First, the mammalian basal transcription factor TFIIH, which contains ERCC3, the closest cellular homolog to A18R, is required for in vitro transcription only when linear templates are used and not when supercoiled templates are used(49, 50) . TFIIH seems to act at some point between open complex formation and elongation in a process called ``promoter clearance''(50) . Interestingly, most if not all purifications of vaccinia transcription factors have utilized supercoiled DNAs as templates for transcription. Although one report indicates that supercoiled and linear templates are used with equal efficiency in a late vaccinia virus transcription reaction in vitro(7) , another report notes that linear templates are 3-5-fold less efficient than supercoiled templates in stimulating late viral transcription in vitro(48) . Poor utilization of linear templates may reflect a deficiency of specific transcription factors in in vitro extracts. Second, although A18R is not contained in purified virion extracts specific for early viral gene transcription, transcription is nevertheless defective when directed by permeabilized but otherwise intact virions containing mutant A18R protein, indicating that A18R does indeed play a role in early viral transcription(17) . Interestingly, the procedure normally used to solubilize transcription enzymes from vaccinia virions does not solubilize A18R(17) , and therefore participation of virion-derived A18R in early transcription could have been overlooked during purification of virion extracts. Third, it is noteworthy that the effects of A18R mutations on late viral transcription in vivo are mostly qualitative rather than quantitative; A18R mutations do not abolish late viral transcription in vivo but rather promote transcription of regions of the viral genome which are normally not transcribed late during infection (19) . Thus, demonstration of a role for A18R in viral transcription may require some redesign of an in vitro transcription system such that qualitative effects of A18R can be detected. Experiments to test these hypotheses are in progress.
Additional indirect evidence implies that the A18R protein may be
part of a larger complex of proteins involved in transcription. The
phenotype of A18R mutant infections is identical to the
phenotype observed when wild type vaccinia infections are treated with
the poxviral inhibitor, isatin--thiosemicarbazone
(IBT)(13, 19, 34) , which suggests that IBT
inhibits A18R activity. Mutants dependent on or resistant to IBT have
been mapped to G2R (14), a gene of unknown function, and A24R (51), the second largest subunit of the viral RNA
polymerase. A likely explanation for these observations is that A18R
interacts with RNA polymerase and that mutation of the RNA polymerase
can compensate for the effects of IBT on A18R.
We have shown that the product of the vaccinia gene A18R, a putative helicase that affects viral transcription and which shows homology to the transcription/repair proteins ERCC3 and RAD25, has DNA-dependent ATPase activity. The vaccinia system thus serves as a model for the role of this family of proteins in DNA repair and transcription.