©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Vaccinia Virus A18R Gene Product Is a DNA-dependent ATPase (*)

(Received for publication, September 15, 1994; and in revised form, November 7, 1994)

Christopher D. Bayliss (§) Richard C. Condit (¶)

From the Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 RNAbulletDNA hybrids, but not by either single-stranded or double-stranded RNA.


INTRODUCTION

Vaccinia virus, the prototype poxvirus, is a large double-stranded (ds) (^1)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.


EXPERIMENTAL PROCEDURES

Eukaryotic Cells, Viruses, Bacterial Hosts

BSC40 cells and wild type vaccinia stain WR were maintained as described previously (34, 35) . VVT7, obtained from B. Moss, is a vaccinia virus recombinant that constitutively expresses the bacteriophage T7 RNA polymerase under control of an early/late vaccinia virus promoter(36) . Escherichia coli DE3 (37) contains an isopropyl-1-thio-beta-D-inducible chromosomal copy of the bacteriophage T7 RNA polymerase gene.

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.

Nucleic Acids

Purified poliovirus virion RNA was a generous gift from Dr. Bert Flanegan. Purified reovirus RNA was a generous gift of Dr. Bill Joklik. Synthetic homopolymeric polynucleotides were purchased from Pharmacia Biotech Inc.. Oligonucleotides (68-mer through 17-mer) were chosen on the basis of size only from a collection of oligonucleotides synthesized by the University of Florida ICBR DNA synthesis core. Each contains all four bases. 6-mer is a random mixture of hexanucleotides.

Cloning and Mutagenesis

p16A18 contains the vaccinia virus A18R coding region cloned in frame downstream from an amino-terminal polyhistidine tag in the vector pET16b (Novagen). The A18R coding region was first cloned by polymerase chain reaction amplification from vaccinia virus WR DNA using primers that placed an XbaI site followed by an NdeI site at the 5` end of the gene and a BamHI site directly following the translation termination codon at the 3` end of the gene. The NdeI site at the 5` end of the gene includes the ATG translation initiation codon of A18R. The polymerase chain reaction product was first cloned as an XbaI-BamHI fragment into the plasmid vector pGEM3ZF(+) (Promega) to obtain the clone pALwt, and the amplified region was sequenced to ensure an absence of polymerase chain reaction errors. Because the A18R coding region contains an internal NdeI site, the A18R gene was subcloned from pALwt into NdeI and BamHI-digested pET16b using two subfragments, NdeI-SnaB1 and SnaB1-BamHI. Expression of the polyhistidine-A18R fusion protein in p16A18 is under the control of a T7 RNA polymerase promoter.

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.

Induction and Preparation of Extract from E. coli

An overnight culture of DE3 cells harboring the p16A18 plasmid was used to inoculate 2 liters of L-broth, containing 100 µg/ml ampicillin. The culture was incubated at 37 °C to an A of 0.6. Isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 1 mM, and the culture was incubated at 29 °C for 3 h. The cells were pelleted and resuspended in 80 ml of 1 times binding buffer (see below) plus proteinase inhibitors (leupeptin, phenylmethylsulfonyl fluoride, and aprotinin). The cells were lysed by two passages through a French press. Insoluble material, which contained approximately 90% of the induced A18R protein, was removed by centrifugation first for 20 min at 10,000 rpm in a Sorvall SS34 rotor at 4 °C and second for 1 h in a Beckman SW 28 at 20,000 rpm at 4 °C. For purification of the soluble A18R protein, the supernatant was then chromatographed on a His-Bind (Novagen) column as described below.

Induction and Preparation of Extract from Vaccinia-infected Cells

Ten confluent 150-mm dishes of BSC40 cells were infected at multiplicity of infection 10 each with VVT7 and the recombinant vaccinia VVTMHisA18. At 10-16 h postinfection the cells were scraped from the dishes and harvested by centrifugation. The cells were resuspended in 10 ml of 20 mM Tris-HCl, pH 8.0, 10 mM NaCl, and then 0.25 volumes of a solution containing 0.5% Triton X-100, 1.5 M NaCl, 1.0 mM phenylmethylsulfonyl fluoride, and 50% glycerol was added. (^2)The cells were vortexed and incubated on ice for 15 min. Nucleic acids in the extract were degraded by the addition of CaCl(2) (0.1 M stock solution) to a final concentration of 1.25 mM, and micrococcal nuclease (Worthington Biochemical, 15,000 units/mg, 1 mg/ml stock solution) to a final concentration of 1.2 µg/ml and incubation for 15 min at 30 °C. The nuclei were pelleted by centrifugation at 2,500 rpm for 10 min in a Sorvall RT6000 at 4 °C. The postnuclear supernatant was clarified by centrifugation at 25,000 rpm for 30 min in a Beckman SW 55-Ti at 4 °C. The supernatant was applied to a His-Bind column.

His-Bind Column

The extract from virus-infected BSC40 cells was chromatographed on a 1.0-ml column of His-Bind resin (Novagen), charged with Ni as described in the Novagen literature. All steps were done at 4 °C unless stated otherwise. Extract was loaded onto the column at a flow rate of 10 ml/h using a peristaltic pump. The column was washed sequentially at a flow rate of 10 ml/h with 10 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5% glycerol), 10 ml of wash buffer 1 (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5% glycerol), and 15 ml of wash buffer 2 (100 mM imidazole, 0.5 M NaCl, 20 mM Tris- HCl, pH 7.9, 5% glycerol). Bound proteins were eluted at a flow rate of 8 ml/h with 10 ml of strip buffer (100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 10% glycerol), collecting 0.5-ml fractions. Peak fractions were identified using the Bradford protein assay (Bio-Rad), pooled, dialyzed for 24 h against 4 changes, 500 ml each, of a solution containing 40 mM Tris-HCl, pH 8.0, 20 mM KCl, and 40% glycerol. The enzyme was stored at -20 °C, where it is stable for at least 6 months. A typical preparation yields 250-300 µg of protein at a concentration of 125-150 µg/ml.

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(2), 5% glycerol) and finally into buffer B (buffer A plus 1 mM dithiothreitol).

ATPase Assay

Unless otherwise stated, ATPase assays (50 µl) contained 40 mM Tris-HCl, pH 8.0, 100 mM KCl, 2 mM dithiothreitol, 4 mM MgCl(2), 4 mM ATP, 10 µg/ml single-stranded M13 DNA, and 1.35 µg/ml enzyme. Reactions were mixed on ice, initiated by the addition of enzyme, incubated at 37 °C for 15 min, and stopped by addition of 5 µl of 0.5 M EDTA. ATPase activity was monitored by assaying release of inorganic phosphate using either a radiochemical (see Fig. 2) or a colorimetric (see Fig. 4Fig. 5Fig. 6Fig. 7) assay. For the radiochemical assay, reactions contained 2 µCi of [-P]ATP, and release of P(i) was measured as described(39) . For the colorimetric assay, release of P(i) was measured as described(40) , except that Sterox was omitted from the assay mixture. One unit of ATPase activity is defined as hydrolysis of 1 nmol of ATP in 15 min.


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(2), 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(4)Cl as indicated. Panel B, the final concentrations of ATP and MgCl(2) 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(2) 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 [alpha-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.

Polyacrylamide Gel Electrophoresis and Western Blot Analysis

Proteins were electrophoresed on 10% polyacrylamide gels and transferred to nitrocellulose using standard techniques. Western blots were probed with a 1:200 dilution of an antisera raised against the carboxylterminal 10 amino acids of the A18R gene(17) , and bound antibody was detected using a goat anti-rabbit IgG conjugated to horseradish peroxidase and the Amersham Enhanced Chemiluminescence Western blotting kit.


RESULTS

Purification of Histidine-tagged A18R

To facilitate purification of the vaccinia virus A18R gene product, the entire A18R open reading frame was cloned in-frame downstream from a DNA sequence that encodes 10 histidine residues. Upon expression in either prokaryotic or eukaryotic cells, the resulting amino-terminal polyhistidine-tagged protein can be affinity purified by binding to Ni immobilized on a His-Bind resin (see ``Experimental Procedures''). Overexpression of the polyhistidine-tagged A18R protein (His-A18R) was tested using both a plasmid vector in E. coli and a vaccinia virus recombinant in virus-infected mammalian cells. Overexpression in E. coli was accomplished using the pET vector system, in which transcription of the His-A18R gene was controlled by a T7 RNA polymerase promoter on a plasmid vector harbored in an E. coli strain that contains an inducible copy of the T7 RNA polymerase gene(37) . Expression in mammalian cells was achieved by coinfecting cells with two vaccinia virus recombinants, one that constitutively expresses T7 RNA polymerase, and one that contains the His-A18R gene under transcriptional control of a T7 RNA polymerase promoter(36) .

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.

His-A18R ATPase Reaction Products and Optima

To determine the specificity of ATP hydrolysis by the His-A18R ATPase, reactions were done with [alpha-P]ATP, and the reaction products were analyzed by thin layer chromatography (Fig. 3). The sole nucleotide product observed was ADP. ADP formation increased with incubation time and was not observed in the absence of His-A18R protein. We conclude that His-A18R cleaves the beta--phosphodiester bond of ATP.


Figure 3: Analysis of His-A18R ATPase reaction products. ATPase reactions were done under standard conditions except that [alpha-P]ATP was included at a concentration of 60 µCi/ml, and the ATP and MgCl(2) 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 NH(4)Cl 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(2) 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(m) for the His-A18R ATPase of 2.4 mM and a V(max) 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).

Substrate Specificity of the His-A18R ATPase

To determine the substrate specificity of the His-A18R ATPase, hydrolysis of all four ribo- and deoxyribonucleotides was tested (Fig. 6). The enzyme exhibited a strong preference for adenosine nucleotides; ATP and dATP were hydrolyzed with equal efficiency. The enzyme displayed some activity on all other ribonucleoside triphosphates; purine nucleotides were preferred over pyrimidine nucleotides. The order of preference for ribonucleotides is ATP GTP > UTP > CTP. The remaining deoxynucleoside triphosphates, dCTP, dGTP, and dTTP, displayed only minimal activity but nevertheless maintained the same order of preference as the ribonucleotides.

Effects of Different Nucleic Acid Cofactors on His-A18R ATPase Activity

To gain additional insight into the nucleic acid cofactor requirement for ATPase activity, a variety of different natural and synthetic RNA and DNA molecules were tested for their effect on the His-A18R ATPase (Fig. 7). All purely RNA molecules, whether single or double-stranded, were inactive in stimulating ATPase activity. These molecules include a natural polyadenylated ssRNA (poliovirus virion RNA), a natural dsRNA (reovirus virion RNA), all four ribohomopolymers, and a synthetic dsRNA (poly(I)bulletpoly(C)). As described above (Fig. 4C), heteropolymeric DNAs which were either single-stranded or double-stranded stimulated ATPase activity. Assay of heteropolymeric oligonucleotides of varying sequence and size showed that ATPase activity could be stimulated by ssDNAs as short as 17 nucleotides, whereas shorter oligonucleotides (a 10-mer and a random mixture of hexanucleotides) were inactive as cofactors (Fig. 7). Of the four deoxyribohomopolymers, only poly(dI) stimulated ATPase activity. However, a synthetic DNAbulletDNA hybrid molecule (poly(dA)bulletpoly(dT)) and a synthetic RNAbulletDNA hybrid molecule (poly(rA)bulletpoly(dT)), both displayed near maximal stimulation of ATPase activity.

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 RNAbulletDNA hybrids but not by ssRNA or dsRNA.


DISCUSSION

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 RNAbulletDNA 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. (^3)

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 beta--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-beta-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI18094. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, U. K. OX1 3RE.

To whom correspondence should be addressed: Dept. of Immunology and Medical Microbiology, University of Florida, Box 100266, Gainesville, FL 32610. Tel.: 904-392-3128; Fax: 904-392-3133; condit{at}icbr.ifas.ufl.edu.

(^1)
The abbreviations used are: ds, double-stranded; ss, singlestranded; IBT, isatin-beta-thiosemicarbazone.

(^2)
S. Shuman, personal communication.

(^3)
D. A. Simpson, C. D. Bayliss, and R. C. Condit, unpublished results.


ACKNOWLEDGEMENTS

We thank Mark Challberg, Dick Moyer, Sue Moyer, Stewart Shuman, and David Simpson for helpful discussions while this work was in progress. We thank Jackie Lewis for technical assistance. We thank David Simpson for a careful reading of the manuscript.


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