Mechanism of Inhibition of Vaccinia Virus DNA Polymerase by Cidofovir Diphosphate

Cidofovir (CDV) is a broad-spectrum antiviral agent that hasbeen approved for clinical use in the treatment of cytomegalovirusretinitis. It has also been used off label to treat a varietyof other viral infections, including those caused by orf andmolluscum contagiosum poxviruses. Because it is a dCMP analog,CDV is thought to act by inhibiting viral DNA polymerases. However,the details of the inhibitory mechanism are not well establishedand nothing is known about the mechanism by which the drug inhibitspoxvirus DNA polymerases. To address this concern, we have studiedthe effect of the active intracellular metabolite of CDV, CDVdiphosphate (CDVpp), on reactions catalyzed by vaccinia virusDNA polymerase. Using different primer-template pairs and purifiedvaccinia virus polymerase, we observed that CDV is incorporatedinto the growing DNA strand opposite template G's but the enzymeexhibits a lower catalytic efficiency compared with dCTP. CDV-terminatedprimers are also good substrates for the next deoxynucleosidemonophosphate addition step, but these CDV + 1 reaction productsare poor substrates for further rounds of synthesis. We alsonoted that although CDV can be excised from the primer 3' terminusby the 3'-to-5' proofreading exonuclease activity of vacciniavirus polymerase, DNAs bearing CDV as the penultimate 3' residueare completely resistant to exonuclease attack. These resultsshow that vaccinia virus DNA polymerase can use CDVpp as a dCTPanalog, albeit one that slows the rate of primer extension.By inhibiting the activity of the proofreading exonuclease,the misincorporation of CDV could also promote error-prone DNAsynthesis during poxvirus replication.

INTRODUCTION

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Introduction

Poxviruses are large, double-stranded DNA viruses that replicatein the cytoplasm of infected cells. Members of this virus familycan cause severe infections, including human smallpox. Smallpoxwas declared eradicated in 1980, but concerns over bioterrorism(12, 27) and a recent outbreak of monkeypox in the midwesternUnited States (19) illustrate some need for the continued developmentof effective new treatment regimens. A number of new treatmentsare currently the subject of active investigation, includingimmunotherapy. However, antiviral drugs offer a combinationof chemical stability and simplicity of delivery that is especiallyattractive from a public health perspective.

One class of drugs that have been shown to inhibit poxvirusreplication are the nucleoside phosphonate analogs of cellulardeoxyribonucleotides that were developed by De Clercq et al.(9). These drugs have been shown to be effective against a widerange of DNA viruses and retroviruses (reviewed in reference8), and one of these compounds, (S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl)]cytosine,also known as cidofovir (CDV), has been granted Food and DrugAdministration approval for the treatment of cytomegalovirus(CMV)-induced retinitis. CDV has been used off label in thetreatment of orf (11) and molluscum contagiosum (21) virus infections.It has also been shown to block the replication of variola andmonkeypox viruses in culture (1) and to protect mice from alethal challenge dose of ectromelia, vaccinia, or cowpox virus(2, 3).

Unfortunately, CDV causes significant problems of nephrotoxicityand can only be administered by intraperitoneal or intravenousinjection. These problems have been addressed by the Hostetlergroup, who have shown that alkoxyglycerol or alkoxypropanediolesters of CDV and cyclic CDV are 50 to 230 times more activethan CDV against vaccinia virus in vitro (17) while also beingorally bioavailable (5). Further studies showed that this familyof CDV analogs also blocked the replication of variola, monkeypox,cowpox, and ectromelia viruses in vitro (3, 15, 17) and whendelivered orally protect mice from a lethal challenge with vaccinia,cowpox, or ectromelia virus (3, 22).

CDV is taken up into cells by fluid phase endocytosis (7), whereasits alkoxy derivatives are more rapidly absorbed through directassociation with the lipid bilayer (5). Regardless of the uptakeroute, the alkoxyester link (if present) is then hydrolyzedto yield CDV and the CDV is phosphorylated in a two-step processto yield the active intracellular metabolite CDV diphosphate(CDVpp). The phosphorylation of CDV is carried out by cellularenzymes and in the absence of viral infection (6). This propertyof CDV results in drug efficacy even against kinase-deficientor mutant viruses. Human DNA polymerases ${alpha}$ , ß, and ${gamma}$ also exhibit some natural resistance to CDVpp, thus partiallyexplaining the antiviral specificity of the drug (4, 14).

Previous studies have examined the effects of CDVpp on the activityof the CMV DNA polymerase (29, 30). This is a member of theB family of DNA polymerases, and it possesses both 5'-to-3'polymerase and 3'-to-5' exonuclease activities encoded withina single polypeptide. These studies have shown that CDVpp doesbehave as a dCTP analog, which CMV DNA polymerase can incorporateinto a growing polynucleotide chain opposite template G's (29,30). Under these circumstances, a single molecule of incorporatedCDV somewhat retarded further DNA synthesis while adding twoconsecutive molecules of CDV opposite a pair of template G'scaused a severe reduction or termination of elongation. In addition,a primer bearing a molecule of CDV at the 3' terminus was refractoryto the CMV enzyme's 3'-to-5' exonuclease activity (29). Theseobservations led Xiong et al. to suggest that a combinationof these two enzymatic effects was responsible for the efficacyof CDV in CMV-infected cells (29).

These observations also suggest a mechanism by which CDV couldinhibit orthopoxvirus DNA replication since poxviruses similarlyencode B family DNA polymerases. The best characterized of thepoxvirus polymerases is the 116-kDa E9L gene product encodedby vaccinia virus (10, 20, 25). This enzyme can be producedin a recombinant form (20) and serves as an excellent surrogatemodel for its variola virus homolog since the two enzymes differby only 18 to 20 amino acids over a polypeptide spanning 1,006amino acids. Vaccinia virus DNA polymerase normally forms partof a larger protein complex (16, 18), and its processivity ismodified by the A20R virus gene product (18), but it is neverthelesscatalytically active in isolation and this permits the characterizationof its substrate specificity. In this study, we have used steady-stateexperimental methods and this purified form of vaccinia virusDNA polymerase to show that the effects of CDV on vaccinia virusDNA polymerase in vitro differ in important ways from the effectsof the drug on betaherpesvirus DNA polymerases. These effectsprovide formal biochemical support for the contention that poxvirusDNA polymerases are an enzymatic target of these drugs. Thesestudies also provide a starting point for the characterizationof viruses exhibiting acquired resistance to CDV (24).

MATERIALS AND METHODS

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Materials and Methods

Chemicals. CDVpp was synthesized by Trilink Biotechnologies, San Diego,CA. Radioactive nucleotides were purchased from Amersham Biosciencesand oligonucleotides from Sigma-Genosys.

Vaccinia virus DNA polymerase. Recombinant vaccinia virus DNA polymerase was purified fromvaccinia virus-infected cells according to the protocol of McDonaldand Traktman (20) and appeared homogeneous as judged by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) analysisand silver staining (data not shown). The enzyme was storedat –20°C in glycerol at a concentration of 230 ng/µl.For use in enzyme assays, the enzyme was freshly diluted in25 mM potassium phosphate (pH 7.4)-5 mM ß-mercaptoethanol-1mM EDTA-10% (vol/vol) glycerol-0.1 mg/ml bovine serum albumin(New England Biolabs).

DNA polymerase and exonuclease assays. Different combinations of oligonucleotide primers and templateswere used to create different enzymatic substrates (Fig. 1).The primer strands were first end labeled using T4 polynucleotidekinase (Fermentas) and [ ${gamma}$ -³²P]ATP, and the unincorporated nucleotideswere removed using MicroSpin G-25 columns (Amersham Biosciences).Each 10-µl reaction mixture contained 1 pmol of end-labeledprimer (approximately 90,000 cpm) and 3 pmol of template strandin a solution containing 30 mM Tris · HCl (pH 7.9), 5mM MgCl₂, 70 mM NaCl, 1.8 mM dithiothreitol, 80 µg/mlbovine serum albumin, and 0 to 25 ng of purified vaccinia virusDNA polymerase. Different concentrations of CDVpp and deoxynucleosidetriphosphates (dNTPs) were added as indicated. The primer andtemplate strands were mixed, heated to 55°C, and allowedto cool slowly to room temperature prior to adding the remainingreagents. The reaction mixtures were incubated at 37°C andstopped by adding 5 µl of gel loading buffer (80% formamide,10 mM EDTA [pH 8.0], 1 mg/ml xylene cyanol FF, 1 mg/ml bromophenolblue). The reaction products were separated using a 10% denaturingpolyacrylamide sequencing gel run at 45 W for 45 min in half-strengthTris-borate-EDTA (23). The gels were fixed in a solution containing10% methanol plus 10% acetic acid and dried under vacuum. Thedried gels were exposed to phosphorimager screens (AmershamBiosciences) and the images analyzed using a Storm 840 or Typhoon8600 phosphorimager (Amersham Biosciences) with ImageQuant software(v. 5.1).

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FIG. 1. Oligonucleotide primer-template pairs used in this study. The P1 primer and templates T1, T2, and T3 were originally described by Xiong et al. (29). Each primer strand was 5' end labeled with ³²P prior to its annealing with a template strand. The melting point under the salt conditions used in this study (70 mM NaCl, 5 mM MgCl₂) is estimated to exceed 70°C. nt, nucleotides.

The large fragment of Escherichia coli DNA polymerase (Fermentas)and dideoxy sequencing reactions were used to generate sizestandards. The relative concentrations of each dideoxynucleosidetriphosphate-dNTP pair were adjusted as needed to generate asuitable distribution of marker fragments.

Determination of K_m and V_max values for CDVpp. Standard 10-µl reaction mixtures were prepared as describedabove containing $~$ 8 pmol of ³²P-labeled primer P1, 32 pmol oftemplate T3, 25 ng of vaccinia virus DNA polymerase, 10 µMdGTP, and various concentrations of dCTP or CDVpp. After incubationfor 0 to 16 min at 37°C, the reactions were stopped andthe amount of primer molecules extended by 1 nucleotide wasdetermined by PAGE and phosphorimager analysis. Initial primerextension rates were calculated for each CDVpp or dCTP concentrationand K_m and V_max determined from a nonlinear regression fit ofthese data to the Michaelis-Menten equation. Prism software(v. 4.0b for Macintosh) was used to perform all the curve fitsfrom which were obtained the relevant kinetic parameters. Theresults are presented as the mean ± the standard errorof the mean.

RESULTS

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Results

Adding CDVpp to DNA polymerase reaction mixtures promotes chain termination. As a first step in determining the effect of CDVpp on vacciniavirus DNA polymerase, we performed a simple analysis of therate of primer extension in the absence of any drug. These assaymixtures contained 10 µM each dATP, dTTP, and dGTP and5 µM dCTP. The dNTP concentrations are similar to thoseestimated to exist in vivo in vaccinia virus-infected cellsand vary from 2- to 10-fold greater than the calculated K_msfor each of the four nucleotides (13, 20). Figure 2 shows theresults of one such assay. Vaccinia virus DNA polymerase hasbeen estimated to incorporate dNTPs at a rate in excess of 30s^–1, and under our steady-state conditions, the majorityof the primers were extended out to near the end of the templatestrand in well under a minute (Fig. 2, top panel). We notedthat the enzyme could never fully extend the primer strand outto the terminus of the duplex and created instead an array ofproducts terminated 1 to 3 nucleotides from the end of the templatestrand, as judged by a comparison with the four sequencing lanes(Fig. 2). Small amounts of other partially extended strandswere also sometimes detected at early times in the reaction.None of these effects were artifacts of the templates used inthese studies and appear to be characteristic features of thenonprocessive form of vaccinia virus DNA polymerase. For example,a different polymerase (Klenow enzyme) extended essentiallyall of the primer strands out to the end of the template inreaction mixtures containing ddTTP (Fig. 2). (Note that theP1-T3 primer-template pair which was used in this reaction mixturecontained no template dA's that could direct chain terminationin the presence of ddTTP [Fig. 1].)

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FIG. 2. CDVpp promotes chain termination in reaction mixtures containing a mixture of CDVpp and dCTP. Both reaction mixtures contained ³²P-labeled primer P1, template T3, and all four dNTPs (10 µM each dATP, dGTP, and dTTP plus 5 µM dCTP). The reactionmixture analyzed in the lower panel was further supplemented with 10 µM CDVpp. The reactions were started by adding vaccinia virus DNA polymerase, and the mixtures were incubated at 37°C. Sampling was conducted over a time scale ranging from 0 to 15 min, and each reaction was stopped by adding gel loading buffer on ice. The products were subjected to PAGE analysis, and ³²P-labeled molecules were detected by phosphorimaging. Note the accumulation of premature termination products in the lower panel. Comparison with dideoxy sequencing ladders (at right) shows that these reaction products are terminated 1 nucleotide past the point where CDV would be expected to be incorporated.

In contrast, adding 10 µM CDVpp to these reaction mixturescaused the accumulation of a significant fraction of products(28% at 1 min) terminated 2 nucleotides beyond the end of theprimer strand (Fig. 2, bottom panel, lanes 3 to 7). When thesize of these products is compared with the sequencing ladder,it is apparent that these CDV-induced stops are located at positionN + 1, where N is the site where a molecule of CDV is expectedto be incorporated opposite a template dG (Fig. 2). In contrast,strands that have avoided suffering this fate seem to have beenextended out to the end of the template. It is important tonote that this process does not irreversibly block DNA synthesis,because prematurely terminated molecules were slowly chasedinto full-length extension products over the 15-min time courseof these experiments.

All of these studies were conducted using primer P1 annealedto template T3. To test whether these effects might be an idiosyncraticfeature of this particular template-primer pair, we repeatedthe experiment using two additional oligonucleotide duplexes.Adding CDVpp to reaction mixtures containing a P1-T1 primer-templatecombination inhibited further chain extension after the primerhad been extended by either 2 or 4 nucleotides (data not shown).Similarly, some termination was seen after primer P1 had beenextended either 2 or 3 nucleotides when annealed to templateT2 (data not shown). In all three situations, these terminationsites were located at positions N + 1 relative to the site wherea template dGMP would direct the incorporation of CDV.

CDV can be incorporated into DNA. These preliminary studies suggested that CDVpp promotes thetermination of DNA synthesis but are complicated by the mixtureof dCTP, CDVpp, and other dNTPs used in the reaction mixtures.We therefore performed a series of assays that examined eachaddition step in these primer extension reaction mixtures. Totest the suitability of CDVpp as a substrate for vaccinia virusDNA polymerase, we investigated the kinetics of incorporationof CDVpp versus dCTP using a combination of primer P1 and templateT9. The reaction was supplied with only dCTP or CDVpp, so weexpected the primer to be extended at most only 1 nucleotidebeyond its 3' end. The results of this experiment are shownin Fig. 3. Under these reaction conditions, we could not detectany great differences between CDVpp and dCTP as substrates forvaccinia virus DNA polymerase, with some conversion of the P1primer into a primer + 1 product occurring in both reactionmixtures even on ice. By the first time point (1 min), the primerwas quantitatively converted into the primer + 1 product inboth reaction mixtures (Fig. 3, lanes 3). We also noted thatby the 5-min time point molecules terminated with CDV or dCMPwere broken down again into much smaller labeled reaction products(Fig. 3, lanes 5 to 7). Presumably, the 5'-to-3' DNA polymeraseand 3'-to-5' proofreading exonuclease activities can consumeall of the dNTPs in these reaction mixtures and, once this hashappened, the proofreading exonuclease can then degrade thesubstrate down to a limit digest composed of hexanucleotidesand other smaller oligonucleotides. CDVpp is a substrate forthe polymerase activity, and CDV-terminated DNAs can be attackedby the proofreading exonuclease.

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FIG. 3. Kinetics of dCTP versus CDVpp incorporation. Primer P1 plus template T9 was incubated with vaccinia virus DNA polymerase and either 10 µM dCTP (left panel) or 10 µM CDVpp (right panel). The reaction mixtures were sampled at 0, 1, 2, 5, 10, and 15 min; the reactions were stopped; and then the mixtures were subjected to PAGE analysis. Both compounds are rapidly incorporated into DNA during the initial stages of the reaction; at later time points, the primers are degraded into molecules as small as hexanucleotides.

CDV-terminated primers are substrates for the vaccinia virus DNA polymerase. The pause sites detected in our primer extension assays containingCDVpp are located at positions N + 1 relative to the site ofincorporation of CDV. This suggests that vaccinia virus DNApolymerase might encounter some difficulties utilizing CDV-terminatedprimers. To test this hypothesis, we first added dCMP or CDVresidues to primer-template pair P1-T9 as described in Fig.3. After sampling the reaction mixture, we added dATP and comparedthe subsequent rate of chain extension. The results are shownin Fig. 4. We noted that a dCMP-terminated primer was a goodsubstrate for adding dAMP (Fig. 4, top, lanes 3 to 8), althougha dynamic interplay between 3'-to-5' proofreading exonucleaseand polymerase activities was apparent and the enzyme eventuallydegraded the reaction products down to oligonucleotides overthe course of the reaction. A CDV-terminated primer also directedthe addition of dAMP with extension kinetics indistinguishable(using these methods) from a dCMP-terminated primer (Fig. 4,bottom panel). However, once extended by the addition of CDVplus dAMP, this reaction product was stable over the 15-mincourse of the experiment.

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FIG. 4. Kinetics of dAMP addition to 3'-CDV- or 3'-dCMP-terminated primer strands. Duplex P1-T9 was incubated with vaccinia virus DNA polymerase and either 10 µM dCTP (top panel) or 10 µM CDVpp (bottom panel) for 1 min at 37°C. The concentration of dATP was then adjusted to 10 µM and further samples taken for gel analysis over another 15-min interval. The first samples (lanes 3) were taken on ice.

Primers bearing CDV as the 3' penultimate base are poor substrates for vaccinia virus DNA polymerase. We next examined what happens when vaccinia virus DNA polymeraseencounters a molecule bearing CDV as the penultimate 3' residue.To do this, we used a P1-T9 primer-template pair and a seriesof sequential assembly reaction mixtures to incorporate CDV(10 µM CDVpp), followed by residue N + 1 (10 µMdATP), into DNA. We then tested the substrate properties ofthis primed structure after adding the remaining three dNTPs(no dCTP). As a control, dCTP was substituted for CDVpp. Theresults of these experiments are shown in Fig. 5. As noted inprevious experiments (Fig. 4), vaccinia virus DNA polymeraseproduced a mixture of extension products terminated in dCMPor dCMP-dAMP (Fig. 5, top, lane 3). These two control reactionproducts were nearly instantaneously extended out to the endsof the template strand in the presence of dGTP, dATP, and dTTP(Fig. 5, top, lanes 4 to 9). Primers terminated in CDV-dAMPwere synthesized in high yield (Fig. 5, bottom, lane 3), butthe rate of extension of this product by vaccinia virus DNApolymerase clearly lagged well behind the rate of extensionof control primers. Based upon the calculated distribution oflabel in substrates and products (data not shown), we estimatedthat the rate of primer extension, measured as a first-orderrate constant, is at least threefold slower when CDV replacesdCMP as the 3' penultimate nucleotide (K = 3 min^–1 versus0.9 min^–1 for extension from dCMP- versus CDV-bearingprimers, respectively). However, there is a large standard errorin the estimate of the control rate (95% confidence intervalsfor K_dCMP = 0 to 24 min^–1) and thus this calculation willprobably underestimate the rate difference by at least a factorof 10.

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FIG. 5. Kinetics of chain extension using a 3'-CDV-dAMP-terminated primer. Substrates were prepared as described in the legend to Fig. 4, consisting of duplex P1-T9 that had been extended 2 nucleotides using dCTP-dATP (top) or CDVpp-dATP (bottom) as illustrated by Fig. 3 and 4. (Note that vaccinia virus DNA polymerase always generates a mixture of dCMP- and dCMP-dAMP-terminated products, whereas only a single CDV-dAMP product is formed.) A mixture of dATP, dGTP, and dTTP was then added and further samples taken for gel analysis over another 15-min interval. The first samples (lanes 4) were taken on ice.

We also looked to see if the enzyme could extend a primer incorporatingtwo CDV molecules. Previous experiments with CMV DNA polymeraseindicated that the presence of two consecutive CDV moleculesdramatically reduced or terminated DNA synthesis (29). To comparethe behavior of the two DNA polymerases, an assay was performedusing primer P1 and template T2. The primer-template pair wasfirst incubated with 10 µM CDVpp or dCTP (as a control),and then 10 µM dATP was added. Samples were taken foranalysis after a brief incubation period, following by the additionof dATP, dGTP, and dTTP (10 µM each) to the remainderof the mixture. This reaction mixture was then sampled overthe next 80 min. The results are shown in Fig. 6. Moleculesterminating with 5'-CC-OH 3' and 5'-CCA-OH 3' were again veryrapidly extended out to the ends of the template within lessthan a minute (Fig. 6, top panel, lanes 4 to 9). In contrast,the rate of elongation of a primer incorporating two CDV moleculesis drastically reduced, as judged by the very slow incorporationof the next (dAMP) residue and the subsequent extension of theprimer out to the end of the template strand. However, it isalso apparent that two CDV molecules do not completely blockDNA synthesis catalyzed by vaccinia virus DNA polymerase. Wesaw the accumulation of primers terminated with 5'-XXA-OH 3'and 5'-XXAG-OH 3' (where X = CDV), but molecules extended immediatelybeyond this point were not seen and appeared to be chased intofull-length products over the 80-min incubation time.

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FIG. 6. The presence of two consecutive template dG's slows, but does not completely block, DNA synthesis. A series of reaction mixtures were used to incorporate dCMP-dAMP (top panel) or CDV-dAMP (bottom panel) onto the 3' end of primer P1. This was accomplished by incubating P1-T2 with DNA polymerase plus 10 µM dCTP (top) or 10 µM CDVpp (bottom) for 1 min at 37°. After sampling the two reaction mixtures, dATP was added to a final concentration of 10 µM in each reaction mixture and the incubation continued for another 1 min at 37°C. Further samples were taken, and then a dNTP mixture was added to both reaction mixtures to produce final concentrations of 10 µM each dATP, dGTP, and dTTP with no additional dCTP. These solutions were incubated at 37°C with sampling at 1, 5, 10, 20, 40, and 80 min. A mixture of primers terminating in 5'-CC-OH 3' or 5'-CCA-OH 3' (top panel, lane 3) are fully extended to the end of the template strand in less than a minute by vaccinia virus DNA polymerase (lanes 4 to 9). However, the enzyme only reluctantly synthesizes molecules encoding two CDV resides (bottom panel, lanes 2 and 3) and takes much longer to assemble a primer terminating in 5'-XXA-OH 3' (where X = CDV; lanes 4 to 9). These molecules do eventually chase into various amounts of full-length products over an 80-min period (lanes 7 to 9).

A single CDV molecule is a substrate for vaccinia virus DNA polymerase 3'-to-5' exonuclease. Vaccinia virus DNA polymerase encodes a 3'-to-5' proofreadingexonuclease activity, and we also tested whether molecules terminatedwith CDV were substrates for this activity. To address thisquestion, we first added a single dCMP or CDV residues to primer-templatepair P1-T9 as described in Fig. 3. We purified these reactionproducts free of any unincorporated dCTP or CDVpp and then incubatedthe two extension products with fresh enzyme in the absenceof any additional dNTPs or CDVpp. The results of this experimentare shown in Fig. 7. Molecules terminated with CDV (Fig. 7,bottom panel) were degraded as quickly as were dCMP-terminatedprimers (Fig. 7, top panel). This shows that a single CDV moleculeis a substrate for the 3'-to-5' proofreading exonuclease activityif it is located at the 3' end of the primer strand.

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FIG. 7. Excision of CDV from a 3'-CDV-terminated strand. Duplex P1-T9 was extended 1 nucleotide using either dCTP (top) or CDVpp (bottom) and then purified free of reaction materials by gel filtration. The purified product was then incubated with vaccinia virusDNA polymerase at 37°C, in the absence of dNTPs, with sampling at 1, 5, 10, 20, 40, and 80 min. Both types of extension products are substrates for the 3'-to-5' proofreading exonuclease.

The CDV + 1 product is not a substrate for the 3'-to-5' exonuclease. In contrast to primers bearing a 3'-terminal CDV molecule, theCDV + 1 reaction product was completely resistant to attackby the proofreading exonuclease. For this assay, primer P1 andtemplate T9 were again used. The primer-template pair was incubatedtogether with 10 µM each CDVpp (or dCTP) and dATP, andvaccinia virus DNA polymerase, to create primers terminatedwith either 5'-CA-OH 3' or 5'-XA-OH 3' residues. The unincorporateddCTP, CDVpp, and/or dATP were removed by gel filtration, andthen a new reaction mixture was prepared containing DNA polymerasebut lacking any dNTPs (as above). Figure 8 shows the resultsof this experiment. Molecules terminated with a 5'-CA-OH 3'sequence were rapidly attacked by the exonuclease, generatinga series of degradation products of which the smallest detectableby these methods were 6 to 7 nucleotides in length (Fig. 8,left panel, lanes 4 to 10). In contrast, the 5'-XA-OH 3'-terminatedprimer was stable over 80 min of incubation. A second molecule13 ± 1 nucleotides long was also resistant to this activity(Fig. 8, right panel). These likely correspond to primer moleculesthat during the initial extension step (with CDVpp and dATPbut without dGTP) were excised back to the next nearest 5'-CA-3'residue. If this sequence motif had been replaced with 5'-XA-OH3', it would create a 13-mer molecule (Fig. 1) which, like the20-mer, would also be resistant to nuclease attack. We concludedthat the CDV + 1 extension product is resistant to attack bythe vaccinia virus DNA polymerase 3'-to-5' proofreading exonuclease.

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FIG. 8. CDV inhibits the 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase. Primer-template pair P1-T9 was incubated with vaccinia virus DNA polymerase at 37°C for 1 min in the presence of 10 µM dCTP and 10 µM dATP (left panel) or 10 µM CDVpp and 10 µM dATP (right panel). The unincorporated nucleotides and CDVpp were removed by gel filtration. The purified product was then incubated with vaccinia virus DNA polymerase at 37°C, in the absence of dNTPs, with sampling at 1, 5, 10, 20, 40, and 80 min. Molecules terminated with 5'-C-OH 3' or 5'-CA-OH 3' are rapidly degraded (left), whereas molecules terminated with 5'-XA-OH 3' are completely resistant to exonucleolytic attack (right).

Determination of the K_m of CDVpp. These observations provided a method for estimating the K_m ofCDVpp. Reaction mixtures were prepared containing a P1-T3 primer-templatepair, 25 ng of vaccinia virus DNA polymerase, and 10 µMdGTP. dGTP was added to maximize the stability of the dGMP-terminatedprimer in the presence of various concentrations of CDVpp. Thekinetics of CDV incorporation were then determined in the presenceof 0.3 to 30 µM CDVpp, monitoring the amount of labeledprimer plus CDV extension product using polyacrylamide gel electrophoresisand phosphorimaging (Fig. 9). The various initial rates of CDVincorporation were then used to estimate that the K_m and V_maxfor CDVpp, under these reaction conditions, are 23 ±6 µM and 3.0 ± 0.4 pmol · min^–1, respectively.Each reaction mixture contained 25 ng (0.22 pmol) of DNA polymeraseand, assuming that the enzyme is fully active, the turnovervalue (k_cat) for CDVpp can be estimated as $~$ 0.2 s^–1. Thesame methods were used to determine K_m (3.8 ± 0.7 µM),V_max (2.4 ± 0.2 pmol · min^–1), and k_cat( $~$ 0.2 s^–1) for dCTP (data not shown).

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FIG. 9. Kinetics of CDVpp incorporation. The rate of CDV incorporation was determined using the primer-template pairs and electrophoretic methods shown in Fig. 3 and initial rates calculated from the data plotted as shown in the upper panel using nonlinear regression analysis and Prism 4.0 software (GraphPad). These data were replotted as shown in the bottom panel and the same nonlinear regression method used to calculate the Michaelis-Menten constants K_m (23 ± 6 µM) and V_max (3.0 ± 0.4 pmol · min^–1).

DISCUSSION

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Discussion

The effect of CDVpp on the activity of vaccinia virus DNA polymerasewas investigated using an in vitro primer extension assay. AddingCDVpp to a pool of ordinary dNTPs caused the DNA polymeraseto pause during chain extension at a point always 1 nucleotidepast the site where CDV would be incorporated opposite dG. Theseobservations provide a straightforward rationale for the antiviralactivity against orthopoxviruses that has been reported in vitroand in vivo. They also explain why CDV-resistant viruses encodemutations in the E9L gene (DNA polymerase), mutations that canbe shown to be responsible for the drug resistance phenotypeby marker rescue methods (D.H.E. et al. and K.Y.H. et al., unpublisheddata).

Vaccinia virus DNA polymerase can use CDVpp as a substrate andcan extend a CDV-terminated primer by 1 more nucleotide (Fig.3, 4, and 9). The differences between the substrate propertiesof dCTP and CDVpp are not readily apparent from the steady-statedata seen in Fig. 3, but a more detailed kinetic analysis clearlyshows that CDVpp is a less favored substrate than dCTP. Thisis illustrated by the fact that the catalytic efficiency isabout fivefold higher for dCTP as a substrate versus CDVpp,with the difference primarily accounted for by differences inthe K_m. The effects of CDV on the relative rate of additionof the next templated nucleotide (dATP) is more difficult tojudge by these methods given the effects of the product on theproofreading exonuclease activity (see below) and the consequentialbiased accumulation of the CDV + 1 product. Nevertheless, thepresence of a CDV molecule linked to the 3' terminus of theprimer strand still permitted the addition of the next nucleotidewith kinetics superficially comparable to dATP addition to adCMP-terminated primer (Fig. 4). CDV is thus not a chain-terminatingdrug in the classical sense, since it can be incorporated intoDNA and still prime chain extension after incorporation intothe nascent strand. From a structural perspective, CDVpp mustbe capable of occupying the nucleotide-binding site and servingas a substrate for the nucleophilic attack on the ${alpha}$ -phosphonate.After translocation of the newly incorporated CDV molecule tothe primer-terminus binding site, vaccinia virus polymerasemust also be able to reposition the hydroxyl group with sufficientaccuracy to permit further polymerization. The crystal structureof tenofovir complexed with human immunodeficiency virus reversetranscriptase illustrates the conformational flexibility ofnucleoside phosphonates (26), which presumably accounts forthis enzymatic behavior.

Although these substrate properties can partially explain theanti-poxvirus activity of CDVpp, more dramatic effects of thedrug are seen at the next step in the polymerization cycle.Molecules bearing a CDV residue at the penultimate 3' primerposition are only very slowly extended into full-length chains(Fig. 5). The rate-limiting factor appears to be the reactionsteps catalyzing the addition of the next nucleotide to theCDV + 1 product (Fig. 5). Although these molecules are poorsubstrates, they are still slowly converted into full-lengthproducts with a half-life of $~$ 0.8 min under our conditions (datanot shown). Generally, the same effect was seen when two moleculesof CDV were incorporated into the primer strand. However, thetransient accumulation of a CDV₂ + 2 product and the much-delayedappearance of full-length product (Fig. 6) suggested that thedeleterious effects of CDV are propagated further and with greatereffect under such circumstances. Base-paired deoxynucleosidemonophosphates are relatively inflexible molecules, and thisproperty, combined with structural distortions imposed by anucleoside phosphonate linkage, could result in misalignmentof the 3'-hydroxyl terminus with deleterious effects on thenext nucleotide addition step. Alternatively, the enzyme mighthave difficulty repositioning the CDV + 1 reaction product withinthe polymerase reaction site, which would also inhibit furtherchain extension.

The same effects are seen when one examines the susceptibilityof CDV-containing molecules to attack by the 3'-to-5' proofreadingexonuclease. CDV is excised from the 3' end of the primer ata rate comparable to dCMP (Fig. 7) and shows that vaccinia viruspolymerase has little difficulty hydrolyzing a phosphonate esterlinkage. This is not true of the CDV + 1 substrate, where the3'-terminal (–1) phosphodiester linkage is completelyresistant to hydrolysis (Fig. 8). These observations can berationalized using the same structural arguments outlined above.CDV-terminated molecules might exhibit sufficient conformationalflexibility to still serve as exonuclease substrates, whilethe constraints imposed by a phosphonate linkage at the –2bond may render the phosphodiester bond at –1 resistantto hydrolysis.

HCMV DNA polymerase exhibits some of the same enzymatic responsesto CDVpp (29, 30). In particular, the two different virus enzymesutilize CDVpp less efficiently that dCTP (30). SubstitutingCDVpp for dCTP also causes the transient accumulation of theCDV + 1 reaction product. However, poxvirus polymerases appearcapable of slowly copying templates encoding a dGpdG motif inthe presence of CDVpp (Fig. 6), while these residues completelyblock readthrough catalyzed by HCMV polymerase. A much morenotable difference is that HCMV polymerase cannot excise CDVfrom a CDV-terminated primer (29), whereas it is the CDV + 1reaction product that creates the greatest difficulties forvaccinia virus DNA polymerase. These differences suggest thatthe two enzymes interact with CDV and CDV-bearing DNAs in sometimesdifferent ways, and caution must be observed extrapolating fromthe molecular genetic properties of CDV-resistant herpesvirusesto poxvirus systems.

In conclusion, these data provide insights into the anti-poxvirusactivity of nucleoside phosphonate drugs. These compounds inhibitchain extension, and since DNA synthesis is a key regulatorof the virus life cycle, they are expected to compromise a diversearray of other processes, including intermediate and late genetranscription and virus assembly. The incorporation of CDV intoDNA also completely inhibits the associated 3'-to-5' exonuclease.The proofreading exonuclease serves a critical role in minimizingreplication errors and probably also catalyzes virus geneticrecombination (28, 31). Future studies will use CDV, and CDV-resistantviruses, as a tool for investigating these two critical viralsystems.

ACKNOWLEDGMENTS

Operating and equipment grants provided to D.H.E. by the AHFMR,NSERC, and CIHR funded this work. K.Y.H. was supported by NEIgrant EY11834, NIAID grant AI29164, and Department of the Armygrant DAMD17-01-2-007. The U.S. Army Medical Research AcquisitionActivity, Fort Detrick, MD, was the awarding acquisition office.W.C.M. is a recipient of a University of Alberta Ph.D. scholarship.

The content of this article does not necessarily reflect theposition or policy of the U.S. Government, and no official endorsementshould be inferred.

FOOTNOTES

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, The University of Alberta, Edmonton, AB T6G 2H7, Canada. Phone: (780) 492-2308. Fax: (780) 492-7521. E-mail: devans{at}ualberta.ca.

REFERENCES

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