©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Photoaffinity Labeling of Herpes Simplex Virus Type-1 Thymidine Kinase (*)

(Received for publication, November 18, 1994; and in revised form, January 25, 1995)

Tammy M. Rechtin (1) Margaret E. Black (2)(§) Feng Mao (1) Marcia L. Lewis (1) Richard R. Drake (1)(¶)

From the  (1)Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 and the (2)Department of Pathology, Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The molecular basis for the treatment of human herpesviruses with nucleoside drugs is the phosphorylation of these drugs by the viral-encoded thymidine kinases. In order to better understand the structural and enzymatic mechanisms by which herpesviral thymidine kinases recognize their substrates, photoaffinity labeling with [alpha-P]5-azido-2`-deoxyuridine-5`-monophosphate and [-P]8-azidoadenosine-5`-triphosphate was used to characterize the thymidine, thymidylate, and ATP active sites of the herpes simplex virus-1 (HSV-1) thymidine kinase. For this study, HSV-1 thymidine kinase and a site-specific mutant enzyme (C336Y, known to confer acyclovir resistance) were expressed in bacteria and purified by a rapid, two-step protocol. The specificity of photoaffinity labeling of these HSV-1 thymidine kinases was demonstrated by the ability of site-directed substrates such as thymidine, thymidylate, acyclovir, 5-bromovinyl-2`-deoxyuridine, and ATP to inhibit photoinsertion. Differences in inhibition patterns of photoaffinity labeling correlated with kinetic differences between the wild-type and C336Y HSV-1 thymidine kinases. Cumulative results suggest that the acyclovir-resistant cysteine 336 mutation primarily affects the ATP binding site; yet it also leads to alteration in the binding affinity of nucleoside drugs in the thymidine site. In this study, azidonucleotide photoaffinity analogs are shown to be effective tools for studying the active-site environment of HSV-1 thymidine kinase and related site-specific mutants.


INTRODUCTION

The herpes simplex virus thymidine kinases (HSV-TKs) (^1)are the pharmacological targets of most herpesvirus treatments, because these kinases catalyze the initial phosphorylation of many anti-herpesvirus nucleoside drugs, such as acyclovir (ACV) and 5-bromovinyldeoxyuridine (BVDU)(1, 2, 3) . This targeting is based primarily on the differences in substrate specificity compared to the cellular TKs. As a highly regulated enzyme of the pyrimidine salvage pathway, the cytosolic cellular TK specifically phosphorylates thymidine with ATP as the phosphoryl donor (4) . The HSV-1 TK has a much broader range of substrates which include most pyrimidine nucleosides, many guanosine derivatives (e.g. ACV), and most purine and pyrimidine nucleoside triphosphates(5) . HSV-TK also possesses a thymidylate kinase activity, and it has been suggested that the thymidine and TMP sites are shared or overlapping(6, 7, 8) . Due to significant amino acid sequence homology between the herpesviral TKs, six putative regions involved in active-site formation have been identified(9, 10) . In addition, drug-resistant and site-directed mutants have been helpful in identifying some regions involved in substrate binding(11, 12, 13, 14) . However, the structural basis for this broad substrate recognition is not known. Because clinical isolates of herpesviruses resistant to ACV are increasing in frequency(15, 16) , this structural information is critical for designing TK molecular models and improving nucleoside drugs. Additionally, HSV-1 TK is currently being delivered to tumor cells as a toxin gene, via retroviral vectors, whereby these cells are killed after administration of ganciclovir(17, 18) . Therefore, a better understanding of the molecular mechanism of HSV-TK could lead to future customized, drug-specific TKs for gene therapy.

Toward these goals, we have expressed HSV-1 TK and a site-specific mutant in Escherichia coli and purified them for use in photoaffinity labeling analysis with base-substituted, azidonucleotide analogs. These analogs have found many applications in the characterization, identification, and purification of nucleotide-binding proteins(19, 20) . Upon UV irradiation, a highly reactive nitrene intermediate of short half-life is generated which allows indiscriminant insertion into active-site amino acids in a nucleotide-binding protein(19) . The most commonly used photoaffinity analogs are the 8-azidopurine nucleotides(19) , and more recently 5-azidouridine derivatives(20) . Both classes of analogs are currently finding wide usage for the identification of active-site (or regulatory-site) peptides and amino acids involved in nucleotide binding(21, 22) . Prior to this report, base substituted azidonucleosides have not been reported in the literature for use as inhibitors of viral replication or in the study of herpesviral-encoded proteins.

In this study, we report the bacterial expression and a rapid, two-step purification of HSV-1 TK and a site-specific mutant TK containing a tyrosine at position 336 in place of cysteine (C336Y). A similar mutant isolated from an acyclovir-resistant HSV-1 strain, in which C336Y was proposed to constitute part of the ATP and nucleoside-binding sites, was shown to have higher K values for thymidine, ATP, and ACV relative to wild-type TK(11) . We have used two photoaffinity analogs, [-P]8-N(3)ATP and [alpha-P]5-N(3)dUMP, for comparative analysis of the wild-type and mutant HSV-1 TKs. This active-site directed technique is shown to be an effective tool for studying HSV-1 TK and related site-specific mutants.


EXPERIMENTAL PROCEDURES

Materials

All reagents and nucleotides were purchased from Sigma unless otherwise indicated. [methyl-^3H]Thymidine (specific activity, 59 Ci/mmol) was purchased from Moravek Biochemicals. [-P]ATP and [P]P(i) (specific activity, 5-10 mCi/µmol) were from ICN Radiochemicals. Whatman DE81 filter paper discs and DE-52 resin were purchased from Fisher. The Bio-Scale Q2 column was from Bio-Rad. Restriction endonucleases, Vent polymerase, and T4 DNA ligase were from New England Biolabs. ^1H NMR spectra were recorded on a GE 300 MHz spectrophotometer using Me(2)SO-d(6) as the solvent and internal standard. IR spectra were recorded on a Perkin-Elmer 475 spectrophotometer and UV spectra were recorded on a Shimadzu UV-1201 spectrophotometer.

Expression Vector Constructions

pHSV106, containing the HSV-1 TK coding sequence of the HSV-1 strain MP, was purchased from Life Technologies, Inc. Synthetic DNA primers, containing NdeI and BamHI restriction sites, were utilized for polymerase chain reaction amplification of the TK coding region with Vent polymerase. This polymerase chain reaction product was subsequently ligated into a T7 expression vector, pET-9a (purchased from Novagen), to form pET-TK1. The recombinant pET-TK1 was sequenced by the method of Sanger et al.(23) and confirmed to be that of the HSV-1 TK MP strain, ligated correctly for expression.

The C336Y mutation was first created by site-directed mutagenesis using a modified version of Kunkel (24) as described in Black and Hruby(25) . The HSV-1 TK gene was cloned into the pET-8c at the NcoI site using the NcoI site at the initiating methionine codon and an NcoI site 3` to the HSV-TK open reading frame(26) . The orientation, site of insertion, and C336Y mutation were confirmed by sequencing.

Enzyme Purification

E. coli SY211 cells (TK, derived from the BL21 cell line and obtained as a gift from Dr. William Summers, Yale University), in which T7 RNA polymerase, under control of the isopropyl-beta-D-thiogalactopyranoside-inducible lacUV5 promoter(27) , were transformed with pET-TK1. These cells were grown to A = 0.6, expression induced by 1 mM isopropyl-beta-D-thiogalactopyranoside for 2.5 h, and lysed in buffer A (20 mM Tris buffer, pH 7.6, 10% glycerol, 1 mM dithiothreitol, 40 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and aprotinin (1 µg/liter)) by sonication for 3 min. The homogenate was then centrifuged at 10,000 times g for 30 min to separate insoluble material. The soluble lysate was loaded onto a 15 times 3-cm column of DE-52 and washed extensively with buffer A. Selective elution of TK activity was achieved with buffer B (50 mM potassium phosphate buffer, pH 7.6, 20% glycerol, 1 mM dithiothreitol, 40 mM KCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) as modified from the procedure of Larder et al.(28) . Fractions with TK activity were pooled and concentrated to 10 ml in an Amicon filtration apparatus with a 20-volume excess of buffer A. Aliquots of 1 mg were loaded onto a Bio-Scale Q2 column equilibrated with buffer A and eluted with a 40-200 mM NaCl gradient for 60 min by FPLC (Pharmacia). Purified HSV-1 TK was stored in buffer B with 0.5 mg/ml bovine serum albumin at -80 °C. This approach has reproducibly led to greater than 95% purified TK, with cumulative yields ranging from 0.5 to 2 mg of protein.

Enzyme Assays

The activity of the purified HSV-1 TK was determined using the following reaction mixture: 0.5-5 ng of protein, 4 µM [methyl-^3H]thymidine (4Ci/mmol), 20 mM potassium phosphate, pH 7.6, 1 mM dithiothreitol, 5 mM ATP, 5 mM MgCl(2), 25 mM NaF, 40 mM KCl, and 0.5 mg/ml bovine serum albumin in a total volume of 25 µl for 10 min at 37 °C. For TMP kinase activity, 10-20 µM [methyl-^3H]TMP was substituted for thymidine. To identify phosphorylated products, 20 µl of the reaction mixture was loaded onto a DE-81 filter, washed three times in 95% ethanol, and counted for radioactivity(29) . Alternatively, an aliquot of the reaction mixture was loaded onto a polyethyleneimine-cellulose TLC plate and developed in 0.5 M LiCl (30) to separate the thymidine from the thymidine monophosphate (TMP) and diphosphate products. Radioactivity was determined using an LKB 1214 Rackbeta liquid scintillation counter and corrected for background using controls with enzyme incubated in the absence of ATP. A unit of enzyme activity is equal to the formation of 1 nmol of TMP or thymidine diphosphate/min/mg at 37 °C.

Synthesis of 5-Azidodeoxyuridine

The basic synthetic protocol is analogous to that reported for the synthesis of 5-N(3)UMP (20) or 5-N(3)dUMP(31) . These reactions involve nitration of UMP with nitrosonium tetrafluoroborate, reduction of the 5-nitro compound to the amine with zinc/HCl, and azide exchange with sodium azide via a diazonium intermediate(20, 31) . To synthesize the 5-azidouridine nucleosides, the only modifications to the basic protocol involve different purification steps of the 5-amine and 5-azido compounds. From 250 mg of deoxyuridine (dU), 5-nitro-dU is quantitatively produced and extracted as described previously(20) . After Zn reduction, the resulting 5-amino-dU is separated from unreacted 5-nitro-dU with a Dowex 50W-H column (1 times 8 cm) resin: 5-nitro-dU does not bind and the 5-amino-dU is eluted with 0.5 M ammonium hydroxide. After the azide exchange reaction (19) , the resulting 5-azido-dU was passed through a Dowex 50W-H column (1 times 4 cm) for desalting and applied to a Waters Sep-Pak C18 cartridge equilibrated with water. 5-N(3)dU was eluted with 1 ml of methanol and stored at -20 °C. Final product yields vary between 10 and 20% of the starting material. This identical procedure was utilized to produce 5-N(3)U and 5-N(3)dU from the corresponding starting nucleosides. Similarly, [6-^3H]deoxyuridine (0.1 mCi) (DuPont NEN) was used in the above protocol to synthesize [6-^3H]5-N(3)dU (0.005 µCi/µmol). IR (KBr) 2090 cm (azido); UV (H(2)O) (max) 289 nm (disappears after photolysis(19) ). ^1H NMR (Me(2)SO-d(6)) 2.10 (m, 2H, 2`-H); 3.55-3.75 (m, 2H, 5`-H); 4.28 (m, 1H, 4`-H); 4.32 (m, 1H, 3`-H); 5.12 (m, 1H, 3`-OH); 5.24 (m, 1H, 5`-OH); 6.10 (m, 1H, 1`-H); 7.84 (s, 1H, 6-H); 11.80 (s, 1H, 3-NH, D(2)O exchangeable). An extinction coefficient of 7,600 M cm previously determined for 5-N(3)dUMP was used(31) .

Synthesis of [alpha-P]5-Azidodeoxyuridine Monophosphate and [-P]8-Azido-ATP

For [P]5-N(3)dUMP, purified HSV-1 TK (0.5 µg), 0.5 µmol of 5-N(3)dU, 40 mM KCl, 10 mM MgCl(2), 1 mCi of [-P]ATP (3000 mCi/µmol) were incubated at 37 °C for 20 min in a total of 0.15 ml. ATP (2 µmol) was added for 5 min prior to column purification. The resulting [P]5-N(3)dUMP was purified by DEAE-cellulose chromatography as described previously for other 5-azidouridine nucleotides(20) . [-P]8-N(3)ATP was synthesized from 8-N(3)ATP (19, 32) using the phosphate exchange reaction originally described by Glynn and Chappel(33) .

Photolabeling of HSV-1 TK

For photolabeling studies(20) , 10 µg of DE-52-purified TK in 20 mM potassium phosphate buffer, pH 7.6, 10% glycerol, 5 mM MgCl(2), 1 mM dithiothreitol, 40 mM KCl, was incubated with the appropriate concentration of the photoprobe, [-P]8-N(3)ATP or [alpha-P]5-N(3)dUMP for 10 s. The sample was then irradiated for 90 s with a hand-held UV lamp (254 nm UVP-11, Ultraviolet Products, Inc.) at a distance of 3 cm. For competition experiments, varying concentrations of nonradiolabled competitors were incubated in the reaction mixture for 20 s prior to the addition of photoprobe. All reactions were terminated by addition of an equal volume of 10% trichloroacetic acid, incubated on ice for 10 min, and pelleted by centrifugation at 13,000 times g for 5 min. The protein was resuspended in a solublization mixture (20) and separated on 10% SDS-polyacrylamide gels. Dried gels were exposed to film for 0.5 to 3 days. The intensity of the bands corresponding to TK on the autoradiographs were determined using a laser densitometer (Bio-Rad Model GS-670 Imaging Densitometer).

Cytoxicity and Antiviral Assays

Vero cells, in Dulbecos's minimum essential medium supplemented with 5% newborn calf serum and 50 µg/ml penicillin/streptomycin, were grown to confluency (approximately 1 times 10^6) in 60 times 15-mm tissue culture plates and infected with HSV-1 (strain KOS) or HSV-2 (strain 333) for 1 h. Medium was subsequently replaced with fresh medium containing the test compounds (0-50 µM range) for plaque reduction assays (300 plaque forming units/plate) after 48 h post-infection as described previously(34) . The cytotoxic effects of the compounds on uninfected cells were determined using a soluble tetrazolium dye, Alamar blue, as directed by the manufacturer (Alamar, Sacramento, CA).


RESULTS

Purification and Expression of HSV-1 TK

The purification and expression scheme was designed to allow rapid isolation of milligram amounts of WT and mutant HSV-1 TK enzyme. A bacterial T7 expression system (Novagen) was utilized to express the native enzyme and resulted in ranges of 4-10% of the total protein being HSV-1 TK. Purification on DEAE-cellulose was achieved by a combination of protocols described previously(28) , with the key modification being the use of 20 mM Tris loading buffer, then selective elution of HSV-1 TK in a 50 mM phosphate buffer (see Fig. 1). As shown in Fig. 2, the final step of purification entailed FPLC using a Bio-Scale Q2 column. The SDS-PAGE profile of this two-step purification of HSV-1 TK is shown in Fig. 1and described in Table 1. The final purification was 74-fold as determined by specific activity (Table 1), although this has varied depending on the initial levels of the induced HSV-1 TK. Simliar results were also found for the C336Y mutant with identical FPLC elution profiles (data not shown). To show that the expression of the native enzyme in E. coli did not result in altered activity, the WT and C336Y enzymes were analyzed kinetically: the K(m) values for thymidine were 1.3 (WT) and 10 (C336Y) µM; for ATP, 30 µM (WT) and 1.3 mM (C336Y) (data not shown). These values agree with previously published kinetic analyses of HSV-1 TK(8, 28) .


Figure 1: SDS-polyacrylamide gel electrophoresis of proteins at different stages of HSV-1 TK purification. Protein was taken from the pooled fractions containing HSV-1 TK activity from crude supernatant (25 µg), DEAE-cellulose column (10 µg), and Bio-Scale Q2 FPLC (2 µg) as described under ``Experimental Procedures'' and separated on a 10% SDS-polyacrylamide gel, and stained with Coomassie Blue.




Figure 2: Purification of HSV-1 TK by Bio-Scale Q2 FPLC. Pooled DEAE-cellulose fraction (1 mg) was applied to a Bio-Scale Q2 column in a Pharmacia FPLC system previously equilibrated with Buffer A. HSV-1 TK was eluted in Buffer A by a 40-200 mM NaCl gradient as indicated by protein absorbance at 280 nm (-). Thymidine kinase activity(- - -) was determined by assaying 15 µl of each fraction as described under ``Experimental Procedures.'' Fractions 40-45 were pooled, concentrated, and analyzed by SDS-PAGE as shown in Fig. 1.





Photoaffinity Labeling of HSV-1 TK with [P]5-N(3)dUMP

For photoaffinity labeling, a photoactive thymidine analog was synthesized, 5-azidodeoxyuridine (5-N(3)dU), and tested as a substrate of HSV-1 TK. Using [^3H]5-N(3)dU, products corresponding to [^3H]5-N(3)dUMP (major) and [^3H]5-N(3)dUDP (<10%) were detected in TLC assays. In kinetic experiments using 5-N(3)dU as an inhibitor of thymidine phosphorylation, it was shown that 5-N(3)dU had complex mixed inhibition patterns due to its dual function as both a competitive inhibitor and substrate. Half-maximal inhibition of thymidine phosphorylation by 5-N(3)dU was 1 µM. 5-N(3)dU was readily converted to [alpha-P]5-N(3)dUMP using [-P]ATP and HSV-1 TK. Since previous kinetic experiments (5) indicated that thymidine and thymidylate kinase activities have a single or overlapping active-site, we utilized [P]5-N(3)dUMP to examine these sites.

By use of the TK assay buffer minus bovine serum albumin and standard procedures(19, 20) , photolabeling of WT and the C336Y HSV-1 TKs by [alpha-P]5-N(3)dUMP was pursued. Although not shown, photoincorporation of the photoprobes with the HSV-1 TKs was always dependent on UV irradiation. As shown in Fig. 3, half-maximal saturation of photoinsertion of [P]5-N(3)dUMP was 6 (WT) and 8 (C336Y) µM. These values are consistent with the reported K(m) values of TMP for both of these HSV-1 TKs(8, 28) , and are indicative of specific active-site incorporation. To further test the active-site specificity of photolabeling, known nucleoside and nucleotide substrates at varying concentrations were included in the photolabeling reactions with [P]5-N(3)dUMP and the two TKs. As shown in Fig. 4, A-C, photolabeling of WT HSV-1 TK was competitively inhibited in a concentration dependent manner by thymidine, TMP, ACV, BVDU, 5-N(3)dU, and 5-N(3)dUMP. The concentrations of inhibitors that reduce photoincorporation by 50% (ICP) were derived from densitometric scanning of the autoradiographs in Fig. 4and are are listed in Table 2.


Figure 3: Saturation of [alpha-P]5-NdUMP photoinsertion into WT and C336Y HSV-1 TKs. HSV-1 TK (10 µg) in a 40-µl reaction buffer was photolyzed with the indicated amount of [alpha-P]5-N(3)dUMP and subjected to SDS-PAGE. Photoincorporation was detected by autoradiography and quantified by laser densitometry. bullet, WT; circle, C336Y.




Figure 4: Effect of various substrates on photolabeling of WT and C336Y HSV-1 TK with [alpha-P]5-NdUMP. Either wild-type or C336Y HSV-1 TKs (10 µg) were incubated in the presence of the indicated concentrations of HSV-1 TK substrates, photolyzed with 15 µM [alpha-P]5-N(3)dUMP, and subjected to SDS-PAGE. A, thymidine and TMP; B, 5-N(3)dU and 5-N(3)dUMP; C, ACV and BVDU; D, ATP. Photoincorporation was detected by autoradiography and quantified by laser densitometry.





Comparisons between the [P]5-N(3)dUMP photolabeling of the C336Y and WT HSV-1 TKs are shown in Fig. 4and Table 2. The C336Y HSV-1 TK consistently resulted in 1.3-fold higher levels of photoincorporation of 15 µM [P]5-N(3)dUMP relative to WT HSV-1 TK (data not shown). Of the compounds tested, ICP values for TMP did not differ between C336Y and WT HSV-1 TKs, and were over 2-fold higher for 5-N(3)dUMP. However, these values significantly differed for the nucleosides tested: thymidine 5.7-fold, 5-N(3)dU 16.7-fold, ACV >6-fold, and BVDU >16.7-fold (Table 2). Addition of ATP (Fig. 4D) to inhibit [P]5-N(3)dUMP photolabeling resulted in ICP values of 150 µM for WT and >1 mM for C336Y HSV-1 TKs.

Photoaffinity Labeling of HSV-1 TK with [P]8-N(3)ATP

To study the nucleoside triphosphate active site of the HSV-1 TKs, the ATP analog, [-P]8-N(3)ATP, was used. As shown in Fig. 5, half-maximal saturation of photoincorporation of [P]8-N(3)ATP for WT was 90 µM, while photolabeling of the C336Y HSV-1 TK did not saturate at concentrations of up to 1 mM. Photoincorporation of 15 µM [P]8-N(3)ATP for the C336Y mutant was 20% of the WT levels (data not shown). As shown in Fig. 6, addition of ATP, GTP, or TTP (ICPs: 80, 185, and 137 µM, respectively) inhibited photoincorporation of [-P]8-N(3)ATP into HSV-1 TK in a concentration-dependent manner, while CTP had little effect. In contrast, only minor inhibition of [P]8-N(3)ATP photolabeling by competing NTPs was observed with the C336Y TK (see Fig. 6). Cumulatively, the photoaffinity data suggest that the C336Y HSV-1 TK mutant is primarily altered at the ATP site.


Figure 5: Saturation of [-P]8-NATP photoinsertion into WT and C336Y HSV-1 TKs. Wild-type HSV-1 TK (10 µg) (A) or C336Y TK (B) were photolyzed with the indicated concentration of [-P]8-N(3)ATP. Proteins were separated by SDS-PAGE followed by autoradiography and laser densitometry quantitation.




Figure 6: Inhibition of [-P]8-NATP photoinsertion into WT and C336Y HSV-1 TKs by nucleoside triphosphates. Wild-type HSV-1 TK (A, ) or C336Y TK (B, ) were preincubated with the indicated concentrations of nucleotide and photolyzed with 15 µM [-P]8-N(3)ATP. The reaction mixture was subjected to SDS-PAGE followed by autoradiography and laser densitometry quantitation. , CTP; bullet, GTP; , TTP.



Anti-herpesvirus Activities of 5-Azidouridine Analogs

The photoaffinity experiments indicated that 5-N(3)dUMP could be used to photolabel the T/TMP active sites of HSV-1 TK. One of the underlying assumptions at the initiation of these studies was that photoactive analogs of known anti-HSV drugs could be used to study cellular and viral drug metabolizing enzymes, and more importantly, that this information would reflect actual intracellular drug metabolism. To test this directly, 5-azidouridine (5-N(3)U), 5-N(3)dU, and 5-azidodideoxyuridine (5-N(3)ddU) were tested as inhibitors of HSV-1 and HSV-2 replication in Vero cells as described under ``Experimental Procedures.'' Compared to previous values obtained for BVDU(3) , 5-N(3)dU was an effective inhibitor of HSV-1 replication leading to 50% inhibition of viral plaque formation at 0.2 µM (Table 3), yet had no activity against HSV-2. 5-N(3)U had no apparent activity or cytoxicity. 5-N(3)ddU was inhibitory, but its antiviral activity was difficult to distinguish from its cytoxic properties. An additional metabolic experiment was done by incubating [^3H]5-N(3)dU with control and HSV-1 infected cells for 1 h, followed by perchloric acid extraction of the cells and TLC analysis of the recovered nucleotides. [^3H]5-N(3)dUMP was only detected in extracts derived from HSV-1 infected cells (data not shown), which is indicative of phosphorylation by HSV-1 TK.




DISCUSSION

In order to facilitate rapid and high-yield purification of HSV-1 TKs expressed in bacteria, a novel purification protocol was developed independent of the conventional thymidine-affinity chromatography resins routinely employed with this enzyme. Because we intend to apply this purification protocol to other HSV-1 TKs mutated in the thymidine or TMP active-sites, the thymidine affinity resins would have had limited utility for these purposes. The rapid purification technique consists of only two steps following sonication of the isopropyl-beta-D-thiogalactopyranoside-induced E. coli cells: a selective, modified DEAE-cellulose chromatography procedure and Q2 FPLC. This purification reproducibly results in greater than 95% purity of HSV-1 TK. Comparison of specific activities obtained with this protocol and thymidine-affinity chromatography indicates little differences(28) .

Site-directed mutagenesis studies of HSV-1 TK have been useful in correlating conserved sites with activity(12, 13, 14) , but the basis of the molecular differences in substrate and drug specificities of the HSV-1 TK remains unclear. In combination with site-specific mutants of HSV-1 TK, the active-site directed photoaffinity analogs described here should aid in elucidating the molecular mechanism underlying HSV-1 TK activity. As a model, we have compared the photoaffinity labeling of the WT and an ACV-resistant TK, C336Y, with 5-N(3)dUMP and 8-N(3)ATP. The experiments presented in Fig. 3Fig. 4Fig. 5Fig. 6indicate specific active-site photoincorporation of these analogs and highlight several important observations regarding the nature of the C336Y mutation and ACV resistance. For WT HSV-1 TK, the most effective inhibitors of [P]5-N(3)dUMP photolabeling are nucleosides, not TMP or 5-N(3)dUMP (Table 2). This observation is consistent with previous kinetic studies which have shown the K(m) values for thymidine and TMP differ by 12-25-fold, and both compounds to be competitive inhibitors of each other, indicating that their sites overlap or are partially shared (6, 7, 8) . However, as shown in Fig. 3and Fig. 4A, the TMP site in the C336Y mutant remains unaffected in contrast to the thymidine and ATP sites (Fig. 4Fig. 5Fig. 6). This suggests that although nucleosides (and nucleoside derived drugs) are preferred in the T/TMP site(s) compared with nucleoside monophosphates, the thymidine kinase and thymidylate kinase activities can be uncoupled and are independent. The data also suggests that a mutation at either the nucleoside/drug-binding site or in the ATP site will lead to increases in K(m) values for both substrates and potentially lead to a drug-resistant enzyme. This is currently being tested with the photoaffinity analogs and other HSV-1 TK site-specific mutants containing amino acid substitutions thought to be involved in either TK or TMP kinase activity.

The photolabeling data with [P]8-N(3)ATP and the C336Y mutant ( Fig. 5and Fig. 6) indicate that the primary effect of this amino acid substitution is in the ATP site. Therefore, an apparent structural conformation change as a result of the mutation somehow alters ATP/nucleoside binding, yet still retains a WT-like ATP/TMP binding. The cysteine 336 of HSV-1 TK is believed to comprise part of the ATP-binding site, as suggested by comparison of homologous domains with other viral TKs and a related motif conserved in porcine adenylate kinase(8, 11) . Interestingly, 8-N(3)ATP was used to identify an ATP active-site peptide 5 amino acids N-terminal to this conserved site in adenylate kinase(21) . Thus, if this homology is reflective of HSV-1 TK function, Cys-336 is likely to be near the adenine base in the ATP site. An interesting question to address in the C336Y mutant is why the altered affinity for ATP binding effects phosphorylation of nucleosides, but does not apparently affect thymidylate kinase activity. The lack of competition by CTP of [P]8-N(3)ATP photolabeling for either HSV-1 TK was not expected. Consistent with previous studies(5) , the purified HSV-1 TK utilizes CTP as a phosphoryl donor very efficiently (data not shown). This lack of CTP inhibition of photolabeling may indicate a distinct binding site for CTP and is currently being studied.

BVDU is one of the most potent inhibitors of HSV-1 replication in cell culture, yet has little effect on the replication of HSV-2(3) . Of the azidonucleosides tested, the best inhibitor of HSV-1 replication was 5-N(3)dU (Table 3). This compound would be the closest structural mimic of BVDU, so it is not surprising that it had anti-HSV-1 activity and little activity against HSV-2. The 5-azido moiety is consistent with the criteria established for determining the effectiveness of a 5-substituted deoxyuridine as an anti-HSV-1 compound (35) : it is an unsaturated, hydrophobic, electronegative 5-substituent of no more than 4 atoms in length. Metabolic labeling experiments indicate that [^3H]5-N(3)dU can be phosphorylated by HSV-1 TK in HSV-1 infected cells. Even though 5-N(3)dU is a potent inhibitor of HSV-1 replication in cell culture, the poor half-maximal cytoxicity doses of 10-20 µM preclude any potential clinical applications. These results, however, do validate the hypothesis that azidonucleosides can act as biological mimics of anti-HSV nucleoside drugs. Therefore, if they are used as photoaffinity analogs and UV-cross-linked to HSV-TK active-site peptides, these are likely to be the same peptide domains that interact with the known antiviral drug. Cumulatively, this study has shown that 5-N(3)dUMP and 8-N(3)ATP are effective tools for determining the active-site environment of HSV-1 TK.


FOOTNOTES

*
This work was supported in part by a grant from the Arkansas Science and Technology Authority (to R. R. D.). 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.

§
Current address: Darwin Molecular Corp., 1631 220th Ave. S.E., Suite 101, Bothell, WA 98021. Tel.: 206-489-8000; Fax: 206-489-8017.

To whom correspondence should be addressed: Dept. of Biochemistry, Slot 516, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Tel.: 501-686-5419; Fax: 501-686-8169.

(^1)
The abbreviations used are: HSV-1, herpes simplex virus type 1; BVDU, 5-bromovinyl-2`-deoxyuridine; ACV, acyclovir; HSV-2, herpes simplex virus type 2; TK, thymidine kinase; 8-N(3)ATP, 8-azidoadenosine-5`-triphosphate; 5-N(3)dUMP, 5-azido-2`-deoxyuridine-5`-monophosphate; 5-N(3)dU, 5-azido-2`-deoxyuridine; TMP, thymidine monophosphate; FPLC, fast protein liquid chromatography; dU, deoxyuridine; PAGE, polyacrylamide gel electrophoresis; WT, wild type.


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