Deoxyribopyrimidine Triphosphatase Activity Specific for Cells Infected

Proc. Natl. Acad. Sci. USA Vol. 77, No. 4, pp. 1872-1876, April 1980 Biochemistry Deoxyribopyrimidine triphosphatase activity specific for cells infected with herpes simplex virus type 1 (deoxyribonucleotide degradation/diphosphohydrolase/viral temperature-sensitive mutants) FRANZ WOHLRAB AND BERTOLD FRANCKE Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Edward A. Adelberg, December 26, 1979

ABSTRACT Nuclei from baby hamster kidney cells infected infection at 320C were disrupted hypotonically in 20 mM with herpes simplex virus type 1 contain a virus-specific deoxy- Hepes, pH 7.8/1 mM dithiothreitol/1 mM MgCl2 (hypotonic ribonucleoside triphosphate degrading activity. The reaction buffer), the hypotonic lysates were diluted 1:6 with the same proceeds at 4°C and can thus be distinguished from host enzymes. Under these conditions the enzyme is specific for buffer, and the resulting suspension was treated with 0.2% deoxyribopyrimidine triphosphates and catalyzes pyrophos- Nonidet P40 for 20 min. Nuclei were isolated by centrifugation, phate cleavage to produce the monophosphates, dUTP being washed twice with hypotonic buffer, and resuspended in 20 the best substrate followed by dCIP and dTIZP. The appearance mM Hepes, pH 7.8/1 mM dithiothreitol/1 mM MgCI2/80 mM of the activity after infection parallels that of viral DNA-syn- potassium acetate. thesis-related functions. Of a series of eight temperature-sen- Triphosphatase Assay. Triphosphatase activity was assayed sitive mutants tested, two (tsD and tsK) exhibit significantly decreased triphosphatase levels after infection at nonpermissive by measuring the production of radiolabeled deoxynucleoside temperature, whereas a viral deoxypyrimidine kinase-deficient monophosphates from the corresponding triphosphates. Stan- mutant induced wild-type levels. dard assays were carried out in a total volume of 50 MA containing: 1 mM dithiothreitol; 1 mM ethylene glycol bis(,B- After infection of cells with herpes simplex virus type 1 aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); 2 mM (HSV-1), large changes in nucleotide pools occur (1). Most af- ATP; 50 MM [3H]dTTP (2 Ci/mmol; 1 Ci = 3.7 X 1010 bec- fected are the pools of deoxypyrimidine nucleoside triphos- querels), 50 MM [3H]dCTP (2 Ci/mmol), or 1 mM [3H]dUTP phates, notably that of dTTP followed by dCTP. Whether these (0.1 Ci/mmol); 3 mM EDTA; and nuclei corresponding to 3 X changes have a function in viral DNA replication is unclear. 106 cells. The reaction was started by adding 5 Ml of 80 mM Several enzymes involved in deoxyribonucleotide metabolism MgCl2 and the incubation temperature was 4°C. The reaction have been shown to be induced by the virus, among which are was terminated by addition of 20,u of 0.1 M EDTA and 112.5 a deoxypyrimidine kinase/thymidylate kinase (2, 3), an altered Ml of cold methanol. Small aliquots (1-4 Ml) of this mixture were ribonucleotide reductase (4), and deoxycytidine deaminase applied to prewashed polyethyleneimine-cellulose strips to- (5). gether with unlabeled marker nucleotides. The strips were In order to understand how HSV-1 DNA is replicated we are developed with 1 M HCOOH/0.5 M LiCl at room temperature, using a cell-free DNA-synthesizing system (6) in an approach dried, and examined under UV light. Spots containing deoxy- similar to the one described for bacteriophage T4 (7). During nucleoside mono-, di-, and triphosphates were each cut out and these studies we made the observation that deoxyribonucleoside radioactivity was determined by liquid scintillation counting triphosphates added to nuclei from HSV-1-infected cells are without prior elution. highly unstable and are degraded to a much greater extent than Other Enzymes. HSV-1 DNA polymerase was determined could be accounted for by their incorporation into newly syn- as described by Weissbach et al. (8); HSV-1 alkaline nuclease, thesized DNA. These findings prompted us to investigate the by the method of Francke (9); and deoxypyrimidine kinase, by dynamics of DNA precursor pools during HSV-1 DNA repli- the procedure of Jamieson et al (2). cation. 'y-32P-Labeled Deoxypyrimidine Triphosphates. Synthesis We report here on a HSV-1-specific enzyme activity that of 'y-32P-labeled deoxypyrimidine triphosphates was carried degrades deoxyribopyrimidinenucleoside triphosphates to the out by phosphorylation of the respective diphosphates with corresponding monophosphates and seems to be firmly bound nucleoside diphosphate kinase and ['y-32P]ATP as phosphate to the nucleus of the infected cell. donor. The reaction mixture contained in a total volume of 320 Al: 80 mM Tris-HCI (pH 7.0), 2 mM ATP (0.3 Ci/mmol), 10 MATERIAL AND METHODS mM MgCl2, 25 mM KCI, 400 AM deoxypyrimidine diphos- Cells and Virus Stocks. Stocks of HSV-1 Glasgow strain 17 phate, and 80 units of nucleoside diphosphate kinase. Incuba- syn+ ts+ and the DNA-negative mutants tsB, tsD, tsE, tsH, tsJ, tion was at 32°C for 2 hr. Aliquots (150,ul) were applied to a tsK, tsS, and tsU were prepared in baby hamster kidney (BHK) 1.5 X 12 cm DEAE-Sephacel column which was subsequently cells as described (6). The strain TK 43 and the corresponding washed with 0.1 M NH4HCO3. Nucleotides were eluted with wild type Cl 101 were kindly provided by W. P. Summers. All a linear gradient of 0.1-1 M NH4HCO3 (total volume of gra- experiments were performed 15 hr after infection at 32°C at dient, 40 ml), and radioactivity in the effluent was determined a multiplicity of infection of 10. by assaying Cerenkov radiation. Fractions containing labeled Preparation of Nuclei. Nuclei were prepared by a modifi- deoxyribopyrimidine triphosphates were checked for purity cation of the described procedure (6). Cells harvested 15 hr after by thin-layer chromatography, pooled, and lyophilized. Separation of Pyrophosphate from Orthophosphate. A The publication costs of this article were defrayed in part by page standard triphosphatase assay was carried out, except that in charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: HSV-1, herpes simplex virus type 1; BHK, baby hamster this fact. kidney. 1872 Downloaded by guest on September 25, 2021 Biochemistry: Wohlrab and Francke Proc. Natl. Acad. Sci. USA 77 (1980) 1873 addition to the 3H-labeled deoxynucleotide y-32P-labeled de- Table 2. Properties of the HSV-1-specific triphosphatase oxynucleotide was also present. The resulting solution was di- % of control activity vided into two parts. One part was assayed on polyethylene- dTTPase dCTPase dUTPase imine-cellulose strips for triphosphatase activity as described above. The other part was made 20 mM in MgCl2 and a 5-pA Complete system 100 100 100 was with marker phosphates on pre- -Mg2+,+ 5 mM EDTA 6 sample spotted together -Nuclei 3 washed Whatman no. 1 filter paper. Ortho- and pyrophosphate Nuclei heated to 1000C 4 were separated by ascending paper chromatography in ace- +PCMB* (5 mM) 26 tone/35% formic acid, 3:2 (vol/vol). Spots were located by + Pyrophosphate (2.5 mM) 38 43 94 spraying the paper with a solution of 1 g of ammonium mo- + Pyrophosphate (10 mM) 20 21 81 lybdate in 5 ml of 60% perchloric acid/10 ml 1 M HCl/85 ml + Orthophosphate (10 mM) 57 85 water and, after drying, reducing the phosphomolybdate complex by irradiation with UV light. Spots were cut out Standard triphosphatase assays were performed with the omissions and additions indicated. Numbers are relative activities (standard and radioactivity was determined by liquid scintillation conditions = 100%1). counting. * p-Chloromercuribenzoate. Chemicals. Nucleotides were from Sigma, nucleotide diphosphate kinase was from Calbiochem, DEAE-Sephacel was from Pharmacia, and polyethyleneimine-cellulose thin-layer divalent cations as indicated by the strong inhibition by EDTA. sheets were from Schleicher & Schuell. [3H]dTTP (47 Ci/ Fig. 1 shows the dependence of reaction rates on the concen- mmol), [3H]dCTP (21 Ci/mmol), [3H]dUTP (13.4 Ci/mmol), tration of Mg2+, Mn2+, and Ca2+. Mn2+ could partially sub- [3H]dGTP (10-30 Ci/mmol), [a-32P]dATP (400 Ci/mmol), stitute for Mg2+, but optimal activity occurred at lower con- [y-32P]ATP (16.3 Ci/mmol), and [3H]dThd (42 Ci/mmol) were centrations and the maximal rate was considerably slower. Ca2+ from Amersham. All chemicals were of the highest purity did not stimulate enzyme activity and had even a slightly in- available. hibitory effect. The enzyme has a broad pH optimum; between pH 6.0 and 9.0 there was no significant change in activity. Like RESULTS other HSV-1-specific enzymes, the triphosphatase is rather insensitive to high salt. At concentrations up to 400 mM KCI no Isolated nuclei from both uninfected and. HSV-1-infected BHK inhibition was observed (data not shown). cells are capable of degrading ribo- and deoxyribonucleotides The fact that deoxyribopyrimidine nucleoside triphosphates at 32°C. Lowering the temperature to 4°C permits the detec- are degraded rapidly at 4°C indicates a high affinity of the tion of virus-specific triphosphatase activities because host enzyme towards its substrates, especially towards dUTP. Al- enzymes are inactive under these conditions. This is illustrated though Km determinations of crude enzyme preparations in by the data in Table 1. Nuclei from HSV-1-infected cells and heterogeneous systems as the one used here are somewhat not from uninfected cells degraded dTTP, dCTP, and dUTP at considerable rates, whereas deoxyribopurine nucleoside triphosphates remained stable. The product of the reaction appears to be the corresponding monophosphate only, and no diphosphate intermediate was observed even after letting the reaction proceed to completion. Small amounts of diphosphate produced can be suppressed by addition of ATP or by lowering the substrate concentration. ATP (2 mM) was added routinely to the assay, although there is no ATP requirement for the reaction. The enzymatic activity is linearly dependent on the concentration of nuclei and proceeds in a linear manner for at S~~~~ m least 30 min for all three substrates under the standard assay I0 conditions (data not shown). Other properties of the reaction are listed in Table 2. The x E * --_ enzymatic activity is heat-labile and requires the presence of UL * 0 Table 1. Degradation of radiolabeled deoxynucleoside triphosphates by nuclei from mock-infected and 1 HSV-1-infected BHK cells f--ttf-to Mock-infected, % HSV-1-infected, % Substrate dNMP dNDP dNTP dNMP dNDP dNTP dATP 2 18 80 4 21 75 dCTP 2 15 83 86 5 9 . I . dGTP 3 14 83 9 11 80 0 2 4 6 8 10 12 14 dTTP 3 15 82 84 8 8 Divalent cation, mM dUTP 2 4 94 92 5 3 FIG. 1. Dependence of triphosphatase activity on Mg2+ (0), Mn2+ (@), and Ca2+ (A) concentrations. Nuclei from infected cells Nuclei from mock-infected and HSV-1-infected cells were prepared were prepared and assayed for dUTPase as described in Materials and triphosphatase activity was determined; assays for dATP and and Methods, except that MgCI2 was omitted in the final buffer. dGTP were performed in a similar manner, except that 50 juM [y- Reactions were started by adding the nuclei containing the indicated 32P]dATP (2 Ci/mmol) or 50MuM [3H]dGTP (2 Ci/mmol) was present amounts of MgCl2, MnCl2, or CaCl2 to the reaction mixture. For the in the reaction mixture. The distribution of label in monophosphate Ca2+ curve, EGTA was omitted. Reactions were terminated and (dNMP), diphosphate (dNDP), and triphosphate (dNTP) after 2 hr monophosphate produced was determined as described for the at 4°C is expressed as percentage of input radioactivity. standard assay procedure.*__a__, Downloaded by guest on September 25, 2021 1874 Biochemistry: Wohlrab and Francke Proc. Natl. Acad. Sci. USA 77 (1980)

Table 3. Competition of substrates with other nucleotides phosphate marker. The presence of pyrophosphate anions in- Competitor Substrate dicates that the enzyme is indeed a diphosphohydrolase, rather (1 mM) dUTP dCTP dTTP than a sequential monophosphatase. Fig. 3 shows triphosphatase activity as a function of time after 100 100 100 infection. The enzyme can be detected first between 4 and 6 dATP 100 95 90 hr. The kinetics of appearance resemble that of the viral DNA dCTP 79 59 polymerase, alkaline nuclease, and deoxypyrimidine kinase. dGTP 105 61 55 The relative activity of the triphosphatase for all three substrates dTTP 94 69 remains constant throughout the time period studied, which dUTP 15 14 is a further for a UTP 78 76 75 indication common enzyme performing three BrdUTP different reactions. 9 11 The results of the experiments shown in Table 1 and Fig. 3 Triphosphatase activity was assayed under standard conditions, strongly suggest that the triphosphatase is virus-coded. To except that the [3HldUTP concentration was 500MgM (0.2 Ci/mmol) further substantiate this point, a series of mutants temperature and the indicated amounts ofcompetitors were present in the reaction mixture. The amount of nucleoside monophosphate generated after sensitive for viral DNA synthesis was investigated. The results 20 min is expressed as percentage of that of the reactions in the ab- are shown in Fig. 4. Nuclei from cells infected with all mutants sence of competitors. were able to degrade deoxypyrimidine triphosphates after infection at permissive temperature; after infection at nonpermissive temperature, six out of eight mutants showed activity problematic, the Km values obtained under these conditions comparable to wild-type virus, but tsD and tsK expressed sig- reflect the high affinity between substrates and enzyme. The nificantly lower levels. These findings exclude the gene prod- apparent Km is 3 X 10-6 M for dTTP, 0.6 X 10-6 M for dCTP, ucts of tsB, tsE, tsH, tsJ, tsS, and tsU as constituents of the and <5 X 10-8 M for dUTP. triphosphatase. The fact that two mutants appear to be defec- The high affinity of dUTP for the triphosphatase is corrob- tive at nonpermissive temperature demonstrates a requirement orated by competition experiments. TableS shows that dUTP competes effectively with dCTP and dTTP at equal concentrations. dCTP and dTTP compete with each other, but only weakly with dUTP even at 50 times the substrate concentration. In addition, these findings indicate, though they do not prove, the existence of a common active site for the three deoxyribopyrimidine triphosphates. It is noteworthy that 5-BrdUTP is a good competitor for all three substrates. In general, all deoxypyrimidine triphosphates compete much better than the deoxypurine triphosphates and the ribonucleoside triphosphates. Among these, dGTP and rUTP are relatively the best competitors. Deoxypyrimidine mono- and diphosphates have only a slight inhibitory effect on triphosphatase activity. As shown in Table 2, pyrophosphate is a better inhibitor than orthophosphate, which supports the idea of the enzyme being a pyrophosphatase. To investigate this point in more detail, the reaction products arising from 3H-base-labeled and -32P-labeled substrates were analyzed by paper chromatography (Fig. 2). Most of the radioactivity migrated together with the pyro-

3 dUTP dCTP dTTP 6 PPj Pi PP1 P1 PP1 P1

Il ~~~~~~~0

3~~~~~13579~ ~ 135791f 5 E -~~~~~c frmoriginm -

of -3P-lbldad3-aelblddTdT n TPb-3 0of d nucei3 ro5 HS-l-nfcte7 9 1 1 3 5cels7 3H-able9 11 3 5doyulsidmo7 9 11 cm from origin 0 2 4 6 8 10 12 14 16 18 FIG. 2. Paper chromatogram of reaction products of degradation Time after infection at 31.50C, hr of -y-32p-labeled and 3H-base-labeled dUTP, dCTP, and dTTP by FIG. 3. Time course of appearance of triphosphatase activity after nuclei from HSV-1-infected cells. 3H-Labeled deoxynucleoside mo- infection. BHK cells were infected with HSV-1 at a multiplicity of nophosphate formed was assayed in parallel on polyethyleneimine- 10 plaque-forming units per cell and harvested at the indicated times. cellulose strips and the reactions were terminated when 95% of dUTP, Nuclei were prepared and assayed for triphosphatase activity under 95% of dCTP, and 62% of dTTP were degraded. After chromatogra- standard conditions. (A) Appearance of viral DNA polymerase, viral phy, the filter paper strip was cut into pieces and radioactivity was alkaline nuclease (alk. DNase), and deoxypyrimidine kinase (dPyK) determined by liquid scintillation counting: 0, 32p; 0, 3H. The shaded (assayed at the same times). (B, C, and D) Formation of deoxynu- areas on top of the panels give the position of the markers: PP;, py- cleoside monophosphates out of the corresponding triphosphates. (B) rophosphate; Pi, orthophosphate. TMP. (C) dCMP. (D) dUMP. Downloaded by guest on September 25, 2021 Biochemistry: Wohlrab and Francke Proc. Natl. Acad. Sci. USA 77(1980) 1875

31-50C El HSV-1-infected cells. Whether this is due to a lack of a com- 1 6rL TMP Infection at 38.50C O parable enzyme in the type 2 virus or whether the type 2 triphosphatase is not able to perform the 40C reaction remains 1 2k subject to further investigations.

8 DISCUSSION Evidence is presented for a HSV-1-specific enzymatic activity 7hF 4 in the nucleus of the infected cell. The enzyme(s) degrades deoxyribopyrirnidine nucleoside triphosphates to the corre-

r _F. I I 4 InI 9 I 1 II -1 sponding monophosphates with dUTP being by far the best 0 substrate, followed by dCTP and dTTP. The appearance of dCMP pyrophosphate in the reaction products suggests that the en- 18- zyme cleaves the a, 3-phosphate bond and that there is no nucleoside diphosphate intermediate. A tentative name for the 12- enzyme would therefore be deoxyribopyrimidine nucleoside triphosphate diphosphohydrolase. The reaction proceeds at low temperatures with considerable rates and this behaviour can be used to distinguish it from host activities. It requires divalent 6 X -9Alhh cations such as Mg2+ and Mn2+ and is strongly suppressed by metal chelators such as EDTA. L dUMP The biological significance of the activity is at the moment 18~ unknown, although the importance of a dUTPase for the pre- vention of uracil misincorporation into DNA (10) makes a 2 similar function for this enzyme in the HSV-1 system seem 4 likely. HSV-1 has been reported not to contain any unusual bases, so that a system that keeps dUTP concentrations far below the Km of the viral DNA polymerase can be postulated. -R S1 The Km value for the triphosphatase towards dUTP is extremely 6; - low (<5 X 10-8 M). This could be sufficient for such a function, Mock ts' tsB tsD tsE tsH tsJ tsK tsS tsU especially when compared to the values obtained for the soluble FIG. 4. Triphosphatase activity in cells infected with wild-type enzyme from Escherichia coli, which lie in the micromolar HSV-1 and DNA-negative temperature-sensitive mutants at per- range (10). missive (o) and nonpermissive (I) temperature. Cells infected with In isolated nuclei from HSV-1-infected cells, not only dUTP HSV-1 (wild type) or the respective mutants were harvested 15 hr after infection; nuclei were prepared and assayed for triphosphatase but also dCTP and dTTP are converted to the monophosphates activity. Activity is expressed as radioactivity in the monophosphate and no definite proof for the involvement of a single enzyme after 30 min at 40C. in all three reactions can be given. However, several findings argue against separate enzymes: the three deoxyribopyrimidine triphosphates compete with each other to a degree corre- for these viral genes for the expression of the triphosphatase. sponding to their respective Km values, and throughout infection the ratios of all three activities remain constant. Whether tsK or tsD is the structural gene for this activity remains questionable, because shifting of tsD- and tsK-infected The time course of appearance of triphosphatase activity cells from permissive to nonpermissive temperature did not after infection together with the findings obtained with viral temperature-sensitive mutants provide strong evidence for result in a drop of triphosphatase activity (data not shown). To the rule out the possibility that the observed enzymatic activity viral origin of the enzyme. Cells infected with two of the mu- an aspect of the viral deoxypyrimidine kinase/thy- tants, tsD and tsK, were defective in triphosphatase activity represents when was carried out at nonpermissive midylate kinase system, we assayed for the enzyme in HSV-1 infection temperature. TK- mutant-infected cells. As shown in Table 4, nuclei from Three interpretations of this observation are possible: (i) Viral cells infected with the TK- strain displayed triphosphatase functions represented by these two mutants induce a modified activity at rates comparable to nuclei from wild type-infected cellular triphosphatase. (ii) The tsD locus, or tsK locus, or both cells. Nuclei from cells infected with herpes simplex virus type loci are in the structural gene(s) for the triphosphatase. (iii) Because these 2 are not able to degrade deoxypyrimidine triphosphates under mutants are known to be of the early regulatory type (11, 12), the expression of a late viral gene for the the same conditions as described in this paper for nuclei from triphosphatase is prevented. In the latter case, the DNA-negative mutants tsB, tsE, tsH, tsJ, tUS, and tsU can be excluded as candidates. Table 4. Triphosphatase activity in nuclei from cells infected This includes the viral DNA polymerase (tsH) (13) TK- strain and argues against these two enzymes being identical, although with wild-type HSV-1 and a HSV-1 the tsH lesion need not necessarily affect the known pyro- Activity* with phosphorolysis reaction of DNA polymerase (14). However, different substrates soluble extracts from HSV-1-infected cells containing viral Strain dUTP dCTP dTTP DNA polymerase, viral alkaline nuclease, and deoxypyrimidine HSV-1 TK+ Cl 101 9,850 5650 3840 kinase showed no detectable triphosphatase activity (unpub- HSV-1 TK- 43 19,990 7070 4262 lished results). We have so far been unable to solubilize the activity from the infected cell nuclei, and it is unclear at this BHK cells were infected with HSV-1 TK+ or HSV-1 TK- at a or one of multiplicity of 10 plaque-forming units per cell and were harvested time whether it is an unknown tightly bound enzyme 15 hr after infection. Standard triphosphatase assays were performed. the above-mentioned enzymes in complex with a nuclear * Results are expressed as cpm in monophosphate after 30 min at 4VC. structure. Downloaded by guest on September 25, 2021 1876 Biochemistry: Wohlrab and Francke Proc. Nati. Acad. Sci. USA 77 (1980)

We thank Dr. J. H. Subak-Sharpe and Dr. W. P. Summers for gen- 7. Alberts, B., Morris, C. F., Mace, D., Sinka, N., Bittner, M. & erously providing their HSV-1 mutants, A. Waldrop for advice on Moran, L. (1975) in DNA Synthesis and Its Regulation, eds. chromatography, and B. Garrett for excellent technical assistance. This Goulian, M. & Hanawalt, P. (Benjamin, Menlo Park, CA), Vol. work was supported by Grants Al 15644 and CA 16038 from the Na- 3, pp. 241ff. tional Institutes of Health. 8. Weissbach, A., Huang, S.-C. C., Aucker, J. & Muller, R. (1973) J. Biol. Chem. 245,6270-6277. 9. Francke, B. (1977) Biochemistry 16,5664-5670. 1. Jamieson, A. T. & Bjursell, G. (1976) J. Gen. Virol. 31, 101- 10. Shlomai, J. & Kornberg, A. (1978) J. Biol. Chem. 253, 3305- 113. 3312. 2. Jamieson, A. T., Gentry, G. A. & Subak-Sharpe, J. H. (1974) J. 11. Bone, D. R., Brown, M., Crombie, I. & Francke, B. (1978) J. Virol. Gen. Virol. 24, 465-480. 28, 14-19. 3. Chen, M. S., Summers, W. P., Walker, J., Summers, W. C. & 12. Marsden, H. S., Crombie, I. K. & Subak-Sharpe, J. H. (1976) J. Prusoff, W. H. (1979) J. Virol. 30, 942-945. Gen. Virol. 31,347-372. 4. Ponce de Leon, M., Eisenberg, R. J. & Cohen, G. H. (1977). J. 13. Crombie, I. (1975) Dissertation (Univ. of Glasgow, Glasgow, Gen. Virol. 36, 163-173. Scotland). 5. Chan, T. (1977) Proc. Nati. Acad. Sci. USA 74,1734-1738. 14. Deutscher, M. P. & Kornberg, A. (1969) J. Biol. Chem. 244, 6. Francke, B. (1977) Biochemistry 16,5655-5664. 3019-028. Downloaded by guest on September 25, 2021