Characteristics of the Induction of a New Protein ... - Semantic Scholar
J. gen. Virol. (1986), 67, 1049-1057. Printed in Great Britain
1049
Key words: HS V-1/PR V/protein phosphorylation
Characteristics of the Induction of a New Protein Kinase in Cells Infected with Herpesviruses By F R A N C E S C. P U R V E S , M A T I L D A K A T A N , t W I L L I A M S. S T E V E L Y AND D A V I D P. L E A D E R * Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K. (Accepted 14 March 1986) SUMMARY
The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gelpermeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this. INTRODUCTION During the infection of cells by herpesviruses there is a change in the profile of phosphorylated proteins, encompassing both proteins absent from uninfected cells, generally virus-coded proteins (Stevely, 1975; Pereira et al., 1977; Marsden et al., 1978; Wilcox et al., 1980), and pre-existing host cell proteins (Fenwick & Walker, 1979; Kennedy et al., 1981). There is as yet no direct evidence that any of these phosphorylations is of functional importance in the life-cycle of herpesviruses, but the diversity of the regulatory systems involving protein phosphorylation would suggest that this is highly likely. The protein phosphorylations occurring during viral infection could, in principle, be catalysed by pre-existing active protein kinases of the host cell, or by protein kinases present or active only in infected cells. We have been directing our attention towards the latter category of enzyme, and have recently reported the partial purification and biochemical characteristics of a new protein kinase (virus-induced protein kinase, ViPK) isolated from the cytosol of hamster fibroblasts infected with pseudorabies virus (PRV) (Katan et al., 1985; Katan, 1985). This enzyme catalyses the transfer of phosphate from ATP (but not GTP) to the seryl residues of basic (but not acidic) proteins, and its activity is not dependent upon molecules that can serve as effectors for the well characterized cellular protein kinases. The feature of the ViPK that perhaps most strikingly distinguishes it from these latter enzymes is a KC1 optimum of approx. 0.5 i . The induction of the enzyme during viral infection, and the difference in its properties from known cellular protein kinases, raised the possibility that the ViPK might be encoded by the viral genome. As a first step towards answering this question (which may ultimately require genetic methods for its resolution), we have examined the relationship of the induction of the ViPK to the virus life cycle, comparing cells infected with PRV with those infected with herpes simplex virus type 1 (HSV-1). t On leave of absence from the Institute for Nuclear Sciences 'Boris Kidri~', Belgrade, Yugoslavia. 0000-6972 © 1986 SGM
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F. C. PURVES AND OTHERS METHODS
Materials. General laboratory chemicals were of analytical grade, where appropriate, and obtained from standard commercial suppliers unless otherwise indicated. Protamine sulphate and cycloheximide were from Sigma; DEAE-Sephacel, Sephacryl S-200 and the Mono Q column were from Pharmacia; 32p~ was from Amersham and was used to synthesize [y-32p]ATP according to Maxam & Gilbert (1980). Viruses. Pseudorabies virus was originally derived from a stock preparation (Kaplan & Vatter, 1959) and has been plaque-purified several times. Virus stocks were prepared from infected BHK monolayer cultures as previously described (Chantler & Stevely, 1973). The tsK mutant of HSV-I Glasgow strain 17 (Marsden et aL, 1976), and the parent strain, were kindly provided by Dr J. Macnab, MRC Virology Unit, Institute of Virology, University of Glasgow. Irradiation of virus. Inactivation by u.v. light was as follows. Stock virus was diluted tenfold with phosphatebuffered saline containing 1~ (w/v) glucose. Aliquots (2.5 ml) received 5 kJ/m 2 of radiation in 50 mm Petri dishes under a UVSL-58 Mineralight (Ultra-Violet Products, San Gabriel, Ca., U.S.A.). Dosage was calculated from the distance between source and sample and the time of irradiation. Cells and injection. BHK-21/C13 cells were maintained in monolayer cultures in modified Eagle's medium containing 10~ calf serum, and infected at or just before confluence with PRV at a multiplicity of approximately 20 p.f.u./cell, or HSV-1 at a multiplicity of approximately 10 p.f.u./cell, unless otherwise indicated. The times of infection were 8 h for PRV and 18 h for HSV-1, except where stated otherwise. Preparation and anion-exchange chromatography of cellular post-ribosomal supernatant. The post-ribosomal supernatant of BHK cells was prepared and subjected to chromatography on DEAE-Sephacel in a 2-5 × 1 cm column, eluting with a linear gradient of 0 to 0.4 M-KC1 exactly as previously described (Katan et al., 1985). Assay of protein kinase activity. The standard assay mixture for protein kinase activity contained, in a total volume of 0.12 ml, 20 mM-Tris-HCl p H 7.4, 50 mM-KCI, 10 mM-MgClz, 10 mM-2-mercaptoethanol, 0-1 mM-ATP containing 0.5 to l ~tCi [7-32P]ATP, and protamine sulphate (0.8 mg/ml). The protein kinase (40 ~tl)was then added and incubation was at 30 °C for 30 rain. At the end of the incubation 100 ~tl samples were spotted onto Whatman 3MM paper discs which were washed for 15 min periods twice in 20~ TCA, four times in 10~o TCA, and then rinsed in absolute ethanol, dried, and their radioactivity was measured by scintiIIation spectrometry. In some cases the products of the phosphorylation reaction were analysed by one-dimensional gel electrophoresis in the presence of SDS, performed according to Parker et al. (1985). Assay o['DNA polymerase. This was performed according to Weissbach et al. (1973). Determination of minimum concentration of cycloheximide for inhibition of protein biosynthesis. This was performed according to Leader & Barry (1971). The percentage inhibitions at 0.2 ~tg, 0-6 ~tg, 2 ~tg, 6 ~tg and 20 ~tg cycloheximide per ml were 74~, 88~o, 94~o, 95~o and 92~, respectively. RESULTS
ViPK is induced by infection with P R V or HSV-1
It is necessary at the outset to explain how we identify and quantify ViPK. In order to distinguish it from other protein kinases (e.g. protein kinase C) that can also phosphorylate protamine (the preferred artificial substrate of ViPK), we routinely subject the post-ribosomal supernatant of infected cells to anion-exchange chromatography and assay the protein kinase activity of column fractions. As previously reported (Katan et al., 1985) the ViPK from BHK cells infected with PRV elutes at a higher ionic strength (approx. 220 mM on DEAE-cellulose) than the other protamine kinases, i.e. protein kinase C (50 to 80 mM) and the so-called protein kinase M, its presumed proteolytic derivative (approx. 150 ram). The relative proportions of protein kinase C and 'protein kinase M' vary in different experiments (compare Fig. 1a and c) because the stability of protein kinase C depends on the concentration of protein applied to the column. However, we stress that viral infection does not influence the yield or relative proportion of these latter enzymes, and that we have previously excluded the possibility that ViPK might be a proteolytic cleavage product of protein kinase C (Katan et al., 1985). Fig. 1 (a, b) illustrates the induction of ViPK 8 h after infection of BHK cells with PRV, the chromatography on DEAE-Sephacel rather than DEAE-cellulose resulting in elution of the enzyme at a slightly higher ionic strength than that cited above. A new protamine kinase activity was also found in cells infected for 18 h with HSV-1, although this eluted at a somewhat lower ionic strength than the enzyme from cells infected with PRV, and hence it was incompletely resolved from the peak of 'protein kinase M'. In view of this difference (further considered below) it was necessary to determine the extent to which these enzymes had similar catalytic
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Fig. 5 Fig. 4 Fig. 4. Temporal relationship of appearance of ViPK to other events during the infection of BHK ceils with PRV. BHK cells were infected with PRV as described in Methods, and the post-ribosomal supernatants isolated at the times indicated. The major portions of these were subjected to chromatography on DEAE-Sephacel as in Fig. l, and column fractions assayed for protein kinase activity. The total ViPK activity ( 0 ) was estimated and expressed as a percentage of the maximum value, as was the D N A polymerase activity (O) assayed on a small portion of the original postribosomal supernatant. (The initial D N A polymerase activity is that of the host enzyme.) The number of p.f.u, of virus in the growth medium was also determined ( i ) . Fig. 5. Effect of multiplicity of infection with PRV ( 0 ) or HSV-1 (©) on the yield of ViPK. B H K cells were infected for 8 h with PRV or 18 h with HSV-1 at the multiplicities indicated; ViPK was isolated and quantified as described in the legend to Fig. 4. I
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1054
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I I I 60 70 80 Fraction number Fig. 8. Induction of ViPK in BHK cells infected with the tsK mutant of HSV-1. Cells were infected with tsK (10 p.f.u./cell) at either 31 °C (a) or 38.5 °C (b), or with the parent wild-type virus at 38.5 °C (c), and harvested 18 h later. The post-ribosomal supernatants were isolated, subjected to chromatography on DEAE-Sephacel using a somewhat shallower gradient than elsewhere, and fractions, the KCI concentrations of which are indicated (---), were assayed for protein kinase activity (O). The DNA polymerase activity of a portion of the post-ribosomal supernatants was also determined, confirming that there was no escape into the synthesis of early viral proteins at 38.5 °C (the restrictive temperature). experiments are consistent with there being a need for viral gene expression for the induction o f ViPK, although neither technique used in the experiments is without its limitations. In the case of HSV-1 a less equivocal a p p r o a c h was possible because of the availability of temperaturesensitive mutants. W e used tsK, in which a mutation in the 175000 mol. wt. immediate-early protein, ICP4, prevents the synthesis of m R N A for the early and late proteins (Watson & Clements, 1980). It can be seen from Fig. 8 that V i P K was induced at the permissive temperature (31 °C) but not at the restrictive temperature (38-5 °C), although the enzyme was induced by infection with the parent virus at the latter temperature. It should be noted that the amount of kinase detected in this experiment was less than in other experiments because circumstances dictated the use o f only 5 x 108 cells (rather than the 5 × 109 cells of the experiment of Fig. 1).
Different physical characteristics of ViPKs The different chromatographic properties on D E A E - S e p h a c e l of V i P K s from cells infected with each of the viruses (Fig. 1) could indicate that the two enzymes are distinct, or it might
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30 40 50 Fraction number Fig. 9. Resolution of ViPK activities by chromatography on Mono Q. Equal activities of ViPK, partially purified from BHK cells infected with PRV or HSV-1 by chromatography of post-ribosomal supernatants on DEAE-Sephacel(Fig. 1) were combined and subjectedto chromatography on Mono Q. Fractions, the NaC1 concentrations of which are indicated (---), were assayed for protein kinase activity (O). The absorbance at 280 nm ( - - ) is also shown. The inset shows the result of phosphorylation of 40S ribosomal subunits by the ViPK in peaks 1 and 2 performed as described in the legend to Fig. 3.
merely be an artefact caused by non-specific association of an identical molecule with different viral proteins. To investigate this point further, we mixed preparations of ViPK isolated by DEAE-Sephacel chromatography of cells infected with PRV or HSV-I, and subjected these to rechromatography on the high-performance Mono Q anion-exchange column. This resolved the mixture into two peaks of activity (Fig. 9), and these eluted at positions similar to the individual preparations analysed separately, ViPK from cells infected with HSV-1 again eluting at lower ionic strength (results not shown). Furthermore, the first peak of activity showed the same ability to phosphorylate both ribosomal proteins $6 and $7 at moderate ionic strength exhibited by cruder preparations from cells infected with HSV-1 (Fig. 3), whereas under the same conditions the second peak of activity only catalysed the phosphorylation of ribosomal protein $7 (see inset). Because the enzymes from the two sources showed apparently genuine differences in anionexchange chromatography, we subjected them (separately) to gel-permeation chromatography on Sephacryl S-200 (Fig. 10). The apparent relative molecular mass (approx. 200000) for ViPK from cells infected with HSV-1 was much greater than that (approx. 90000) from cells infected with PRV. [This latter value is somewhat greater than that of 68 000 obtained previously by three different methods (Katan et al., 1985), perhaps reflecting different behaviour of the enzyme on this resin.] These values, especially that for the enzyme from cells infected with HSV1, may, of course, reflect different oligomeric structures in the two cases, and the subunit size could be quite similar. However, the fact that the chromatography was conducted at 0-5 M-KC1 would argue against the differences being due to non-specific aggregation.
1056
F. C. PURVES AND OTHERS I
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15 20 Elution volume (ml) Fig. 10. Gel-permeation chromatography of ViPKs on Sephacryl S-200. Partially purified preparations (see Fig. 1) of ViPK from BHK cells infected with PRV ( 0 ) or HSV-1 (O) were separately subjected to chromatography on Sephacryl S-200 in a buffer containing 500 mM-KCI, 1 mM-EDTA, 10 mM-2mercaptoethanol, 20 mM-Tris HCI pH 7.6. The positions of elution of proteins of known relative molecular mass (MT) are indicated, and the inset shows a semilogarithmic plot of Mr x 10-~ against Ka,[(V~ - V0)/(V~ - Vo)], where V~ is the elution volume, V0 the void volume and Vt the column volume.
One trivial explanation of the different physical properties of the ViPK preparations is partial proteolytic cleavage of one of the enzymes during extraction from the cells and subsequent chromatography. However, when isolation was performed in the presence of protease inhibitors (2 mM-EGTA, 40 gg/ml phenylmethylsulphonyl fluoride, 100 gg/ml leupeptin) no difference in the position of elution from DEAE-Sephacel was observed (results not shown). Specific intracellular partial proteolysis cannot, however, be excluded (see Discussion). DISCUSSION
We have previously shown on the basis of its catalytic properties that ViPK is distinct from known cellular protein kinases (Katan et al., 1985). The present study demonstrates that the appearance of ViPK in cells infected with PRV or HSV-1 both requires and correlates with the expression of the viral genome. Thus, there can be little doubt that ViPK must play a role in the interaction of these alpha herpesviruses with their host cells. This is a new finding of considerable potential importance. The appearance in infected cells of new proteins dependent on viral gene expression does not necessarily indicate that the proteins are viral. Examples of cellular proteins in this category include stress proteins (Notarianni & Preston, 1982) and the proteins induced by interferon (Friedman et al., 1984), although these are unlikely to be relevant in this particular case. Comparison of the SDS-polyacrylamide gel mobilities of similar proteins from different strains of HSV has frequently been used to provide an indication that these are of viral origin. As we have not yet obtained homogeneous preparations of ViPK that would allow us to identify them on polyacrylamide gels, we were obliged to adopt a modification of this strategy using alpha herpesviruses that infect different species and comparing the elution of enzyme activity on different chromatographic media (Fig. 9 and 10). This approach has the limitation that interaction of the kinase with other proteins could affect its chromatographic behaviour. Nevertheless, the results of the co-chromatography on Mono Q (Fig. 9) provide a strong indication that the ViPK activities from cells infected with each of the two viruses represent
Protein kinase induced by herpesviruses
1057
different species. T h e simplest i n t e r p r e t a t i o n o f this result is t h a t the V i P K is e n c o d e d by the viral g e n o m e . F u r t h e r e x p e r i m e n t s are nevertheless r e q u i r e d before a viral origin for the e n z y m e c a n be c o n s i d e r e d absolutely proven. F o r e x a m p l e , it could be argued t h a t the two different V i P K species m i g h t arise f r o m proteolytic a c t i v a t i o n o f an i n a c t i v e cellular p r o t e i n kinase catalysed by viral proteases ( D i e r i c h et al., 1979) of different c l e a v a g e specificity. A l t h o u g h such a possibility m a y a p p e a r unlikely, it is i m p o r t a n t that we o b t a i n rigorous i m m u n o c h e m i c a l and genetic e v i d e n c e before c o n c l u d i n g t h a t V i P K is a v i r u s - e n c o d e d p r o t e i n kinase. A p a r t f r o m the protein kinases o f o n c o g e n i c retroviruses, no virat p r o t e i n k i n a s e has yet b e e n r e p o r t e d t h a t m e e t s these criteria. We acknowledge preliminary studies with HSV by Mr Charles Shearer and thank Miss Jennifer Leppard for performing the experiment of Fig. 6. We are grateful to Dr Joan Macnab and Mrs M. McNamara for invaluable assistance with viruses and cells. Part of this work was funded by a grant from the Wellcome Trust (to D.P.L.). F.C.P. was supported by an SERC postgraduate studentship. REFERENCES CHANTLER,J. K. &STEVELY,W. S. (1973). Virus-induced proteins in pseudorabies-infected cells. I. Acid-extractable proteins of the nucleus. Journal of Virology 11, 815-822. DIERICH,M. P., LANDEN,B., SCHULZ,TH. & FALKE,D. (1979). Protease activity on the surface of HSV-infectec[ cells. Journal of General Virology 45, 241 244. FENWlCK,M. L. & WALKER,M. J. (1979). Phosphorylation of a ribosomal protein and of virus-specific proteins in cells infected with herpes simplex virus. Journal of General Virology 45, 397-405. FRIEDMAN, R. L., MANLY, S. P., McMAHON, M., KERR, I. M. & START, G. R. (1984). Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 38, 745-755. KAPLAN,A. S. & VATTER,A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394407. KATAN, M. (1985). A protein kinase induced by pseudorabies virus in infected cells. Ph.D. thesis, University of Glasgow. KATAN, M., STEVELY, W. S. & LEADER, D. P. (1985). Partial purification a n d c h a r a c t e r i s a t i o n of a n e w p h o s p h o p r o t e i n
kinase from ceils infected with pseudorabies virus. European Journal of Biochemistry 152, 57-65. KENNEDY, I. M., STEVELY, W. S. & LEADER, D. P. (1981). Phosphorylation of ribosomal proteins in hamster fibroblasts
infected with pseudorabies virus or herpes simplex virus. Journal of Virology 39, 359-366. LEADER,D. P. & BARRY,J. M. (1971). Increase in activity of glucose 6-phosphate dehydrogenase in mouse mammary tissue cultured with insulin. Biochemical Journal 113, 175-182. MARSDEN, H. S., CROMBIE, I. K. & SUBAK-SHARPE, J. H. (1976). Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17. Journal of General Virology 31, 347-372. MARSDEN, H. S., STOW, N. D., PRESTON, V. G., TIMBURY, M. C. & WlLKIE, N. M. (1978). P h y s i c a l m a p p i n g o f h e r p e s
simplex virus-induced polypeptides. Journal of Virology 28, 624-642. MAXAM,A. & GILBERT,W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65, 499-560. NOTARIANNI,E. L. & PRESTON,C. M. (1982). Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides. Virology 123, 113 122. PARKER, P. J., KATAN, M., WATERFIELD, M. D. & LEADER, D. P. (1985). T h e p h o s p h o r y l a t i o n of e u k a r y o t i c r i b o s o m a l
protein $6 by protein kinase C. European Journal of Biochemistry 148, 579-586. PEREIRA, L. M., WOLFF, H., FENWlCK, M. L. & ROIZMAN, B. (1977). R e g u l a t i o n of h e r p e s v i r u s m a c r o m o l e c u l a r
synthesis. V. Properties of polypeptides made in HSV-I and HSV-2 infected cells. Virology 77, 733-749. PERERA, P. A. J. (1970). Induction of enzymes in mammalian cells infected with herpes simplex virus. Ph.D. thesis, University of Glasgow. STEVELY, W. S. (1975). Virus-induced proteins in pseudorabies-infected cells. II. Proteins of the virion and nucleocapsid. Journal of Virology 16, 944-950. STEVELY, W. S., KATAN, M., STIRLING, V., SMITH, G. & LEADER, D. P. (1985). P r o t e i n k i n a s e a c t i v i t i e s a s s o c i a t e d w i t h
the virions of pseudorabies and herpes simplex virus. Journal of General Virology 66, 661-673. WATSON,R. J. & CLEMENTS,J. B. (1980). A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature, London 285, 329 330. WEISSBACH, A., HONG, S-C.L., AUCKER, J. & MULLER, R. (1973). C h a r a c t e r i z a t i o n of h e r p e s s i m p l e x v i r u s - i n d u c e d
deoxyribonucleic acid polymerase. Journal of Biological Chemistry 248, 6270-6277. WILCOX, K. W., KOHN, A., SKLYANSKAYA, E. & ROIZMAN, B. (1980). H e r p e s s i m p l e x virus p h o s p h o p r o t e i n s . I.
Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. Journal of Virology 33, 167-182.
(Received 26 November 1985)
1049
Key words: HS V-1/PR V/protein phosphorylation
Characteristics of the Induction of a New Protein Kinase in Cells Infected with Herpesviruses By F R A N C E S C. P U R V E S , M A T I L D A K A T A N , t W I L L I A M S. S T E V E L Y AND D A V I D P. L E A D E R * Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K. (Accepted 14 March 1986) SUMMARY
The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gelpermeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this. INTRODUCTION During the infection of cells by herpesviruses there is a change in the profile of phosphorylated proteins, encompassing both proteins absent from uninfected cells, generally virus-coded proteins (Stevely, 1975; Pereira et al., 1977; Marsden et al., 1978; Wilcox et al., 1980), and pre-existing host cell proteins (Fenwick & Walker, 1979; Kennedy et al., 1981). There is as yet no direct evidence that any of these phosphorylations is of functional importance in the life-cycle of herpesviruses, but the diversity of the regulatory systems involving protein phosphorylation would suggest that this is highly likely. The protein phosphorylations occurring during viral infection could, in principle, be catalysed by pre-existing active protein kinases of the host cell, or by protein kinases present or active only in infected cells. We have been directing our attention towards the latter category of enzyme, and have recently reported the partial purification and biochemical characteristics of a new protein kinase (virus-induced protein kinase, ViPK) isolated from the cytosol of hamster fibroblasts infected with pseudorabies virus (PRV) (Katan et al., 1985; Katan, 1985). This enzyme catalyses the transfer of phosphate from ATP (but not GTP) to the seryl residues of basic (but not acidic) proteins, and its activity is not dependent upon molecules that can serve as effectors for the well characterized cellular protein kinases. The feature of the ViPK that perhaps most strikingly distinguishes it from these latter enzymes is a KC1 optimum of approx. 0.5 i . The induction of the enzyme during viral infection, and the difference in its properties from known cellular protein kinases, raised the possibility that the ViPK might be encoded by the viral genome. As a first step towards answering this question (which may ultimately require genetic methods for its resolution), we have examined the relationship of the induction of the ViPK to the virus life cycle, comparing cells infected with PRV with those infected with herpes simplex virus type 1 (HSV-1). t On leave of absence from the Institute for Nuclear Sciences 'Boris Kidri~', Belgrade, Yugoslavia. 0000-6972 © 1986 SGM
1050
F. C. PURVES AND OTHERS METHODS
Materials. General laboratory chemicals were of analytical grade, where appropriate, and obtained from standard commercial suppliers unless otherwise indicated. Protamine sulphate and cycloheximide were from Sigma; DEAE-Sephacel, Sephacryl S-200 and the Mono Q column were from Pharmacia; 32p~ was from Amersham and was used to synthesize [y-32p]ATP according to Maxam & Gilbert (1980). Viruses. Pseudorabies virus was originally derived from a stock preparation (Kaplan & Vatter, 1959) and has been plaque-purified several times. Virus stocks were prepared from infected BHK monolayer cultures as previously described (Chantler & Stevely, 1973). The tsK mutant of HSV-I Glasgow strain 17 (Marsden et aL, 1976), and the parent strain, were kindly provided by Dr J. Macnab, MRC Virology Unit, Institute of Virology, University of Glasgow. Irradiation of virus. Inactivation by u.v. light was as follows. Stock virus was diluted tenfold with phosphatebuffered saline containing 1~ (w/v) glucose. Aliquots (2.5 ml) received 5 kJ/m 2 of radiation in 50 mm Petri dishes under a UVSL-58 Mineralight (Ultra-Violet Products, San Gabriel, Ca., U.S.A.). Dosage was calculated from the distance between source and sample and the time of irradiation. Cells and injection. BHK-21/C13 cells were maintained in monolayer cultures in modified Eagle's medium containing 10~ calf serum, and infected at or just before confluence with PRV at a multiplicity of approximately 20 p.f.u./cell, or HSV-1 at a multiplicity of approximately 10 p.f.u./cell, unless otherwise indicated. The times of infection were 8 h for PRV and 18 h for HSV-1, except where stated otherwise. Preparation and anion-exchange chromatography of cellular post-ribosomal supernatant. The post-ribosomal supernatant of BHK cells was prepared and subjected to chromatography on DEAE-Sephacel in a 2-5 × 1 cm column, eluting with a linear gradient of 0 to 0.4 M-KC1 exactly as previously described (Katan et al., 1985). Assay of protein kinase activity. The standard assay mixture for protein kinase activity contained, in a total volume of 0.12 ml, 20 mM-Tris-HCl p H 7.4, 50 mM-KCI, 10 mM-MgClz, 10 mM-2-mercaptoethanol, 0-1 mM-ATP containing 0.5 to l ~tCi [7-32P]ATP, and protamine sulphate (0.8 mg/ml). The protein kinase (40 ~tl)was then added and incubation was at 30 °C for 30 rain. At the end of the incubation 100 ~tl samples were spotted onto Whatman 3MM paper discs which were washed for 15 min periods twice in 20~ TCA, four times in 10~o TCA, and then rinsed in absolute ethanol, dried, and their radioactivity was measured by scintiIIation spectrometry. In some cases the products of the phosphorylation reaction were analysed by one-dimensional gel electrophoresis in the presence of SDS, performed according to Parker et al. (1985). Assay o['DNA polymerase. This was performed according to Weissbach et al. (1973). Determination of minimum concentration of cycloheximide for inhibition of protein biosynthesis. This was performed according to Leader & Barry (1971). The percentage inhibitions at 0.2 ~tg, 0-6 ~tg, 2 ~tg, 6 ~tg and 20 ~tg cycloheximide per ml were 74~, 88~o, 94~o, 95~o and 92~, respectively. RESULTS
ViPK is induced by infection with P R V or HSV-1
It is necessary at the outset to explain how we identify and quantify ViPK. In order to distinguish it from other protein kinases (e.g. protein kinase C) that can also phosphorylate protamine (the preferred artificial substrate of ViPK), we routinely subject the post-ribosomal supernatant of infected cells to anion-exchange chromatography and assay the protein kinase activity of column fractions. As previously reported (Katan et al., 1985) the ViPK from BHK cells infected with PRV elutes at a higher ionic strength (approx. 220 mM on DEAE-cellulose) than the other protamine kinases, i.e. protein kinase C (50 to 80 mM) and the so-called protein kinase M, its presumed proteolytic derivative (approx. 150 ram). The relative proportions of protein kinase C and 'protein kinase M' vary in different experiments (compare Fig. 1a and c) because the stability of protein kinase C depends on the concentration of protein applied to the column. However, we stress that viral infection does not influence the yield or relative proportion of these latter enzymes, and that we have previously excluded the possibility that ViPK might be a proteolytic cleavage product of protein kinase C (Katan et al., 1985). Fig. 1 (a, b) illustrates the induction of ViPK 8 h after infection of BHK cells with PRV, the chromatography on DEAE-Sephacel rather than DEAE-cellulose resulting in elution of the enzyme at a slightly higher ionic strength than that cited above. A new protamine kinase activity was also found in cells infected for 18 h with HSV-1, although this eluted at a somewhat lower ionic strength than the enzyme from cells infected with PRV, and hence it was incompletely resolved from the peak of 'protein kinase M'. In view of this difference (further considered below) it was necessary to determine the extent to which these enzymes had similar catalytic
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Fig. 5 Fig. 4 Fig. 4. Temporal relationship of appearance of ViPK to other events during the infection of BHK ceils with PRV. BHK cells were infected with PRV as described in Methods, and the post-ribosomal supernatants isolated at the times indicated. The major portions of these were subjected to chromatography on DEAE-Sephacel as in Fig. l, and column fractions assayed for protein kinase activity. The total ViPK activity ( 0 ) was estimated and expressed as a percentage of the maximum value, as was the D N A polymerase activity (O) assayed on a small portion of the original postribosomal supernatant. (The initial D N A polymerase activity is that of the host enzyme.) The number of p.f.u, of virus in the growth medium was also determined ( i ) . Fig. 5. Effect of multiplicity of infection with PRV ( 0 ) or HSV-1 (©) on the yield of ViPK. B H K cells were infected for 8 h with PRV or 18 h with HSV-1 at the multiplicities indicated; ViPK was isolated and quantified as described in the legend to Fig. 4. I
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I I I 60 70 80 Fraction number Fig. 8. Induction of ViPK in BHK cells infected with the tsK mutant of HSV-1. Cells were infected with tsK (10 p.f.u./cell) at either 31 °C (a) or 38.5 °C (b), or with the parent wild-type virus at 38.5 °C (c), and harvested 18 h later. The post-ribosomal supernatants were isolated, subjected to chromatography on DEAE-Sephacel using a somewhat shallower gradient than elsewhere, and fractions, the KCI concentrations of which are indicated (---), were assayed for protein kinase activity (O). The DNA polymerase activity of a portion of the post-ribosomal supernatants was also determined, confirming that there was no escape into the synthesis of early viral proteins at 38.5 °C (the restrictive temperature). experiments are consistent with there being a need for viral gene expression for the induction o f ViPK, although neither technique used in the experiments is without its limitations. In the case of HSV-1 a less equivocal a p p r o a c h was possible because of the availability of temperaturesensitive mutants. W e used tsK, in which a mutation in the 175000 mol. wt. immediate-early protein, ICP4, prevents the synthesis of m R N A for the early and late proteins (Watson & Clements, 1980). It can be seen from Fig. 8 that V i P K was induced at the permissive temperature (31 °C) but not at the restrictive temperature (38-5 °C), although the enzyme was induced by infection with the parent virus at the latter temperature. It should be noted that the amount of kinase detected in this experiment was less than in other experiments because circumstances dictated the use o f only 5 x 108 cells (rather than the 5 × 109 cells of the experiment of Fig. 1).
Different physical characteristics of ViPKs The different chromatographic properties on D E A E - S e p h a c e l of V i P K s from cells infected with each of the viruses (Fig. 1) could indicate that the two enzymes are distinct, or it might
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merely be an artefact caused by non-specific association of an identical molecule with different viral proteins. To investigate this point further, we mixed preparations of ViPK isolated by DEAE-Sephacel chromatography of cells infected with PRV or HSV-I, and subjected these to rechromatography on the high-performance Mono Q anion-exchange column. This resolved the mixture into two peaks of activity (Fig. 9), and these eluted at positions similar to the individual preparations analysed separately, ViPK from cells infected with HSV-1 again eluting at lower ionic strength (results not shown). Furthermore, the first peak of activity showed the same ability to phosphorylate both ribosomal proteins $6 and $7 at moderate ionic strength exhibited by cruder preparations from cells infected with HSV-1 (Fig. 3), whereas under the same conditions the second peak of activity only catalysed the phosphorylation of ribosomal protein $7 (see inset). Because the enzymes from the two sources showed apparently genuine differences in anionexchange chromatography, we subjected them (separately) to gel-permeation chromatography on Sephacryl S-200 (Fig. 10). The apparent relative molecular mass (approx. 200000) for ViPK from cells infected with HSV-1 was much greater than that (approx. 90000) from cells infected with PRV. [This latter value is somewhat greater than that of 68 000 obtained previously by three different methods (Katan et al., 1985), perhaps reflecting different behaviour of the enzyme on this resin.] These values, especially that for the enzyme from cells infected with HSV1, may, of course, reflect different oligomeric structures in the two cases, and the subunit size could be quite similar. However, the fact that the chromatography was conducted at 0-5 M-KC1 would argue against the differences being due to non-specific aggregation.
1056
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15 20 Elution volume (ml) Fig. 10. Gel-permeation chromatography of ViPKs on Sephacryl S-200. Partially purified preparations (see Fig. 1) of ViPK from BHK cells infected with PRV ( 0 ) or HSV-1 (O) were separately subjected to chromatography on Sephacryl S-200 in a buffer containing 500 mM-KCI, 1 mM-EDTA, 10 mM-2mercaptoethanol, 20 mM-Tris HCI pH 7.6. The positions of elution of proteins of known relative molecular mass (MT) are indicated, and the inset shows a semilogarithmic plot of Mr x 10-~ against Ka,[(V~ - V0)/(V~ - Vo)], where V~ is the elution volume, V0 the void volume and Vt the column volume.
One trivial explanation of the different physical properties of the ViPK preparations is partial proteolytic cleavage of one of the enzymes during extraction from the cells and subsequent chromatography. However, when isolation was performed in the presence of protease inhibitors (2 mM-EGTA, 40 gg/ml phenylmethylsulphonyl fluoride, 100 gg/ml leupeptin) no difference in the position of elution from DEAE-Sephacel was observed (results not shown). Specific intracellular partial proteolysis cannot, however, be excluded (see Discussion). DISCUSSION
We have previously shown on the basis of its catalytic properties that ViPK is distinct from known cellular protein kinases (Katan et al., 1985). The present study demonstrates that the appearance of ViPK in cells infected with PRV or HSV-1 both requires and correlates with the expression of the viral genome. Thus, there can be little doubt that ViPK must play a role in the interaction of these alpha herpesviruses with their host cells. This is a new finding of considerable potential importance. The appearance in infected cells of new proteins dependent on viral gene expression does not necessarily indicate that the proteins are viral. Examples of cellular proteins in this category include stress proteins (Notarianni & Preston, 1982) and the proteins induced by interferon (Friedman et al., 1984), although these are unlikely to be relevant in this particular case. Comparison of the SDS-polyacrylamide gel mobilities of similar proteins from different strains of HSV has frequently been used to provide an indication that these are of viral origin. As we have not yet obtained homogeneous preparations of ViPK that would allow us to identify them on polyacrylamide gels, we were obliged to adopt a modification of this strategy using alpha herpesviruses that infect different species and comparing the elution of enzyme activity on different chromatographic media (Fig. 9 and 10). This approach has the limitation that interaction of the kinase with other proteins could affect its chromatographic behaviour. Nevertheless, the results of the co-chromatography on Mono Q (Fig. 9) provide a strong indication that the ViPK activities from cells infected with each of the two viruses represent
Protein kinase induced by herpesviruses
1057
different species. T h e simplest i n t e r p r e t a t i o n o f this result is t h a t the V i P K is e n c o d e d by the viral g e n o m e . F u r t h e r e x p e r i m e n t s are nevertheless r e q u i r e d before a viral origin for the e n z y m e c a n be c o n s i d e r e d absolutely proven. F o r e x a m p l e , it could be argued t h a t the two different V i P K species m i g h t arise f r o m proteolytic a c t i v a t i o n o f an i n a c t i v e cellular p r o t e i n kinase catalysed by viral proteases ( D i e r i c h et al., 1979) of different c l e a v a g e specificity. A l t h o u g h such a possibility m a y a p p e a r unlikely, it is i m p o r t a n t that we o b t a i n rigorous i m m u n o c h e m i c a l and genetic e v i d e n c e before c o n c l u d i n g t h a t V i P K is a v i r u s - e n c o d e d p r o t e i n kinase. A p a r t f r o m the protein kinases o f o n c o g e n i c retroviruses, no virat p r o t e i n k i n a s e has yet b e e n r e p o r t e d t h a t m e e t s these criteria. We acknowledge preliminary studies with HSV by Mr Charles Shearer and thank Miss Jennifer Leppard for performing the experiment of Fig. 6. We are grateful to Dr Joan Macnab and Mrs M. McNamara for invaluable assistance with viruses and cells. Part of this work was funded by a grant from the Wellcome Trust (to D.P.L.). F.C.P. was supported by an SERC postgraduate studentship. REFERENCES CHANTLER,J. K. &STEVELY,W. S. (1973). Virus-induced proteins in pseudorabies-infected cells. I. Acid-extractable proteins of the nucleus. Journal of Virology 11, 815-822. DIERICH,M. P., LANDEN,B., SCHULZ,TH. & FALKE,D. (1979). Protease activity on the surface of HSV-infectec[ cells. Journal of General Virology 45, 241 244. FENWlCK,M. L. & WALKER,M. J. (1979). Phosphorylation of a ribosomal protein and of virus-specific proteins in cells infected with herpes simplex virus. Journal of General Virology 45, 397-405. FRIEDMAN, R. L., MANLY, S. P., McMAHON, M., KERR, I. M. & START, G. R. (1984). Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 38, 745-755. KAPLAN,A. S. & VATTER,A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394407. KATAN, M. (1985). A protein kinase induced by pseudorabies virus in infected cells. Ph.D. thesis, University of Glasgow. KATAN, M., STEVELY, W. S. & LEADER, D. P. (1985). Partial purification a n d c h a r a c t e r i s a t i o n of a n e w p h o s p h o p r o t e i n
kinase from ceils infected with pseudorabies virus. European Journal of Biochemistry 152, 57-65. KENNEDY, I. M., STEVELY, W. S. & LEADER, D. P. (1981). Phosphorylation of ribosomal proteins in hamster fibroblasts
infected with pseudorabies virus or herpes simplex virus. Journal of Virology 39, 359-366. LEADER,D. P. & BARRY,J. M. (1971). Increase in activity of glucose 6-phosphate dehydrogenase in mouse mammary tissue cultured with insulin. Biochemical Journal 113, 175-182. MARSDEN, H. S., CROMBIE, I. K. & SUBAK-SHARPE, J. H. (1976). Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17. Journal of General Virology 31, 347-372. MARSDEN, H. S., STOW, N. D., PRESTON, V. G., TIMBURY, M. C. & WlLKIE, N. M. (1978). P h y s i c a l m a p p i n g o f h e r p e s
simplex virus-induced polypeptides. Journal of Virology 28, 624-642. MAXAM,A. & GILBERT,W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65, 499-560. NOTARIANNI,E. L. & PRESTON,C. M. (1982). Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides. Virology 123, 113 122. PARKER, P. J., KATAN, M., WATERFIELD, M. D. & LEADER, D. P. (1985). T h e p h o s p h o r y l a t i o n of e u k a r y o t i c r i b o s o m a l
protein $6 by protein kinase C. European Journal of Biochemistry 148, 579-586. PEREIRA, L. M., WOLFF, H., FENWlCK, M. L. & ROIZMAN, B. (1977). R e g u l a t i o n of h e r p e s v i r u s m a c r o m o l e c u l a r
synthesis. V. Properties of polypeptides made in HSV-I and HSV-2 infected cells. Virology 77, 733-749. PERERA, P. A. J. (1970). Induction of enzymes in mammalian cells infected with herpes simplex virus. Ph.D. thesis, University of Glasgow. STEVELY, W. S. (1975). Virus-induced proteins in pseudorabies-infected cells. II. Proteins of the virion and nucleocapsid. Journal of Virology 16, 944-950. STEVELY, W. S., KATAN, M., STIRLING, V., SMITH, G. & LEADER, D. P. (1985). P r o t e i n k i n a s e a c t i v i t i e s a s s o c i a t e d w i t h
the virions of pseudorabies and herpes simplex virus. Journal of General Virology 66, 661-673. WATSON,R. J. & CLEMENTS,J. B. (1980). A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature, London 285, 329 330. WEISSBACH, A., HONG, S-C.L., AUCKER, J. & MULLER, R. (1973). C h a r a c t e r i z a t i o n of h e r p e s s i m p l e x v i r u s - i n d u c e d
deoxyribonucleic acid polymerase. Journal of Biological Chemistry 248, 6270-6277. WILCOX, K. W., KOHN, A., SKLYANSKAYA, E. & ROIZMAN, B. (1980). H e r p e s s i m p l e x virus p h o s p h o p r o t e i n s . I.
Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. Journal of Virology 33, 167-182.
(Received 26 November 1985)
