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Identification of a protein kinase activity associated with human adenoviruses
VIROLOGY
86,
157-166
Identification
(1978)
of a Protein
Kinase Activity Adenoviruses
G. E. BLAIR’ Division
of Virology,
National
Institute
for
Medical
with Human
W. C. RUSSELL
AND
Accepted
Associated
Research,
December
Mill
Hill,
London
NW7
IAA,
England
3, 1977
The identification of a cyclic nucleotide-independent protein kinase in purified adenovirus types 2 and 5 is described. Enzyme activity was detected only in virus preparations which had been mildly disrupted by dialysis against low ionic strength buffers and not in intact virus preparations. Virus polypeptides IIIa, V. VI, VII, and X were phosphorylated in vitro. The enzyme transferred the y-phosphate of ATP to serine and threonine residues of virus proteins. Other proteins (histones, casein, and protamine sulphate) were phosphorylated by the virion protein kinase.
priate to study the protein kinase enzymes which might be involved in these events. During the course of this work, we used the sensitive technique of SDS gel electrophoresis and autoradiography and were able to detect a protein kinase enzyme in highly purified preparations of adenovirus types 2 and 5. This enzyme is capable of phosphorylating both exogenous protein substrates and adenovirus structural proteins. In the latter case, a pattern of in vitro polypeptide phosphorylation resembling that observed in vivo was obtained. The adenovirus protein kinase may represent the first instance of a kinase enzyme tightly bound to a nonenveloped virus.
INTRODUCTION
Protein kinase enzymes have been detected in many enveloped viruses (reviewed in Rubin and Rosen, 1975). Nonenveloped viruses such as poliovirus and adenovirus (Strand and August, 1971) and SV40 (Tan and Sokol, 1972) have been reported to contain no protein kinase activity in their virions, on the basis of undetectable incorporation of the labelled y-phosphate of ATP into trichloroacetic acid (TCA)-insoluble material. The question of the host cell or virus origin of particle-bound protein kinases has proved difficult to resolve. In frog virus 3, the enzyme has been purified and shown to be a structural protein of the virus (Silberstein and August, 1976a,b). In contrast, Imblum and Wagner (1974) found evidence to suggest that the protein kinase of vesicular stomatitis virus may originate from the host cell. The true function of virion protein kinases in infection remains a matter for speculation. Our previous work has identified phosphorylated derivatives of both structural and nonstructural adenovirus proteins (Russell et al., 1972; Russell and Blair, 1977). Specific phosphorylation of a host cell ribosomal protein was also detected (Blair and Horak, 1977). It seemed appro’ To dressed.
whom
requests
for
reprints
should
MATERIALS
AND
METHODS
cells. Adenovirus types 2 and 5 were propagated in KB cells growing in suspension culture. Cells were maintained in minimal essential medium (Joklik modified) supplemented with 5% calf serum. Virus seeds were prepared and titrated as previously described (Russell et al., 1967). Cells were infected with virus seed at a multiplicity of lo-20 PFU/cell and incubated at 37O for 48 hr. Virus purification. Virus was purified from infected cells by extraction with fluorocarbon followed by one velocity and two equilibrium centrifugations in caesium chloride gradients. Purified virus was either Virus
be ad-
and
157 0042~6822/78/0861-0157$02.00/O Copyright 0 1978 by Academic Press, Im. All rights of reproduction in any form reserved.
158
BLAIR
AND RUSSELL
dialysed against 500 vol of 10 m&f Tris-maleate (pH 6.4), 10% glycerol for 15 hr at 4”, or rapidly desalted by gel chromatography on a column (1 x 15 cm) of coarse Sephadex G-25, equilibrated, and eluted with 10 mM Tris-maleate (pH 6.4), 10% glycerol. “Desalted virus” was assayed immediately for protein kinase activity, whereas “dialysed virus” was stored at -70” for several months without loss of activity. Protein kinase assay. Protein kinase activity was assayed by mixing aliquots of virus (5-20 pg) with 5 @i of [Y-~‘P]ATP in a buffer containing 20 mM Tris-maleate (pH 6.4), 5 r&f MgC12, 6 mM 2-mercaptoethanol, 5% glycerol. The total volume was 25 ~1. Incubation was at 37” for 30 min. Reactions were terminated by the addition of SDS denaturing mix (15 mM Tris-HCl, pH 6.8, 8 M urea, 1% SDS, 1% 2-mercaptoethanol), heated at 100” for 2 min and then analysed by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis. Analysis of phosphorylated polypeptides was performed on discontinuous slab gels as previously described (Russell and Blair, 1977) containing either 15 or 25% acrylamide or a linear gradient of 10 to 25% acrylamide in the separating gel. Analysis ofphosphothreonine andphosphoserine. In experiments where the in vitro product was hydrolysed in acid, the [y32P]ATP was increased to 20 @i per reaction. After incubation, three 25-~1 reaction mixtures were pooled and dialysed against 2 liters of distilled water for 18 hr. The dialysed sample was lyophilized and sealed under vacuum with either 2 or 6 M HCI. Hydrolysis was performed at 105” for 6 hr. The hydrolysate was lyophilized, resuspended in distilled water, lyophilized again, and resuspended in 10 ~1 of 1 mJ4 HCl containing 1 mg/ml of standard phosphoserine and phosphothreonine. The sample was applied to Whatman 3MM paper and high-voltage electrophoresis was performed in a pH 1.9 buffer (containing 2.5% formic acid, 7.8% acetic acid) at 3 kV for 3 hr. Marker phosphoamino acids were located by cadmium-ninhydrin staining. The paper
was exposed to X-ray film for 7 days. Virus concentration. The concentration of virus was determined spectrophotometrically (Maize1 et al., 1968). Aliquots of virus were suspended in 0.1 x SSC, 0.5% SDS. The concentration was calculated assuming that 1 ODpso unit is equivalent to 0.28 mg of virus protein, and that adenovirus type 5 is composed of 87.7% protein, 12.3% DNA. This procedure was verified using the method of Lowry et al. (1951). Nomenclature. The nomenclature proposed by Ginsberg et al. (1966) for the major capsid components (hexon, penton base, and fibre) is used. The remainder of the structural polypeptides are referred to according to the terminology proposed by Maize1 et al. (1968) and Anderson et al. (1973), as discussed in relation to adenovirus type 5 by Russell and Blair (1977). RESULTS
Identification and Characteristics of the Protein Kinase Activity Initial experiments involving incubation of aliquots of virus with [Y-~~P]ATP, followed by precipitation with TCA and scintillation counting of samples, failed to reveal any protein kinase activity in the virus preparations. This confirmed a previous report (Strand and August, 1971). However, before concluding that no protein kinase enzyme was present in adenovirus particles, we felt it appropriate to investigate methods of disruption of the virus structure and to apply the more sensitive technique of discontinuous SDS gel electrophoresis and autoradiography to the detection of phosphorylated products of the protein kinase reaction. Previous investigations had shown that adenovirus particles could be disrupted under mild conditions by dialysis against either distilled water (Laver et al., 1967) or low ionic strength buffers (Prage et al., 1970), for example, 10 mJ4 Tris-maleate, pH 6.4. This treatment caused pentons and fibres to dissociate from the particles, leaving spaces at the vertices which could be observed by electron microscopy. Such disruption also renders the DNA of the adenovirus “core” accessible to degradation by micrococcal nuclease (Corden et al., 1976),
ADENOVIRUS
PROTEIN
indicating that protein molecules of low molecular weight (16,800 for micrococcal nucleate; Taniuchi et al., 1967) can pass within the capsid. Preparations of “dialysed virus” and “desalted virus” (see Materials and Methods) were assayed in vitro for protein kinase activity by incubation with [y-32P]ATP, followed by electrophoresis on SDS-polyacrylamide gels. Both preparations were also examined under an electron microscope after negative staining. Almost all particles in the “dialysed virus” preparation were disrupted (that is, had holes at the vertices of the particle) while the “desalted
KINASE
159
virus” showed good integrity of virus capsids. The phosphorylation of virus polypeptides in vitro in each virus preparation was examined over a concentration range (Fig. 1). Phosphorylation of a discrete number of polypeptides was observed only in the dialysed virus preparation, at a concentration as low as 12 pg/assay (Fig. 1). The degree of phosphorylation was then approximately proportional to virus concentration. However no phosphorylation of virus polypeptides was observed in the preparation of desalted virus even at a concentration (12 pg, Fig. 1) where labelling was clearly evident in the dialysed virus preparation. Be-
FIG. 1. Identification of protein kinase activity in adenovirus type 5. “Dialysed” virus and “desalted” virus (referred to as disrupted and intact, respectively) were prepared as described under Materials and Methods. Increasing amounts of disrupted (12-84 pg) and intact (2-12 pg) virus were assayed for protein kinase activity and analysed on a 15% acrylamide gel. (“‘S IC) An aliquot of adenovirus-infected HeLa cells labelled with [?S]methionine late in infection.
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AND RUSSELL
cause of the approximate lo-fold dilution incurred in preparing desalted virus by gel filtration, comparison over a similar concentration range proved impossible. Mild disruption of the virus structure was therefore necessary for detection of the protein kinase suggesting that the enzyme or substrate is located inside the capsid. More vigorous conditions of disruption, such as treatment with sodium dodecyl sulphate or heating in the presence of deoxycholate to form adenovirus cores (Russell et al., 1971), resulted in loss of protein kinase activity. The tight association of the protein kinase with virus was demonstrated by fractionating the second equilibrium density caesium chloride gradient (see Materials and Methods), dialysing each fraction against 10 mM Tris-maleate (pH 6.4), 10% glycerol, and assaying each fraction for protein kinase activity. Only fractions containing adenovirus were found to have protein kinase activity when casein was used as substrate (Fig. 2). The same results were obtained when “endogenous” activity was assayed and when calf thymus histones were used as substrate. The identity of the phosphorylated polypeptides was established by comparison with an extract of adenovirus-infected HeLa cells, pulse labelled with [35S]methionine late in infection (Fig. 1). Virus polypeptides IIIa, V, VI, and VII were phosphorylated in vitro. The major capsid proteins, hexon, penton base, and fibre, were not phosphorylated in uitro. Labelled species which did not comigrate with virus polypeptides were rarely observed. Alterations in the stoichiometry of virus polypeptide phosphorylation were sometimes observed. In many experiments, some labelled material migrated with the buffer front on 15% gels. In order to resolve this fraction, dialysed virus phosphorylated in vitro was analysed on linear lo-25% gradient SDS gels. Dialysed virus of adenovirus type 2 was also assayed for protein kinase activity and analysed in parallel (Fig. 3). The results of the gradient gel analysis confirm those obtained with 15% gels (Fig. 1) and also indicate that polypeptide X is phosphorylated in vitro. Adenovirus type 2 also possesses a virion protein kinase which shows similar
specificity to the type 5 enzyme for virus polypeptides, that is, polypeptides IIIa, V, VI, VII, and X were phosphorylated (Fig. 3). However, smaller amounts of phosphorylated polypeptide X were found in type 2. In addition, phosphorylated species corresponding in mobility to polypeptides IVai and IVa2 were detected in type 2 but not in type 5. It may also be noted that the mobilities of the virus structural polypeptides of types 2 and 5 are essentially similar in these experiments (shown in the stained gels of Fig. 3), the major exception being the hexon polypeptide which migrates more slowly in type 2 than in type 5. The requirement for monovalent and divalent cations and for cyclic nucleotide cofactors was examined. Although MgCh did not stimulate the kinase reaction, inhibition was obtained on addition of 5 mM EDTA. Divalent cations previously bound to virus may therefore be sufficient for observed activity. Addition of NaCl or KC1 was not required for optimal kinase activity. Neither CAMP nor cGMP, tested over a concentration range of low4 to low5 M stimulated in vitro phosphorylation. The specificity of phosphorylation of virus polypeptides was not altered by addition of cyclic nucleotides or variation in the ionic conditions. Nature of the Phosphoprotein Bond When in vitro reactions were terminated without incubation, no labelled phosphate was incorporated into virion polypeptides suggesting that the [y-32P]ATP was not simply bound to protein under the conditions of incubation and analysis. Definitive evidence for transfer and covalent bonding of the y-phosphate group of ATP to protein was provided by acid hydrolysis of the in vitro products. After phosphorylation in vitro, the whole virus preparation was dialysed and hydrolysed in acid (see Materials and Methods). Labelled phosphoamino acids were separated by high-voltage paper electrophoresis and located by autoradiography (Fig. 4). In parallel, a control reaction was performed using bovine heart muscle protein kinase (assayed in the presence of 10 fl CAMP) and adenovirus type 5, previously heated at 60’ for 15 min. (This
FIG. 2. Copurification of protein kinase activity with adenovirus. After purification by one velocity and one equilibrium caesium chloride centrifugation, purified adenovirus was centrifuged on a second (4.5 ml) gradient to equilibrium (see Materials and Methods). Fractions (0.5 ml) were collected and dialysed against 500 vol of 10 mM Tris-maleate (pH 6.4), 10% glycerol for 18 hr at 4”, and 15-/d aliquots of each fraction were assayed for protein kinase activity using 10 pg of casein as substrate. The in vitro products were analysed on a 15% gel. The upper panel shows the gradient profile. CM) EZM; (A-----A) density (grams per cubic centimeter). The products of in vitro phosphorylation of each fraction are displayed immediately below, so that fractions from the upper panel correspond to the slots on the gel in the lower panel. V, The products of phosphorylation of casein by 5 ~1 of purified adenovirus; PK, the products of phosphorylation of casein by 2 1.18 of bovine heart muscle protein kinase. The additional bands in this slot are due to endogenous activity of the protein kinase preparation. 161
162
BLAIR
AND
RUSSELL
FIG. 3. Protein kinase of adenovirus types 2 and 5. Forty micrograms of either dialysed adenovirus type 5 (ii) or type 2 (iii) were assayed for protein kinase activity and analysed on the same IO-25% linear gradient acrylamide gel. (i) Coomassie blue-stained gel of adenovirus type 5 proteins; (ii) in vitro phosphorylated polypeptides of adenovirus type 5; (iii) in vitro phosphorylated polypeptides of adenovirus type 2; (iv) Coomassie blue-stained gel of adenovirus type 2 proteins.
ADENOVIRUS
PROTEIN
163
KINASE
FIG. 4. Analysis of in vitro phosphorylated virus polypeptides for phosphoamino acids. Whole adenovirus type 5, phosphorylated in uitro, was hydrolysed in either 2 or 6 M HCl under vacuum for 6 hr at 105’. In parallel, in vitro reactions using a CAMP-dependent protein kinase from bovine heart muscle and heated adenovirus as substrate were also performed and similarly hydrolysed (see Materials and Methods). Highvoltage paper electrophoresis was performed using standard phosphoserine and phosphothreonine as markers. The phosphoamino acids were located by cadmium-ninhydrin staining and marked, and the paper was exposed _ to X-ray film for 7 days.
procedure inactivated the virion protein kinase.) Labelled phosphoserine and phosphothreonine were detected as products of the in vitro adenovirus protein kinase reaction after hydrolysis in either 2 or 6 M HCl. Phosphoserine and phosphothreonine were also labelled in the control bovine heart muscle protein kinase reaction. The y-phosphate is thus transferred and covalently linked to protein. We are unable to exclude the possibility that the y-phosphate is transferred to amino acids other than serine or threonine since only the phosphoester bonds of phosphoserine and phosphothreonine are relatively stable under the conditions of acid hydrolysis. However the label in all of the phosphoproteins formed in vitro was sensitive to hydrolysis by alkaline phosphatase, an enzyme specific for phosphomonoester bonds. It would appear therefore that phosphoserine and phosphothreonine residues account for most of the amino acids phosphorylated in vitro.
Substrate Specificity Kinase
of the Virion Protein
The virion protein kinase showed a wide substrate specificity in vitro. Figure 5 shows that, in addition to phosphorylating virus proteins, added calf thymus histones, casein, and protamine sulphate were phosphorylated. However no substrate outcompeted virus proteins as phosphate acceptor. For comparison, these exogenous proteins were also used as substrates for a cellular
CAMP-dependent protein kinase. It may be noted that the level of phosphorylation of substrates obtained with adenovirus as source of protein kinase was much lower than that observed with the cell protein kinase, perhaps by several orders of magnitude. Direct comparison between the adenovirus and cell protein kinases (with a view to estimating the number of copies of kinase per virion) is impossible since at present we know little about the enzymatic parameters of the adenovirus protein kinase. However the data shown in Fig. 5 do suggest that the protein kinase is present as a minor component in the adenovirus particle, perhaps only at the level of a few molecules per virion. DISCUSSION
The sensitive technique of SDS gel electrophoresis and autoradiography has been used to detect a protein kinase in purified preparations of adenovirus types 2 and 5. In common with many other virus protein kinases (Rubin and Rosen, 1975), the adenovirus enzyme shows no dependence on CAMP or cGMP for optimal activity. Low concentrations of Mg2+ ions are required, but no stimulation was obtained in the presence of KC1 or NaCl. The substrate specificity of the protein kinase is broad, phosphorylating histones, casein, and protamine sulphate in addition to certain virus structural proteins. The transfer of the y-phosphate of ATP to the serine and threonine residues of virus proteins was demon-
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RUSSELL
FIG. 5. The substrate specificity of the adenovirus protein kinase. Thirty micrograms of adenovirus (i-iv) and 2 pg of CAMP-dependent bovine heart muscle protein kinase (v-viii) were assayed for protein kinase activity either alone (i and v) or with added calf thymus histones (ii and vi), casein (iii and vii), or protamine sulphate (iv and viii). The phosphorylated polypeptides were analysed on a 25% acrylamide gel.
&rated. The enzyme thus shows many similarities to those identified in enveloped viruses. Since adenovirus is a nonenveloped virus, this report suggests that the presence of virion protein kinases may be more widespread in animal viruses than previously believed (see, however, Strand and August, 1971; and Hatanaka et al., 1972). Although the protein kinase showed wide substrate specificity, the pattern of adenovirus polypeptide phosphorylation in vitro resembled that previously described in uivo (Russell and Blair, 1977). In uivo, phosphorylated polypeptides IIIa, V, VI, and IX
were detected, with IIIa being the major phosphoprotein. In u&o, polypeptides IIIa, V, VI, VII, and X were phosphorylated, with IIIa again being the major phosphoprotein. Amino acid analysis has shown that polypeptide VI is rich in serine (Ever&t and Philipson, 1974), which would be consistent with its role as a phosphate acceptor protein both in uiuo and in vitro. Polypeptides VIII and IX were also found to contain at least 17% serine (Everitt and Philipson, 1974). Since neither polypeptide VIII nor IX was phosphorylated in uitro, perhaps a specific amino acid sequence is required for
ADENOVIRUS
PROTEIN
recognition by the protein kinase (Williams, 1976). It is possible that virus polypeptides are only partially phosphorylated after assembly in the infected cell and therefore have sites available for phosphorylation in uitro. The action of host cell phosphoprotein phosphatases may be important in achieving this partially phosphorylated state. Alternatively, new sites may be phosphorylated in vitro. Tao and Doerfler (1972) found that all adenovirus structural proteins had sites available for phosphorylation in vitro by a CAMP-dependent protein kinase isolated either from rabbit reticulocytes or KB cells (the host cell for adenovirus growth; see Materials and Methods). In contrast, the virion protein kinase described in this paper shows discrimination, phosphorylating only minor polypeptides (IIIa, VI, and X) and core proteins (V and VII). Since mild disruption was required for detection of protein kinase activity, this implied that the enzyme or substrate was located internally. In agreement with this, most of the virus polypeptides phosphorylated in vitro are all internally located. Polypeptides V and VII form part of the internal nucleoprotein core (Russell et al., 1971) while polypeptides IIIa, VI, and X are either wholly or partly internal (Eve&t et al., 1975). However since the external proteins of the capsid are not substrates for the virion protein kinase, we cannot exclude the possibility that the enzyme is present on the outside of the virus and gains access to the internal proteins on disruption. A tight binding of kinase to virus is implied since the enzyme remains specifically associated with virus particles after several caesium chloride gradient centrifugations. Therefore if the kinase is a cellular, rather than a virus protein, the enzyme must be closely integrated into the structure of the virus, and may be specifically incorporated during virus assembly. The method of virus purification used yields a preparation of adenovirus which is at least 99.5% pure (McIntosh et al., 1971). The sensitive techniques used in this study might detect a cellular protein kinase present in the remaining 0.5% of the preparation. We have no evidence for a functional role
165
KINASE
for the protein kinase in infection or virus assembly. Our previous work identified a number of phosphorylated polypeptides of both host cell and virus origin in adenovii-us-infected cells (Russell and Blair, 1977). The phosphorylated host cell polypeptide has since been shown to be a derivative of the S6 protein of the 40 S ribosomal subunit (Blair and Horak, 1977). It is possible that the virion kinase is involved in virus-specific phosphorylations in infected cells and may be important in the initiation of virus infection. By analogy with the role of phosphorylation of influenza nucleoprotein in transcription (Kamata and Watanabe, 1977), it might also be suggested that phosphorylation of polypeptides V and VII, the core proteins, may be important in adenovirus transcription. Such modification by phosphorylation may significantly alter the binding of proteins to nucleic acid (Sen et al., 1977), constituting a possible control element in virus replication. ACKNOWLEDGMENTS We thank Dr. M. V. Nermut for electron microscopic analysis of virus preparations. The skilled assistance of Susan Jefferies was greatly appreciated. REFERENCES ANDERSON, C. W., BAUM, P. R., and GF,STELANI~, Ii. F. (1973). Processing of adenovirus 2-induced proteins. J. Virol. 12, 241-252. BLAIR, G. E., and HOHAK, I. (1977). Phosphorylation of ribosomes in adenovirus infection. Biochem. Sot. Trans. 5,660-661. CORDEN, J., ENGELKING, H. M., and PEARSON, G. II. (1976). The chromatin-like organization of the adenovirus chromosome. Proc. Nat. Acad. S’ci. USA 73,401-404. EVERITT, E., L~JTTER, L., and PHKIPSON, L. (1975). Structural proteins of adenoviruses. XII. Location and neighbour relationship among proteins of adenovirion type 2 as revealed by enzymatic iodination, immunoprecipitation and chemical cross-linking. Virology 67, 197-208. EVRRITT, E., and PHII,IPSON, L. (1974). Structural proteins of adenoviruses. XI. Purification of three low molecular weight virion proteins of adenovirus type 2 and their synthesis during productive infection. Virology 62, 253-269. GINSRENG, H. S., P~REIRA, H. G., VALENTINE, R. C., and WILCOX, W. C. (1966). A proposed terminology for the adenovirus antigens and virion morphological subunits. Virology 28, 782-783.
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HATANAKA, M., TWIDDY, E., and GILDEN, R. V. (1972). Protein kinase associated with RNA tumor viruses and other budding RNA viruses. Virology 47,536-538.
IMBLUM, R. L., and WAGNER, R. R. (1974). Protein kinase and phosphoproteins of vesicular stomatitis virus. J. Viral. 13, 113-124. KAMATA, T., and WATANABE, Y. (1977). Role for nucleocapsid protein phosphorylation in the transcription of influenza virus genome. Nature (London)
267,460-462.
LAVER, W. G., WRIGLEY, N. G., and PEREIRA, H. G. (1967). Removal of pentons from particles of adenovirus type 2. Virology 39,599-605. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem 193, 265-275.
MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and comparison of type 2, 7A and 12. Virology 36, 115-125. MCINTOSH, K., PAYNE, S., and RUSSELL, W. C. (1971). Studies on lipid metabolism in cells infected with adenovirus. J. Gen. Viral. 10,251-265. PRAGE, L., PETTERSSON, U., HOGLUND, S., LONBERGHOLM, K., and PHILIPSON, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology 42, 341-358. RUBIN, C. S., and ROSEN, 0. M. (1975). Protein phosphorylation. Annu. Rev. Biochem. 44,841-843. RUSSELL, W. C., and BLAIR, G. E. (1977). Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol. 34, 19-35. RUSSELL, W. C., HAYASHI, K., SANDERSON, P. J., and PEREIRA, H. G. (1967). Adenovirus antigens-A
study of their properties and sequential development in infection, J. Gen. Virol. 1, 495-508. RUSSELL, W. C., MCINTOSH, K., and SKEHEL, J. J. (1971). The preparation and properties of adenoviNS cores. J. Gen. Viral. 11, 35-45. RUSSELL, W. C., SKEHEL, J. J., MACHADO, R., and PEREIRA, H. G. (1972). Phosphorylated polypeptides in adenovirus-infected cells. Virology 50, 931-934. SEN, A., SHERR, C. J., and TODARO, G. J. (1977). Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell 10,489-496. SILBERSTEIN, H., and AUGUST, J. T. (1976a). Purification and properties of a virion protein kinase. J. Biol. Chem 261,3176-3184. SILBERSTEIN, H., and AUGUST, J. T. (1976b). Characterization of a virion protein kinase as a virusspecified enzyme. J. Biol. Chem. 251,3185-3190. STRAND, M., and AUGUST, J. T. (1971). Protein kinase and phosphate acceptor proteins in Rauscher murine leukaemia virus. Nature New Biol. 233, 137-140. TAN, K. B., and SOKOL, F. (1972). Structural proteins of simian virus 40: Phosphoproteins. J. Viral. 10, 985-994. TANIUCHI, H., ANFINSEN, C. B., and SODJA, A. (1967). The amino acid sequence of an extracellular nuclease of Staphylococcus aureus. III. Compelete amino acid sequence. J. Biol. Chem. 242,4752-4763. TAO, M., and DOERFLER, W. (1972). Phosphorylation of adenovirus polypeptides. Eur. J. Biochem. 27, 448-452.
WILLIAMS, R. E. (1976). Phosphorylated sites in substrates of intraceUular protein kinases: A common feature in amino acid sequences. Science 162, 473-474.
86,
157-166
Identification
(1978)
of a Protein
Kinase Activity Adenoviruses
G. E. BLAIR’ Division
of Virology,
National
Institute
for
Medical
with Human
W. C. RUSSELL
AND
Accepted
Associated
Research,
December
Mill
Hill,
London
NW7
IAA,
England
3, 1977
The identification of a cyclic nucleotide-independent protein kinase in purified adenovirus types 2 and 5 is described. Enzyme activity was detected only in virus preparations which had been mildly disrupted by dialysis against low ionic strength buffers and not in intact virus preparations. Virus polypeptides IIIa, V. VI, VII, and X were phosphorylated in vitro. The enzyme transferred the y-phosphate of ATP to serine and threonine residues of virus proteins. Other proteins (histones, casein, and protamine sulphate) were phosphorylated by the virion protein kinase.
priate to study the protein kinase enzymes which might be involved in these events. During the course of this work, we used the sensitive technique of SDS gel electrophoresis and autoradiography and were able to detect a protein kinase enzyme in highly purified preparations of adenovirus types 2 and 5. This enzyme is capable of phosphorylating both exogenous protein substrates and adenovirus structural proteins. In the latter case, a pattern of in vitro polypeptide phosphorylation resembling that observed in vivo was obtained. The adenovirus protein kinase may represent the first instance of a kinase enzyme tightly bound to a nonenveloped virus.
INTRODUCTION
Protein kinase enzymes have been detected in many enveloped viruses (reviewed in Rubin and Rosen, 1975). Nonenveloped viruses such as poliovirus and adenovirus (Strand and August, 1971) and SV40 (Tan and Sokol, 1972) have been reported to contain no protein kinase activity in their virions, on the basis of undetectable incorporation of the labelled y-phosphate of ATP into trichloroacetic acid (TCA)-insoluble material. The question of the host cell or virus origin of particle-bound protein kinases has proved difficult to resolve. In frog virus 3, the enzyme has been purified and shown to be a structural protein of the virus (Silberstein and August, 1976a,b). In contrast, Imblum and Wagner (1974) found evidence to suggest that the protein kinase of vesicular stomatitis virus may originate from the host cell. The true function of virion protein kinases in infection remains a matter for speculation. Our previous work has identified phosphorylated derivatives of both structural and nonstructural adenovirus proteins (Russell et al., 1972; Russell and Blair, 1977). Specific phosphorylation of a host cell ribosomal protein was also detected (Blair and Horak, 1977). It seemed appro’ To dressed.
whom
requests
for
reprints
should
MATERIALS
AND
METHODS
cells. Adenovirus types 2 and 5 were propagated in KB cells growing in suspension culture. Cells were maintained in minimal essential medium (Joklik modified) supplemented with 5% calf serum. Virus seeds were prepared and titrated as previously described (Russell et al., 1967). Cells were infected with virus seed at a multiplicity of lo-20 PFU/cell and incubated at 37O for 48 hr. Virus purification. Virus was purified from infected cells by extraction with fluorocarbon followed by one velocity and two equilibrium centrifugations in caesium chloride gradients. Purified virus was either Virus
be ad-
and
157 0042~6822/78/0861-0157$02.00/O Copyright 0 1978 by Academic Press, Im. All rights of reproduction in any form reserved.
158
BLAIR
AND RUSSELL
dialysed against 500 vol of 10 m&f Tris-maleate (pH 6.4), 10% glycerol for 15 hr at 4”, or rapidly desalted by gel chromatography on a column (1 x 15 cm) of coarse Sephadex G-25, equilibrated, and eluted with 10 mM Tris-maleate (pH 6.4), 10% glycerol. “Desalted virus” was assayed immediately for protein kinase activity, whereas “dialysed virus” was stored at -70” for several months without loss of activity. Protein kinase assay. Protein kinase activity was assayed by mixing aliquots of virus (5-20 pg) with 5 @i of [Y-~‘P]ATP in a buffer containing 20 mM Tris-maleate (pH 6.4), 5 r&f MgC12, 6 mM 2-mercaptoethanol, 5% glycerol. The total volume was 25 ~1. Incubation was at 37” for 30 min. Reactions were terminated by the addition of SDS denaturing mix (15 mM Tris-HCl, pH 6.8, 8 M urea, 1% SDS, 1% 2-mercaptoethanol), heated at 100” for 2 min and then analysed by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis. Analysis of phosphorylated polypeptides was performed on discontinuous slab gels as previously described (Russell and Blair, 1977) containing either 15 or 25% acrylamide or a linear gradient of 10 to 25% acrylamide in the separating gel. Analysis ofphosphothreonine andphosphoserine. In experiments where the in vitro product was hydrolysed in acid, the [y32P]ATP was increased to 20 @i per reaction. After incubation, three 25-~1 reaction mixtures were pooled and dialysed against 2 liters of distilled water for 18 hr. The dialysed sample was lyophilized and sealed under vacuum with either 2 or 6 M HCI. Hydrolysis was performed at 105” for 6 hr. The hydrolysate was lyophilized, resuspended in distilled water, lyophilized again, and resuspended in 10 ~1 of 1 mJ4 HCl containing 1 mg/ml of standard phosphoserine and phosphothreonine. The sample was applied to Whatman 3MM paper and high-voltage electrophoresis was performed in a pH 1.9 buffer (containing 2.5% formic acid, 7.8% acetic acid) at 3 kV for 3 hr. Marker phosphoamino acids were located by cadmium-ninhydrin staining. The paper
was exposed to X-ray film for 7 days. Virus concentration. The concentration of virus was determined spectrophotometrically (Maize1 et al., 1968). Aliquots of virus were suspended in 0.1 x SSC, 0.5% SDS. The concentration was calculated assuming that 1 ODpso unit is equivalent to 0.28 mg of virus protein, and that adenovirus type 5 is composed of 87.7% protein, 12.3% DNA. This procedure was verified using the method of Lowry et al. (1951). Nomenclature. The nomenclature proposed by Ginsberg et al. (1966) for the major capsid components (hexon, penton base, and fibre) is used. The remainder of the structural polypeptides are referred to according to the terminology proposed by Maize1 et al. (1968) and Anderson et al. (1973), as discussed in relation to adenovirus type 5 by Russell and Blair (1977). RESULTS
Identification and Characteristics of the Protein Kinase Activity Initial experiments involving incubation of aliquots of virus with [Y-~~P]ATP, followed by precipitation with TCA and scintillation counting of samples, failed to reveal any protein kinase activity in the virus preparations. This confirmed a previous report (Strand and August, 1971). However, before concluding that no protein kinase enzyme was present in adenovirus particles, we felt it appropriate to investigate methods of disruption of the virus structure and to apply the more sensitive technique of discontinuous SDS gel electrophoresis and autoradiography to the detection of phosphorylated products of the protein kinase reaction. Previous investigations had shown that adenovirus particles could be disrupted under mild conditions by dialysis against either distilled water (Laver et al., 1967) or low ionic strength buffers (Prage et al., 1970), for example, 10 mJ4 Tris-maleate, pH 6.4. This treatment caused pentons and fibres to dissociate from the particles, leaving spaces at the vertices which could be observed by electron microscopy. Such disruption also renders the DNA of the adenovirus “core” accessible to degradation by micrococcal nuclease (Corden et al., 1976),
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indicating that protein molecules of low molecular weight (16,800 for micrococcal nucleate; Taniuchi et al., 1967) can pass within the capsid. Preparations of “dialysed virus” and “desalted virus” (see Materials and Methods) were assayed in vitro for protein kinase activity by incubation with [y-32P]ATP, followed by electrophoresis on SDS-polyacrylamide gels. Both preparations were also examined under an electron microscope after negative staining. Almost all particles in the “dialysed virus” preparation were disrupted (that is, had holes at the vertices of the particle) while the “desalted
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virus” showed good integrity of virus capsids. The phosphorylation of virus polypeptides in vitro in each virus preparation was examined over a concentration range (Fig. 1). Phosphorylation of a discrete number of polypeptides was observed only in the dialysed virus preparation, at a concentration as low as 12 pg/assay (Fig. 1). The degree of phosphorylation was then approximately proportional to virus concentration. However no phosphorylation of virus polypeptides was observed in the preparation of desalted virus even at a concentration (12 pg, Fig. 1) where labelling was clearly evident in the dialysed virus preparation. Be-
FIG. 1. Identification of protein kinase activity in adenovirus type 5. “Dialysed” virus and “desalted” virus (referred to as disrupted and intact, respectively) were prepared as described under Materials and Methods. Increasing amounts of disrupted (12-84 pg) and intact (2-12 pg) virus were assayed for protein kinase activity and analysed on a 15% acrylamide gel. (“‘S IC) An aliquot of adenovirus-infected HeLa cells labelled with [?S]methionine late in infection.
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cause of the approximate lo-fold dilution incurred in preparing desalted virus by gel filtration, comparison over a similar concentration range proved impossible. Mild disruption of the virus structure was therefore necessary for detection of the protein kinase suggesting that the enzyme or substrate is located inside the capsid. More vigorous conditions of disruption, such as treatment with sodium dodecyl sulphate or heating in the presence of deoxycholate to form adenovirus cores (Russell et al., 1971), resulted in loss of protein kinase activity. The tight association of the protein kinase with virus was demonstrated by fractionating the second equilibrium density caesium chloride gradient (see Materials and Methods), dialysing each fraction against 10 mM Tris-maleate (pH 6.4), 10% glycerol, and assaying each fraction for protein kinase activity. Only fractions containing adenovirus were found to have protein kinase activity when casein was used as substrate (Fig. 2). The same results were obtained when “endogenous” activity was assayed and when calf thymus histones were used as substrate. The identity of the phosphorylated polypeptides was established by comparison with an extract of adenovirus-infected HeLa cells, pulse labelled with [35S]methionine late in infection (Fig. 1). Virus polypeptides IIIa, V, VI, and VII were phosphorylated in vitro. The major capsid proteins, hexon, penton base, and fibre, were not phosphorylated in uitro. Labelled species which did not comigrate with virus polypeptides were rarely observed. Alterations in the stoichiometry of virus polypeptide phosphorylation were sometimes observed. In many experiments, some labelled material migrated with the buffer front on 15% gels. In order to resolve this fraction, dialysed virus phosphorylated in vitro was analysed on linear lo-25% gradient SDS gels. Dialysed virus of adenovirus type 2 was also assayed for protein kinase activity and analysed in parallel (Fig. 3). The results of the gradient gel analysis confirm those obtained with 15% gels (Fig. 1) and also indicate that polypeptide X is phosphorylated in vitro. Adenovirus type 2 also possesses a virion protein kinase which shows similar
specificity to the type 5 enzyme for virus polypeptides, that is, polypeptides IIIa, V, VI, VII, and X were phosphorylated (Fig. 3). However, smaller amounts of phosphorylated polypeptide X were found in type 2. In addition, phosphorylated species corresponding in mobility to polypeptides IVai and IVa2 were detected in type 2 but not in type 5. It may also be noted that the mobilities of the virus structural polypeptides of types 2 and 5 are essentially similar in these experiments (shown in the stained gels of Fig. 3), the major exception being the hexon polypeptide which migrates more slowly in type 2 than in type 5. The requirement for monovalent and divalent cations and for cyclic nucleotide cofactors was examined. Although MgCh did not stimulate the kinase reaction, inhibition was obtained on addition of 5 mM EDTA. Divalent cations previously bound to virus may therefore be sufficient for observed activity. Addition of NaCl or KC1 was not required for optimal kinase activity. Neither CAMP nor cGMP, tested over a concentration range of low4 to low5 M stimulated in vitro phosphorylation. The specificity of phosphorylation of virus polypeptides was not altered by addition of cyclic nucleotides or variation in the ionic conditions. Nature of the Phosphoprotein Bond When in vitro reactions were terminated without incubation, no labelled phosphate was incorporated into virion polypeptides suggesting that the [y-32P]ATP was not simply bound to protein under the conditions of incubation and analysis. Definitive evidence for transfer and covalent bonding of the y-phosphate group of ATP to protein was provided by acid hydrolysis of the in vitro products. After phosphorylation in vitro, the whole virus preparation was dialysed and hydrolysed in acid (see Materials and Methods). Labelled phosphoamino acids were separated by high-voltage paper electrophoresis and located by autoradiography (Fig. 4). In parallel, a control reaction was performed using bovine heart muscle protein kinase (assayed in the presence of 10 fl CAMP) and adenovirus type 5, previously heated at 60’ for 15 min. (This
FIG. 2. Copurification of protein kinase activity with adenovirus. After purification by one velocity and one equilibrium caesium chloride centrifugation, purified adenovirus was centrifuged on a second (4.5 ml) gradient to equilibrium (see Materials and Methods). Fractions (0.5 ml) were collected and dialysed against 500 vol of 10 mM Tris-maleate (pH 6.4), 10% glycerol for 18 hr at 4”, and 15-/d aliquots of each fraction were assayed for protein kinase activity using 10 pg of casein as substrate. The in vitro products were analysed on a 15% gel. The upper panel shows the gradient profile. CM) EZM; (A-----A) density (grams per cubic centimeter). The products of in vitro phosphorylation of each fraction are displayed immediately below, so that fractions from the upper panel correspond to the slots on the gel in the lower panel. V, The products of phosphorylation of casein by 5 ~1 of purified adenovirus; PK, the products of phosphorylation of casein by 2 1.18 of bovine heart muscle protein kinase. The additional bands in this slot are due to endogenous activity of the protein kinase preparation. 161
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FIG. 3. Protein kinase of adenovirus types 2 and 5. Forty micrograms of either dialysed adenovirus type 5 (ii) or type 2 (iii) were assayed for protein kinase activity and analysed on the same IO-25% linear gradient acrylamide gel. (i) Coomassie blue-stained gel of adenovirus type 5 proteins; (ii) in vitro phosphorylated polypeptides of adenovirus type 5; (iii) in vitro phosphorylated polypeptides of adenovirus type 2; (iv) Coomassie blue-stained gel of adenovirus type 2 proteins.
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FIG. 4. Analysis of in vitro phosphorylated virus polypeptides for phosphoamino acids. Whole adenovirus type 5, phosphorylated in uitro, was hydrolysed in either 2 or 6 M HCl under vacuum for 6 hr at 105’. In parallel, in vitro reactions using a CAMP-dependent protein kinase from bovine heart muscle and heated adenovirus as substrate were also performed and similarly hydrolysed (see Materials and Methods). Highvoltage paper electrophoresis was performed using standard phosphoserine and phosphothreonine as markers. The phosphoamino acids were located by cadmium-ninhydrin staining and marked, and the paper was exposed _ to X-ray film for 7 days.
procedure inactivated the virion protein kinase.) Labelled phosphoserine and phosphothreonine were detected as products of the in vitro adenovirus protein kinase reaction after hydrolysis in either 2 or 6 M HCl. Phosphoserine and phosphothreonine were also labelled in the control bovine heart muscle protein kinase reaction. The y-phosphate is thus transferred and covalently linked to protein. We are unable to exclude the possibility that the y-phosphate is transferred to amino acids other than serine or threonine since only the phosphoester bonds of phosphoserine and phosphothreonine are relatively stable under the conditions of acid hydrolysis. However the label in all of the phosphoproteins formed in vitro was sensitive to hydrolysis by alkaline phosphatase, an enzyme specific for phosphomonoester bonds. It would appear therefore that phosphoserine and phosphothreonine residues account for most of the amino acids phosphorylated in vitro.
Substrate Specificity Kinase
of the Virion Protein
The virion protein kinase showed a wide substrate specificity in vitro. Figure 5 shows that, in addition to phosphorylating virus proteins, added calf thymus histones, casein, and protamine sulphate were phosphorylated. However no substrate outcompeted virus proteins as phosphate acceptor. For comparison, these exogenous proteins were also used as substrates for a cellular
CAMP-dependent protein kinase. It may be noted that the level of phosphorylation of substrates obtained with adenovirus as source of protein kinase was much lower than that observed with the cell protein kinase, perhaps by several orders of magnitude. Direct comparison between the adenovirus and cell protein kinases (with a view to estimating the number of copies of kinase per virion) is impossible since at present we know little about the enzymatic parameters of the adenovirus protein kinase. However the data shown in Fig. 5 do suggest that the protein kinase is present as a minor component in the adenovirus particle, perhaps only at the level of a few molecules per virion. DISCUSSION
The sensitive technique of SDS gel electrophoresis and autoradiography has been used to detect a protein kinase in purified preparations of adenovirus types 2 and 5. In common with many other virus protein kinases (Rubin and Rosen, 1975), the adenovirus enzyme shows no dependence on CAMP or cGMP for optimal activity. Low concentrations of Mg2+ ions are required, but no stimulation was obtained in the presence of KC1 or NaCl. The substrate specificity of the protein kinase is broad, phosphorylating histones, casein, and protamine sulphate in addition to certain virus structural proteins. The transfer of the y-phosphate of ATP to the serine and threonine residues of virus proteins was demon-
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FIG. 5. The substrate specificity of the adenovirus protein kinase. Thirty micrograms of adenovirus (i-iv) and 2 pg of CAMP-dependent bovine heart muscle protein kinase (v-viii) were assayed for protein kinase activity either alone (i and v) or with added calf thymus histones (ii and vi), casein (iii and vii), or protamine sulphate (iv and viii). The phosphorylated polypeptides were analysed on a 25% acrylamide gel.
&rated. The enzyme thus shows many similarities to those identified in enveloped viruses. Since adenovirus is a nonenveloped virus, this report suggests that the presence of virion protein kinases may be more widespread in animal viruses than previously believed (see, however, Strand and August, 1971; and Hatanaka et al., 1972). Although the protein kinase showed wide substrate specificity, the pattern of adenovirus polypeptide phosphorylation in vitro resembled that previously described in uivo (Russell and Blair, 1977). In uivo, phosphorylated polypeptides IIIa, V, VI, and IX
were detected, with IIIa being the major phosphoprotein. In u&o, polypeptides IIIa, V, VI, VII, and X were phosphorylated, with IIIa again being the major phosphoprotein. Amino acid analysis has shown that polypeptide VI is rich in serine (Ever&t and Philipson, 1974), which would be consistent with its role as a phosphate acceptor protein both in uiuo and in vitro. Polypeptides VIII and IX were also found to contain at least 17% serine (Everitt and Philipson, 1974). Since neither polypeptide VIII nor IX was phosphorylated in uitro, perhaps a specific amino acid sequence is required for
ADENOVIRUS
PROTEIN
recognition by the protein kinase (Williams, 1976). It is possible that virus polypeptides are only partially phosphorylated after assembly in the infected cell and therefore have sites available for phosphorylation in uitro. The action of host cell phosphoprotein phosphatases may be important in achieving this partially phosphorylated state. Alternatively, new sites may be phosphorylated in vitro. Tao and Doerfler (1972) found that all adenovirus structural proteins had sites available for phosphorylation in vitro by a CAMP-dependent protein kinase isolated either from rabbit reticulocytes or KB cells (the host cell for adenovirus growth; see Materials and Methods). In contrast, the virion protein kinase described in this paper shows discrimination, phosphorylating only minor polypeptides (IIIa, VI, and X) and core proteins (V and VII). Since mild disruption was required for detection of protein kinase activity, this implied that the enzyme or substrate was located internally. In agreement with this, most of the virus polypeptides phosphorylated in vitro are all internally located. Polypeptides V and VII form part of the internal nucleoprotein core (Russell et al., 1971) while polypeptides IIIa, VI, and X are either wholly or partly internal (Eve&t et al., 1975). However since the external proteins of the capsid are not substrates for the virion protein kinase, we cannot exclude the possibility that the enzyme is present on the outside of the virus and gains access to the internal proteins on disruption. A tight binding of kinase to virus is implied since the enzyme remains specifically associated with virus particles after several caesium chloride gradient centrifugations. Therefore if the kinase is a cellular, rather than a virus protein, the enzyme must be closely integrated into the structure of the virus, and may be specifically incorporated during virus assembly. The method of virus purification used yields a preparation of adenovirus which is at least 99.5% pure (McIntosh et al., 1971). The sensitive techniques used in this study might detect a cellular protein kinase present in the remaining 0.5% of the preparation. We have no evidence for a functional role
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for the protein kinase in infection or virus assembly. Our previous work identified a number of phosphorylated polypeptides of both host cell and virus origin in adenovii-us-infected cells (Russell and Blair, 1977). The phosphorylated host cell polypeptide has since been shown to be a derivative of the S6 protein of the 40 S ribosomal subunit (Blair and Horak, 1977). It is possible that the virion kinase is involved in virus-specific phosphorylations in infected cells and may be important in the initiation of virus infection. By analogy with the role of phosphorylation of influenza nucleoprotein in transcription (Kamata and Watanabe, 1977), it might also be suggested that phosphorylation of polypeptides V and VII, the core proteins, may be important in adenovirus transcription. Such modification by phosphorylation may significantly alter the binding of proteins to nucleic acid (Sen et al., 1977), constituting a possible control element in virus replication. ACKNOWLEDGMENTS We thank Dr. M. V. Nermut for electron microscopic analysis of virus preparations. The skilled assistance of Susan Jefferies was greatly appreciated. REFERENCES ANDERSON, C. W., BAUM, P. R., and GF,STELANI~, Ii. F. (1973). Processing of adenovirus 2-induced proteins. J. Virol. 12, 241-252. BLAIR, G. E., and HOHAK, I. (1977). Phosphorylation of ribosomes in adenovirus infection. Biochem. Sot. Trans. 5,660-661. CORDEN, J., ENGELKING, H. M., and PEARSON, G. II. (1976). The chromatin-like organization of the adenovirus chromosome. Proc. Nat. Acad. S’ci. USA 73,401-404. EVERITT, E., L~JTTER, L., and PHKIPSON, L. (1975). Structural proteins of adenoviruses. XII. Location and neighbour relationship among proteins of adenovirion type 2 as revealed by enzymatic iodination, immunoprecipitation and chemical cross-linking. Virology 67, 197-208. EVRRITT, E., and PHII,IPSON, L. (1974). Structural proteins of adenoviruses. XI. Purification of three low molecular weight virion proteins of adenovirus type 2 and their synthesis during productive infection. Virology 62, 253-269. GINSRENG, H. S., P~REIRA, H. G., VALENTINE, R. C., and WILCOX, W. C. (1966). A proposed terminology for the adenovirus antigens and virion morphological subunits. Virology 28, 782-783.
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HATANAKA, M., TWIDDY, E., and GILDEN, R. V. (1972). Protein kinase associated with RNA tumor viruses and other budding RNA viruses. Virology 47,536-538.
IMBLUM, R. L., and WAGNER, R. R. (1974). Protein kinase and phosphoproteins of vesicular stomatitis virus. J. Viral. 13, 113-124. KAMATA, T., and WATANABE, Y. (1977). Role for nucleocapsid protein phosphorylation in the transcription of influenza virus genome. Nature (London)
267,460-462.
LAVER, W. G., WRIGLEY, N. G., and PEREIRA, H. G. (1967). Removal of pentons from particles of adenovirus type 2. Virology 39,599-605. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem 193, 265-275.
MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and comparison of type 2, 7A and 12. Virology 36, 115-125. MCINTOSH, K., PAYNE, S., and RUSSELL, W. C. (1971). Studies on lipid metabolism in cells infected with adenovirus. J. Gen. Viral. 10,251-265. PRAGE, L., PETTERSSON, U., HOGLUND, S., LONBERGHOLM, K., and PHILIPSON, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology 42, 341-358. RUBIN, C. S., and ROSEN, 0. M. (1975). Protein phosphorylation. Annu. Rev. Biochem. 44,841-843. RUSSELL, W. C., and BLAIR, G. E. (1977). Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol. 34, 19-35. RUSSELL, W. C., HAYASHI, K., SANDERSON, P. J., and PEREIRA, H. G. (1967). Adenovirus antigens-A
study of their properties and sequential development in infection, J. Gen. Virol. 1, 495-508. RUSSELL, W. C., MCINTOSH, K., and SKEHEL, J. J. (1971). The preparation and properties of adenoviNS cores. J. Gen. Viral. 11, 35-45. RUSSELL, W. C., SKEHEL, J. J., MACHADO, R., and PEREIRA, H. G. (1972). Phosphorylated polypeptides in adenovirus-infected cells. Virology 50, 931-934. SEN, A., SHERR, C. J., and TODARO, G. J. (1977). Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell 10,489-496. SILBERSTEIN, H., and AUGUST, J. T. (1976a). Purification and properties of a virion protein kinase. J. Biol. Chem 261,3176-3184. SILBERSTEIN, H., and AUGUST, J. T. (1976b). Characterization of a virion protein kinase as a virusspecified enzyme. J. Biol. Chem. 251,3185-3190. STRAND, M., and AUGUST, J. T. (1971). Protein kinase and phosphate acceptor proteins in Rauscher murine leukaemia virus. Nature New Biol. 233, 137-140. TAN, K. B., and SOKOL, F. (1972). Structural proteins of simian virus 40: Phosphoproteins. J. Viral. 10, 985-994. TANIUCHI, H., ANFINSEN, C. B., and SODJA, A. (1967). The amino acid sequence of an extracellular nuclease of Staphylococcus aureus. III. Compelete amino acid sequence. J. Biol. Chem. 242,4752-4763. TAO, M., and DOERFLER, W. (1972). Phosphorylation of adenovirus polypeptides. Eur. J. Biochem. 27, 448-452.
WILLIAMS, R. E. (1976). Phosphorylated sites in substrates of intraceUular protein kinases: A common feature in amino acid sequences. Science 162, 473-474.