MARVIN J. GRUBMAN,* BARRY BAXT, JOSE L. LA TORRE,t AND HOWARD L. BACHRACH. Plum Island Animal Disease Center, Agricultural Research, ...
JOURNAL OF VIROLOGY, Aug. 1981, p. 455-462 0022-538X/81/080455-08$02.00/0
Vol. 39, No. 2
Identification of a Protein Kinase Activity in Purified Footand-Mouth Disease Virus MARVIN J. GRUBMAN,* BARRY BAXT, JOSE L. LA TORRE,t AND HOWARD L. BACHRACH Plum Island Animal Disease Center, Agricultural Research, Science and Education Administration, U.S. Department ofAgriculture, Greenport, New York 11944
Received 20 Feburary 1981/Accepted 24 April 1981
Purified preparations of foot-and-mouth disease virus types A, 0, and C contain a protein kinase activity which can transfer the gamma phosphate of [32P]ATP to virion structural proteins VP2 and VP3 and exogenous acceptor proteins. Utilizing protamine sulfate as an acceptor, the kinase activity can be demonstrated in disrupted virus but not in intact virus. The enzyme is heat labile with optimal activity at pH 7 or greater. Serine residues of protamine sulfate were identified as the amino acid phosphorylated by the protein kinase. Treatment of purified virus with typsin, which cleaves VP3, did not affect the protein kinase activity. The results indicate that the protein kinase activity found in FMDV is present in an internally located protein of viral or host origin. Protein kinases are present in many enveloped RNA and DNA viruses (19), and recently this enzyme activity was found in a nonenveloped DNA virus, human adenovirus (1, 7), and in a DNA insect virus, iridescent virus, which contains an internal lipid membrane (13, 17). In most cases, the origin of the enzyme is uncertain, although Silberstein and August (21, 22) found that the kinase in frog virus 3 is viral coded, whereas Imblum and Wagner (11) and Moyer and Summers (18) suggested that the protein kinase in vesicular stomatitis virus is host coded. The function of protein kinases in viral infection is obscure; however, recent evidence indicates that phosphorylation may alter the binding of proteins to nucleic acid (20) and the activity of proteins in transcription (9, 12, 14). We have recently reported that the structural polypeptides of foot-and-mouth disease virus (FMDV) are phosphorylated, constituting the first observation of a post-translational modification of amino acid residues in picornavirus proteins. The degree of phosphorylation of the FMDV proteins varies (15a). Virion proteins VP3 and VP4 were more highly phosphorylated than the other two major structural proteins (VP, and VP2). Picomavirus structural proteins originate from a polyprotein that is subsequently cleaved into mature virion polypeptides (2). To assess whether phosphorylation occurs on the nascent polyprotein or during virus maturation, we anat Permanent address: Centro de Virologia Animal (CEVAN), Serrano 661 1414, Capital Federal, Argentina.
lyzed FMDV-specific proteins from infected cell cultures for 32P label and examined purified virus for protein kinase activity. No labeling of any virus-specific proteins with 32P was detected during the infectious cycle; however, purified virus preparations were found to contain a protein kinase activity. This activity can phosphorylate both viral structural proteins and exogenous substrates and appears to be located within the virus particle. MATERIALS AND METHODS Materials. [y-YP]ATP (2,000 Ci/mmol), 'Pi, and
[3S]methionine (900 to 1,000 Ci/mmol) were purchased from New England Nuclear Corp., Boston, Mass. Protamine sulfate, phosvitin, casein, histones, cyclic AMP, cyclic GMP, and rabbit muscle protein kinase were purchased from Sigma Chemical Co., St. Louis, Mo. Tosylphenylalanyl chloromethyl ketonetrypsin was obtained from Worthington Biochemical Corp., Freehold, N.J. Cells and virus. Baby hamster kidney cells (BHK21, clone 13) were grown in 2-liter roller-bottle cultures and maintained as previously described (5). Bovine kidney (BK) cells were used in the second to fourth passage. FMDV type An strain ll9ab (A12), type 01 strain Campos (O1c^,,.p), and type C3 strain Resende (C3Me.) were grown in BHK cells and purified by CsCl or sucrose gradient centrifugation as described (3, 15a, 23). Radioactive labeling of infected cells. Monolayers of BHK-21 cells were infected at a multiplicity of 100 PFU of A12 per cell. After 1 h at 37°C, the medium was removed and replaced with either medium minus phosphate or medium containing 1/20 the normal concentration of methionine. Two hours after infection, the cells were either continuously labeled 455
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for 3 h with 56 uCi of 32p per ml or 5 ,uCi of [35S]methionine per ml or were pulse-labeled for 30 min at 0.5-h intervals with 110 ,uCi of 32p per ml or 20 uCi of [35S]methionine per ml. Cells were washed, lysed with sample preparation buffer (0.063 M Tris-hydrochloride, pH 6.8; 2% sodium dodecyl sulfate; 0.68 M ,Bmercaptoethanol; 4 M urea; 10% glycerine; 0.005% bromophenol blue), heated at 100°C for 10 min, and analyzed on 10%o polyacrylamide slab gels containing sodium dodecyl sulfate and 8 M urea (15, 16). Protein kinase assay. Protein kinase activity was usually assayed in 50-,ul reaction volumes containing 30 ,ug of virus, 10 or 20 ,ug of protamine sulfate or other substrates as described in the text, and 3 to 6 uCi of [_y-_32P]ATP in a buffer containing 20 mM Tris-hydrochloride (pH 7.0), 6 mM MgCl2; and 10 mM dithiothreitol. Other additions were made as indicated in the text, and the reaction mixture was incubated at 30°C for the periods stated in the figure legends. To determine the kinetics of radiolabeling, samples were removed at various times into tubes containing 1 ml of 20% trichloroacetic acid, 1 ml of 0.125 M sodium pyrophosphate, and 2 drops of 0.5% bovine gammna globulin at 4°C and centrifuged at 1,700 rpm for 5 min. The precipitates were suspended in 100 ,lI of water, dissolved by the addition of 100 ul of 1 N NaOH, reprecipitated by the addition of 1 ml of 0.125 M sodium pyrophosphate plus 1 ml of 20% trichloroacetic acid, washed with 5% trichloroacetic acid, and collected on Whatman GF/A filters (4). The samples were counted in a toluene-based scintillation cocktail in a Beckman liquid scintillation spectrometer. Polyacrylamide gel electrophoresis. To analyze incubation mixtures for phosphorylated proteins, the reactions were terminated by the addition of an equal volume of twice-concentrated (2x) sample preparation buffer. Alternatively, 10 volumes of acetone was added to the reaction mixtures, and the precipitates were suspended in sample preparation buffer. The samples were heated at 100°C for 3 min and analyzed on a 15% or 12.5% sodium dodecyl sulfate-polyacrylamide slab gel containing 8 M urea or on a 10% gel in the absence of urea in Tris-glycine buffer (15, 16). The gels were electrophoresed at 120 or 150 V for 3 to 4 h, stained, destained, and dried onto Whatman 3MM paper. Radioactivity was detected by exposure to Kodak XR-5 X-ray film using a Du Pont Cronex Lightning-Plus intensifying screen at -700C. Detection of phosphorylated amino acids. Ten volumes of acetone was added to the incubation mixtures. The precipitates were suspended in 1 ml of 6 N HCl, hydrolyzed at 105°C for 5 h, and analyzed for phosphorylated amino acids by high-voltage paper electrophoresis as described by Bitte and Kabat (6).
RESULTS cells for phosphorylof infected Analysis ated viral proteins. Cells infected with FMDV at a high multiplicity were pulsed with 32P or [35S]methionine for 30-min intervals from 2 to 5 h postinfection or continuously labeled with 12p or [35S]methionine. Control uninfected cells were similarly labeled with 32p. Under these
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conditions of infection, virus particles are produced and released from cells by 4 to 5 h (unpublished data). After labeling, the cells were washed, lysed with lx sample preparation buffer, boiled, and electrophoresed on a 10% polyacrylamide slab gel. No virus-specific proteins labeled with 32P were detectable at any time during the infectious cycle (data not shown), indicating that phosphorylation may be a late event in virus maturation, possibly as a result of a virion-associated protein kinase. Identification of protein kinase activity in purified virus. It was considered that demonstration of protein kinase activity might require disruption of the virus particle. Since FMDV is the most acid sensitive of the picornaviruses (2) and dissociates in weak acid solutions, purified virus preparations were either first disrupted by treatment at pH 6.0 and then assayed at pH 8.0 or directly assayed at pH 8.0 at 30°C in the presence of [-y-32P]ATP and the exogenous acceptor protein protamine sulfate. Protein kinase activity was barely detectable in intact virus preparations; however, prior disruption of virus at pH 6.0 resulted in the incorporation of 32p into trichloroacetic acid-precipitable material (Fig. la). The enzyme present in disrupted virus particles was essentially inactive when assayed at pH 6.0 or 6.5, but was active at pH 7.0 and displayed a broad pH range (Fig. lb). Incorporation of isotope was barely detectable during 180 min of incubation in the absence of protamine sulfate (Fig. 2a). Virus types A12, C3R1,, and Olcamp, in the presence of protamine sulfate, all stimulated incorporation of 32p into trichloroacetic acid-precipitable material. Labeling, which continued for more than 4 h (data not shown), was dependent upon the presence of virus (Fig. 2b), and the activity was heat labile (Fig. 2c). Lysine- and arginine-rich histones and casein stimulated incorporation to a significantly lower extent than did protamine sulfate (Fig. 2d), as did other exogenous acceptors such as total histones and phosvitin (data not shown). Protein kinase activity was dependent upon the presence of MgCl2 and was optimal at 6 to 9 mM (Fig. 3a). Manganese chloride could substitute for MgCl2, but optimal activity occurred at 1 mM and decreased markedly at higher MnCl2 concentrations (Fig. 3b). Cyclic AMP stimulated rabbit muscle protein kinase, but neither cyclic AMP nor cyclic GMP at 10-4 to 10-6 M stimulated the viral kinase (data not shown). To further demonstrate that the protein kinase was associated with virus, two methods of virus purification were used. The first procedure involved polyethylene glycol precipitation followed by CsCl isodensity centrifugation (3, 23),
A PROTEIN KINASE IN FMDV
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TIME (MIN) FIG. 1. Identification of a protein kinase in disrupted FMDV. (a) Reaction mixtures (50 td) containing 20 pg of protamine sulfate and 30 pLg of intact type A,2 virus (0) or 30 pg of pH 6.0 disrupted virus (-) were incubated at 30°C at pH 8.0, and 10-,ul samples were removed at the indicated times and assayed for incorporation of 32p. (b) Reaction mixtures (50 ,ul) containing 30 pug of pH 6.0 disrupted virus and 20 pg of protamine sulfate were incubated at 30°C at various pH values, and 10-gIl samples were removed at the indicated times and assayed for incorporation of 32p: 0, pH 6.0; 0, pH 6.5; IA, pH 7.0; A, pH 8.0; LI, pH 8.5.
and the second procedure involved treating infected cell fluids with Sarkosyl and EDTA, pelleting the virus by centrifugation, treating again with Sarkosyl, and purifying the virus by sucrose-gradient centrifugation (15a). Both purified virus preparations possessed nearly equivalent enzyme specific activities (data not shown). In addition, protein kinase activity was detected in four independent preparations of virus, one of which was prepared by another researcher (D. M. Moore) at this Center. Type A12 virus was grown in both BHK cells and BK cells, and the purified viruses were assayed for protein kinase activity. The data in Table 1 show that virus grown in both cell types had protein kinase activity, and furthermore that virus grown in BK cells had three to four times higher specific activity after a 2-h incubation than that prepared from BHK cells. The same results were found when the kinetics of incorporation were followed (data not shown). Analysis of phosphorylated proteins by gel electrophoresis. Reactions with and without the exogenous substrates protamine sulfate or casein were terminated by the addition of 2x sample preparation buffer and analyzed by polyacrylamide gel electrophoresis. Figure 4 (lane 1) shows that structural proteins VP2 and VP3, which were about equally labeled, were the major proteins phosphorylated. VP4 and a highmolecular-weight protein of unknown origin were labeled to a very small extent. Analysis of
the stained structural proteins revealed that VP2 consists of two components, a major protein and a minor polypeptide which migrated slightly slower (Fig. 4, lane 6). Comparison of the in vitro-phosphorylated proteins (Fig. 4, lane 7) and the stained bands appears to indicate that the label in VP2 is situated principally in the minor component. Similar results were obtained for virus grown in BHK or BK cells (data not shown). The labeling of viral proteins and exogenous substrates was dependent upon the presence of virus (Fig. 4, lanes 3, 8, and 9), and the activity was heat labile (Fig. 4, lanes 2, 5, and 11) as shown previously (Fig. 2c).
Identification of the phosphoprotein bond. Reactions incubated in the presence of protamine sulfate were terninated by heating at 100°C for 3 min. Pronase treatment removed approximately 95% of the trichloroacetic acidprecipitable radioactivity, indicating that the phosphate was attached to protein (Table 2). Treatment with hot acid (succinic acid at pH 5.5, or succinic acid plus hydroxylamine, which removed 5 to 16% of the precipitable counts) indicated that incorporation was not into phosphoacyl bonds of amino acid residues (6). However, 98% of the trichloroacetic acid-precipitable
counts were labile to hot 1 N NaOH, which is characteristic of phosphoserine or phosphothreonine bonds (6). In addition, a reaction mixture incubated in the presence of protamine sulfate was precipitated with acetone, hydrolyzed for 5
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TIME (MIN) FIG. 2. Characterization of the FMD Vprotein kinase. (a) Reaction mixtures (100 Il) containing 60 pg ofpH 6 dissociated virus were incubated at 30°C, and 10-,ul samples were removed at the indicated times. 0, Type A12 minus protamine sulfate, *, type A12 plus 40 pg ofprotamine sulfate; A, type C3Res plus 40 pug ofprotamine sulfate; A, type Oica,p plus 40 pg of protamine sulfate. (b) Reaction mixtures (50 ,l) containing 20 ug of protamine sulfate were incubated in the absence (0) or presence (0) of 30 pg ofpH 6 dissociated type A12 virus, and 10-,Il samples were removed at the indicated times. (c) Reaction mixtures contained 20 pg of protamine sulfate in the presence of 30 pg of heat-treated (30 min at 65°C) type A12 virus (0) or 30 pg of unheated pH 6-dissociated type A12 virus (0). (d) Reaction mixtures contained 30 pg ofpH 6 dissociated type A12 virus and 20 pg of exogenous acceptors: 0, protamine sulfate; *, casein; A, lysine-rich histones; A,
arginine-rich histones.
h at 105°C in 6 N HCI, and analyzed by highvoltage paper electrophoresis. Phosphoserine was found to be the radioactive amino acid (Fig. 5).
Localization of protein kinase activity in the virus particle. Trypsin treatment of type A FMDV results in cleavage of VP3, decreases the ability of virus to bind to cells, and produces
a 100-fold reduction in infectivity (2, 8, 24; unpublished data). However, the virus particle remains intact and retains its sedimentation constant of 140S (2). Therefore, virus was treated with trypsin, and 140S virus was isolated from sucrose gradients. Analysis of the trypsintreated virus by polyacrylamide gel electrophoresis confirmed that VP3 was the only protein
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FIG. 3. Effect of cation concentration on activity of the FMDV protein kinase. Reaction mixtures (50 1,u) containing 30 ,g of type A12 virus, 20 pg of protamine sulfate, and varying concentrations of MgCl2 (a) or MnCl2 (b) were incubated at 30°C for 120 min, and samples were assayed for incorporation of 32p. 1
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FIG. 4. Polyacrylamide gel analysis of proteins phosphorylated by the FMDV protein kinase. Reaction mixtures containingpH 6 dissociated virus were incubated for 30 or 120 min, treated with an equal volume of 2x sample preparation buffer, boiled for 3 min, and analyzed on 15% (lanes 1 to 5) or 12.5% (lanes 6 to 8) polyacrylamide slab gels containing 8 M urea, or 10% polyacrylamide slab gels without urea (lanes 9 to 11). Reaction mixtures: lane 1, with type A12 virus; lane 2, with heated virus (65°C, 30 min); lane 3, lacking virus; lane 4, with virus and protamine sulfate; lane 5, with heated virus and protamine sulfate; lane 6, with virus but stained with Coomassie brilliant blue; lane 7, with virus; lane 8, lacking virus; lane 9, lacking virus but with casein; lane 10, with virus and casein; lane 11, with heated virus and casein. TABLE 1. Comparison of specific activities of FMDV grown in different cell types Protein kinase sp act (pmol/mg of virus X 10-2)a Cell type Assay 2 Assay 1 8.2 BHK 19 75 32.8 BK aProteini kinase assays were incubated at 300C for 120 min in the presence of 44 ug of type A12 virus and 30 ,g of protamine sulfate. Samples were removed and processed as described in the text.
cleaved (data not shown). Control virus and the trypsin-treated virus were assayed for protein kinase activity in the presence of protamine sulfate. The results in Fig. 6 demonstrate that both virus preparations had similar enzyme activities.
DISCUSSION The results demonstrate that purified preparations of FMDV types A, 0, and C contain a protein kinase activity that phosphorylates vir-
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rylated polypeptides, whereas the other proteins are labeled to a lesser extent (15a). In contrast, VP2 and VP3 are phosphorylated in vitro to essentially the same degree, whereas VP4 is la1. Control ....................... 29,106 (100) beled to only a very low level. The difference in 1,279 (4.4) 2. Pronase ..................... labeling in vitro and in vivo may be related to 3. 10% trichloroacetic acid ........ 26,541 (91.2) the residual sites available for in vitro phospho27,621 (94.9) 4. 1 M succinic acid (pH 5.5) ...... rylation, to a rearrangement of the structural 5. 1 M succinic acid + 1 M 24,312 (83.5) hydroxylamine (pH 5.5) .... proteins with respect to the kinase after in vitro 398 (1.4) 6. 1 N NaOH ................... disruption of the virus, or to both. The minor aProtein kinase assays were incubated at 300C for component of VP2 appears to be phosphoryl30 min, and the reactions were terminated by heating ated. This is similar to the results found with at 100°C for 3 min and chilled at 40C. Samples were the NS protein of VSV, which could be sepaincubated with either (1) 0.01 M Tris-hydrochloride rated into two components, one of which was TABLE 2. Characterization of the reaction product of con~cpm (% trol) Treatment' Treatment"
(pH 7.4) at 370C for 1 h; (2) 100 ,ug of predigested pronase per ml in 0.01 M Tris-hydrochloride (pH 7.4) at 370C for 1 h; (3) 10% trichloroacetic acid at 90°C for 20 min; (4) 1 M succinic acid (pH 5.5) at 370C for 1 h; (5) 1 M succinic acid and 1 M hydroxylamine (pH 5.5) at 370C for 1 h; (6) 1 N NaOH at 900C for 20 min. After these treatments, 1 ml of 0.125 M sodium pyrophosphate, 1 ml of 20% trichloroacetic acid, and 2 drops of 0.5% bovine gamma globulin were added, and the samples were processed as described in the text.
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ion structural proteins VP2 and VP3 and exogenous substrates. The enzyme activity was detected readily with the basic protein protamine sulfate as exogenous acceptor and to a lesser extent with histones, casein, and phosvitin. The y-phosphate of ATP was transferred to serine residues in protamine sulfate (Fig. 5). The virion kinase stimulated incorporation for more than 4 -PHOSPHOSERINE h, and, like kinases associated with other viruses, it is not dependent upon cyclic nucleotides (19). The enzyme is relatively inactive below pH 7, it has a broad pH optimum above 7 (Fig. 1), and -PHOSPHOTHREONINE activity is dependent upon the presence of a divalent cation (Fig. 3). FMDV and adenovirus types 2 and 5 appear to be the only nonenveloped viruses thus far examined which contain a protein kinase (1, 7). Iridescent viruses (types 6, 22, and 23) that possess internal lipid envelopes -ORIGIN also have protein kinase activity (13, 17). The protein kinase appears to reside internally FIG. 5. Identification of the protein-phosphate in FMDV because: (i) detectable activity oc- linkage in protamine sulfate. An incubation mixture curred only after disruption of virus under mild (50 ,ul) containing 30 ,ug of type A12 virus and 20 lAg of acidic conditions (Fig. 1) and (ii) trypsin treat- protamine sulfate was precipitated with 10 volumes ment of virus, which cleaves VP3 and markedly of acetone. The pellet was suspended in 1 ml of 6 N decreases the infectivity and cell-binding capac- HCI and hydrolyzed in vacuo for 5 h at 105°C, and ity of virus, had little effect on the protein kinase the HCI was evaporated. The residue was suspended lyophilized, resuspended in electrophoresis activity (Fig. 6). In addition, the alternative pu- in water, formic acid and 7.8% acetic acid), and (2.5% buffer of proriflcation methods used and the presence on Whatman 3MM paper at 3,000 V electrophoresed tein kinase activity in virus grown in two cell for 2 h. Unlabeled phosphoserine and phosphothretypes strongly indicate that the activity does not onine were used as markers and were identified by reside in an extemal contaminating cellular pro- ninhydrin staining. The paper strips were then extein that copurifies with virus. posed to Kodak X-Omat R X-ray film with an intenIn vivo, VP3 and VP4 are the major phospho- sifying screen.
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A PROTEIN KINASE IN FMDV
is unknown. The observation that the protein kinase in virus grown in BK cells has 3 to 4 times higher specific activity than that in virus grown in BHK cells appears to indicate that the protein kinase is a cellular coded enzyme (11). However, it is possible that the enzyme could be a viral coded protein which is modified in one cell type. Purification of this enzyme may determine its origin.
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ACKNOWLEDGMEENTS We thank Jo Ann Henry, Ann Tuthill, and A. J. Franke for excellent technical assistance.
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TIME (MIN) FIG. 6. Effect of trypsin treatment on protein kinase activity of FMDV. FMDV type A12 (944 pg) was incubated at 37°C for 15 min with 21.5 ug of tosylphenylalanyl chloromethyl ketone-trypsin in 1 ml of 0.15 M NaCI-0.01 M Tris-hydrochloride (pH 7.4) buffer, chilled at 4°C, and centrifuged in a 15 to 30% (wt/wt) sucrose gradient in an SW41 rotor at 35,000 rpm for 2.5 h at 4°C. During gradient fractionation, the optical density at 254 nm of the gradient was monitored with an Altex recording spectrophotometer. Fractions corresponding to 140S virus were pooled, pelleted, and suspended in buffer. A control virus sample was treated identically except for trypsin digestion. The trypsin-treated and control virus samples were assayed in the standard protein kinase assay containing protamine sulfate. 0, Control virus; 0, trypsin-treated virus.
highly phosphorylated than the other (9, 10, 14). Preliminary evidence indicates that both intracellular and extracellular viruses have protein kinase activity. It appears, however, that phosphorylation of the virion proteins in vivo occurs within the particle, possibly just before, during, or immediately after exit from the cell, since no evidence of phosphorylation of virus-specific proteins was obtained in infected cells. It is conceivable that the level of phosphorylation of virus-specific proteins in infected cells is low and is undetectable by the methods used. Analysis of the structural proteins of 32P-labeled intracellular and extracellular virus might resolve this question. The role of a protein kinase and of phosphorylated structural proteins in FMDV replication more
UITERATURE CITED 1. Akusjarvi, G., L Philipson, and U. Pettersson. 1978. A protein kinase associated with adenovirus type 2. Virology 87:276-286. 2. Bachrach, H. L. 1977. Foot-and-mouth disease virus: properties, molecular biology, and immunogenicity, p. 3-32. In J. A. Romberger (ed.), Beltsville Symposia in Agricultural Research. I. Virology in agriculture. Allanheld, Osmun and Co., Montclair, N.J. 3. Bachrach, H. L, R. Trautman, and S. S. Breese, Jr. 1964. Chemical and physical properties of virtually pure foot-and-mouth disease virus. Am. J. Vet. Res. 25:333342. 4. Baxt, B., and R. Bablanian. 1976. Mechanisms of vesicular stomatitis virus-induced cytopathic effects. I. Early morphologic changes induced by infectious and defective-interfering particles. Virology 72:370-382. 5. Baxt, B., and H. L. Bachrach. 1980. Early interactions of foot-and-mouth disease virus with cultured celas. Virology 104:42-55. 6. Bitte, L., and D. Kabat. 1974. Isotopic labeling and analysis of phosphoproteins from mammal ribosomes. Methods Enzymol. 30F:568-580. 7. Blair, G. E., and W. C. Russell. 1978. Identification of a protein kinase activity associated with human adenoviruses. Virology 86:157-166. 8. Cavanagh, D., D. V. Sangar, D. J. Rowlands, and F. Brown. 1977. Immunogenic and cell attachment sites of FMDV: further evidence for their location in a single capsid polypeptide. J. Gen. Virol. 35:149-158. 9. Clinton, G. M., B. W. Burge, and A. S. Huang. 1978. Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. J. Virol. 27:340-346. 10. Clinton, G. M., B. W. Burge, and A. S. Huang. 1979. Phosphoproteins of vesicular stomatitis virus: identity and interconversion of phosphorylated forms. Virology 99:84-94. 11. Imblum, R. I., and R. R. Wagner. 1974. Protein kinase and phosphoproteins of vesicular stomatitis virus. J. Virol. 13:113-124. 12. Kamata, T., and Y. Watanabe. 1977. Role for nucleocapsid protein phosphorylation in the transcription of influenza virus genome. Nature (London) 267:460-462. 13. Kelly, D. C., R. M. Elliot, and G. E. Blair. 1980. Phosphorylation of iridescent virus polypeptides in vitro. J. Gen. Virol. 48:205-211. 14. Kingsford, L., and S. U. Emerson. 1980. Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells. J. Virol. 33:1097-1105. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 15a.La Torre, J. I, M. J. Grubman, B. Baxt, and H. L
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Bachrach. 1980. The structural polypeptides of aphthovirus are phosphoproteins. Proc. Natl. Acad. Sci. U.S.A. 77:7444-7447. 16. Matheka, H. D., and H. L. Bachrach. 1975. N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, 0, and C. J. Virol. 16:1248-1253. 17. Monnier, C., and G. Devauchelle. 1980. Enzyme activities associated with an invertebrate iridovirus: protein kinase activity associated with iridescent virus type 6 (chilo iridescent virus). J. Virol. 35:444-450. 18. Moyer, S. A., and D. F. Summers. 1974. Phosphorylation of vesicular stomatitis virus in vivo and in vitro. J. Virol. 13:455-465. 19. Rubin, C. S., and 0. M. Rosen. 1975. Protein phosphorylation. Annu. Rev. Biochem. 44:831-887. 20. Sen, A., C. J. Sherr, and G. J. Todaro. 1977. Phospho-
rylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell 10:489-496. 21. Silberstein, H., and J. T. August. 1976. Purification and properties of a virion protein kinase. J. Biol. Chem.
251:3176-3184. 22. Silberstein, H., and J. T. August. 1976. Characterization of a virion protein kinase as a virus-specified enzyme. J. Biol. Chem. 251:3185-3190. 23. Wagner, G. G., J. L. Card, and K. M. Cowan. 1970. Immunochemical studies of foot-and-mouth disease. VII. Characterization of foot-and-mouth disease virus concentrated by polyethylene glycol precipitation. Arch. Gesamte Virusforsch. 30:343-352. 24. Wild, T. F., and F. Brown. 1967. Nature of the inactivating action of trypsin on foot-and-mouth disease virus. J. Gen. Virol. 1:247-250.
