Properties of a human cytomegalovirus-induced protein kinase

VIROLOGY

134,259-268 (1984)

Properties

of a Human Cytomegalovirus-Induced

S. MICHELSON,’ Unite’ de Virobgie

M. TARDY-PANIT,

Protein Kinase 0. BARZU*

AND

ilG&xle and *Unite’ de Biochimie des Regulations CeUulaires, Institut Pasteur, 75724 PARIS Cedex 15, France

Received July 18, 1989; accepted January 16, 1984 A human cytomegalovirus (HCMV)-induced polypeptide of 68,666 Da (~68) with protein kinase activity was identified using a monoclonal antibody (F6b) produced against HCMVinfected cell proteins. ~68 was detected by immunoprecipitation from 3 to 126 hr after infection and was induced by several strains of human and not simian CMV. Protein kinase activity was associated almost exclusively with nuclear HCMV-induced ~63. Enzyme activity with ATP and casein as phosphate donor and acceptor, respectively, exhibited an optimum pH between 6 and 6.5. It was Mgc+ dependent and CAMP independent. The Kin’ of 45 j&f at pH 6.5 indicated a relatively high affinity of p68 for the nucleotide. ~68 also transferred phosphate to phosvitin and light chains of F6b, as well as autophosphorylating at threonine and serine residues.

rected against HCMV-infected cell proteins. Among the McAb developed in our laboratory (Amadei et al, 1983), two are reactive with an early nuclear virus-induced protein. Using one of these (F6b), we were thus able to isolate and partially characterize the corresponding protein, the results of which are reported herein. This protein is shown to be a phosphoprotein with protein kinase activity. This observation is of particular interest in light of the fact that HCMV has recently been shown to contain a transforming DNA fragment (Nelson et a& 1982), and that transformation-related gene products in other systems so far described appear to be protein kinases (Collett et uL, 1978; Griffin et al, 1979; Smith et cd, 1979).

Human cytomegalovirus (HCMV) has a genome of 1.5-1.6 X lo* Da (Geelen et aL, 19’78; De Marchi et al, 19’78; Lakeman et a& 1979; Stinski et a& 1979; Westrate et aL, 1980) capable of coding for some 70 to 80 polypeptides. Some 35 viral structural polypeptides have already been described (Stinski et al, 1980; Schmidz et cd, 1980). Moreover, HCMV has a global stimulatory effect on host cell metabolism which renders discriminating between virus-specific proteins and host cell proteins stimulated by infection difficult. It is even more difficult to obtain individual HCMV-infected cell proteins in native form for biochemical analysis. Since monoclonal antibodies (McAb) are, by definition, directed against a single antigenic determinant, they provide a valuable tool for studying the structure of biologically important antigenic determinants, such as neutralizing epitopes, for characterizing viral proteins biochemically and for determining their functional role. Within the last year or so, several groups (Pereira et a,!., 1982; Goldstein et cd, 1982; Amadei et uL, 1983; R. Colimon, personal communication) have developed McAb dii To whom

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MATERIAL

AND

METHODS

untibody Establishment of the mxmmbnd Fs~. The details of the establishment and preliminary characterization of the McAb, F6b, used in this study are given elsewhere (Amadei et al, 1983). Mice were immunized by an intraperitoneal injection of whole, frozen-thawed, Ad-169 HCMV-infected cells and boosted at 4 weeks. Spleen cells

be addressed. 269

0042~6822134 $3.00 Copyright All rights

0 lSS4 by Academic Press. Inc. of reproduction in any form reserved.

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were fused with SP O/Ag 14 myeloma cells. Hybrids were selected in AT medium. F6b was detected by specific immunofluorescence of infected cells. Cells and virus. The following strains of CMVs were used. Human strains; Davis and Towne (generously supplied by Dr. S. Plotkin), Ad-169 (generously provided by Dr. J. Geelen), simian strain; Vervet (SGC) (American Type Cell Collection) and simian-like strain; Colburn (Huang et a& 1978) (provided by Dr. S. Plotkin). All viruses were maintained in MRC-5 human lung fibroblasts (Jacobs et aL, 1970) grown in Eagle’s basal medium (BME) supplemented with 10% aseptic calf serum (Tissue Culture Service, Slough, U.K.), 0.11% bicarbonate, and 40 mMTricine; (N-[2-hydroxy1,1-bis(hydroxymethyl)ethyllglycine) buffer. Antibiotics (50 pg streptomycin and 200 IU penicillin/ml) were only used during radioactive labeling periods (see below).

AND

Antigen extraction. Infected or uninfected cells were washed twice in situ with phosphate-buffered saline (PBS) containing calcium and magnesium ions (complete PBS), scraped into complete PBS containing lop4 1M phenylmethyl sulfonyl fluoride (PMSF) and low4 diisopropylfluorophosphate (DFP), and extracted in high salt, high pH buffer containing 0.5% NP-40 (Michelson et aL, 1979). In some instances, nuclei were separated from cytoplasm by swelling cells in hypotonic buffer, adding NP-40 (final concentration, 0.5%) and douncing ten times (Michelson et al, 1979). Nuclei were sedimented off at 800~ for 5 min at 4” and the resulting supernatant was taken as the cytoplasmic fraction. Nuclei were extracted as above. All extracts were centrifuged at 15,000~ for 15-30 min at 4” and frozen at -70” until used. For phosphotransferase activity experiments antigen was immediately immunoprecip-

Time

a

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post-Infection

(hours)

b

FIG. 1. Kinetics of appearance of p68 (A) and associated protein kinase activity (B). (a) SDSPAGE analysis of polypeptides immunoprecipitated with F6b from nuclear extracts of [&?3]methionine-labeled, uninfected (M) cells and cells infected for the times indicated under each lane. “P” after the time in hours indicates cells which were maintained in the presence of phosphonoacetic acid. (b) Protein kinase activity detected using immunoprecipitates of whole, infected cell extracts sampled at various times after infection and expressed as picomoles minute-’ milligram-’ of the original extract used in precipitation (all extracts were precipitated in a ratio of 0.2 mg extract/5 ~1 of F6b ascites fluid/50 pl of a 10% suspension of S WW~W).

HUMAN

CYTOMEGALOVIRUS-INDUCED

itated and stored bound to Staph&coccus aurew. Immurwprecipitaticm Precipitation was done using 5 ~1 of ascites fluid per 200 pg of extracted protein. Antigen and F6b were incubated with shaking for 1.5 hr at room temperature. Protein A was added as a 10% suspension of formaldehyde-fixed, heatinactivated Cowan serotype I S. aureus (50 ~1/5 ~1 of ascites fluid). Antigen-antibody complexes were washed 3X in NETS (Kessler et aL, 1975) and stored in this buffer as a 10% suspension at 4”, or were dissociated in electrophoresis buffer by heating for 5 min at 100”. Immunoprecipitates were analyzed on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) resolving gels migrated at 30-35 mA. When necessary, fluorography was performed according to Bonnar and Laskey (1974), except that only one DMSO (dimethyl sulfoxide) bath of 15 min at 37” and one DMSO f 20% PPO (2,5-diphenyloxazole) bath of 30 min at 37” were used. Radioactive label&~ of cells Infected cells sampled 1 hr p.i. (postinfection) were deprived in methionine for 15 min, and then adsorbed with virus in a suspension containing 10 &i/ml of [35S]methionine. For the other infected cell samples, virus was adsorbed for 1 hr, and then growth medium was added and all cells were deprived in methionine for 15 min before being labeled for 2 hr, prior to sampling at 3, 9, 24, 48, 72, and 96 hr p.i. Uninfected cells were labeled in a similar fashion. Labeling medium consisted of methionine-free Eagle’s minimum essential medium containing 10 &Yrnl of [?S]methionine (Amersham, sp act 1300 CVmM). Labeling of phosphoproteins was carried out for 3 hr in phosphate-free Dulbecco’s modified medium containing 100 &i/ml of [32P]orthophosphate. Protein k&use reaction Unless otherwise stipulated, testing for protein kinase ,activity was carried out using 50 ~1 of bacteria-bound enzyme in a mixture (100 ~1 final volume) consisting of 22 mlK potassium phosphate buffer, pH 6.8 (Kpi), 5 mM MgS04,0.15 mMEDTA, 3 mM@Me, 0.1 miV ATP, 3 &i [-Y-~“P]ATP, and 1 mg/ml casein. When immunoprecipitates were to be an-

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alyzed in SDS-PAGE, casein was not included in the reaction mixture. Immunoprecipitates were washed as indicated above, suspended in the reaction mixture, and incubated for 30 min at 30”. The reaction was stopped with 10 ~1 of 0.3 m&f EDTA and immunoprecipitates were removed by centrifugation at 5OOOgfor 4 min at 4O. Supernatants were precipitated with cold 5% TCA (trichloroacetic-acid). The precipitates were dissolved in 1 N NaOH, reprecipitated with TCA, and washed by filtration on Whatman GF/C filters. After a further wash in ethanol, filters were dried and counted in Packard scintillation liquid. Immunoprecipitates for gel analysis were washed 3X in KPi buffer, resuspended

I

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FIG. 2. Turnover of p68. (a) Cells infected for 96 hr were deprived in methionine for 10 min and then were pulse-labeled with [SsS]methionine (100 &i/ml in methionine-free medium without serum) for 1 min (1) and chased in methionine-complete medium for 1.5 (2), 4 (3), and 9 min (4). The same amount of protein (determined according to Bradford, 1976) was immunoprecipi$ated for each time. (h) Ninety six hour infected cells were deprived in methionine for 15 min, and then puke-labeled for 30 min (100 pCi/ml [85S]methionine in methionine-free medium), at the end of which time they were placed in complete growth medium with 10% calf serum and chased for 7 (l), 24 (2), and 48 hr (3) before being extracted and immunoprecipitated with F6b. The same amount of protein was precipitated for each time.

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in electrophoresis buffer, and analyzed in 10% SDS-PAGE as above. Estimation of sedimentation coq@ient. pS]Methionine-labeled antigen was prepared and extracted as above from cells infected for 120 hr. The antigen was layered on top of a 5-25s sucrose gradient made in PBS and spun in a Beckman SW-41 rotor at. 35,000 rpm for 17.25 hr at 4”. Fractions were collected from the bottom of the tube. Each fraction was immunoprecipitated with F6b and then analyzed by SDSPAGE. Electropkesis of ‘2P-labeled amino acids. Following the protein kinase reaction using immunoprecipitates in the absence of casein, 32P-labeled material was detached with 0.1% NH4HC03 containing 0.1% SDS. The sample was vacuum-dried, resuspended in 6 N HCl, and hydrolyzed overnight at 110”. After evaporating off the HCl, preparations were resuspended in electrophoresis buffer. Phosphoamino acids were separated at pH 3.5 (50 ml acetic acid and 5 ml pyridineiliter) for 45 min at 1000 V on cellulose-coated thin-layer plates

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(Macherey-Nagel & Co). Standards were stained with cadmium-ninhydrin reagent (Drickamer and Mamon, 1982), and plates were exposed to Kodak X-Omat film for 1 week. RESULTS

Ioknt$icution of the Polypeptide(s) with F6b

Reuctive

To find out where and when the polypeptide recognized by F6b could first be detected by immunoprecipitation, infected cells were pulse-labeled with r5S]methionine and sampled at 1, 3, 9, 24, 43, 72, and 96 hr p.i. (see Materials and Methods for details). In addition, one infected sample was maintained in the presence of phosphonoacetic acid (PAA, 200 pg/ml) for 70 hr p.i., then was labeled for 2 hr in the presence of PAA in order to examine the “early” nature of the protein. Cells were separated into nuclear and cytoplasmic fractions before being immunoprecipitated.

FIG. 3. Influence of Mg2+ concentration on ~68 protein kinase activity. F6b immunoprecipitates were incubated in a protein kinase reaction mixture at pH 6.8 in which the concentration of M%+ (in the form of MgSO,) varied from 0 to 20 mAK Results are expressed as picomoles minute-’ milligram-’ of cell extract immunoprecipitated.

HUMAN

CYTOMEGALOVIRUS-INDUCED

F6b immunoprecipitated a polypeptide of 68 kDa (~68) from the nuclei of infected cells as early as 3 hr p.i., but not at 1 hr p.i. (Fig. la), confirming previous observations by immunofluorescence (Amadei et a& 1983). No polypeptides were precipitated by F6b from cytoplasmic preparations until 48 hr p.i., at which time only ~68 was found (results not shown). ~68 could also be detected in PAA-blocked cells. The amount of ~68 in both cellular compartments increased with time after infection, and represented as much as 0.5% of the protein mass 96 hr p.i., as estimated from the percentage of radioactivity recovered by immunoprecipitation. F6b did not react with uninfected cells. When cells infected for 120 hr were labeled with $P]ortophosphate for 3 hr, F6b immunoprecipitated a =Pi-labeled, 68-kDa protein from such cells (results not shown). Extracts of [%]methionine-labeled cells infected for 120 hr were separated by centrifugation in 525% sucrose gradients. F6bimmunoprecipitated protein from fractions corresponding to a sedimentation coefficient (s&J of 6.9 using catalase and bovine serum albumin as markers. To estimate the turnover of ~68, cells infected for 96 hr were labeled with [%S]methionine in methionine-free medium for 1 min, and either extracted immediately or chased in methionine-complete medium for 1.5, 4, or 9 min before extraction and immunoprecipitation. ~68 had already incorporated labeled precursor after 1 min of labeling and, following 1.5 min of chase, the incorporation had already reached a maximum (Fig. 2a). To determine the halflife of ~68, cells were pulse-labeled for 30 min at 96 hr p.i. and chased for 7, 24, and 48 hr. As can be seen in Fig. 2b, ~68 is extremely stable.

PROTEIN

(1.5 mg/ml), accepted phosphate. No phosphate transfer occurred when the following material was used in the protein kinase reaction mixture, S. aureUS alone, bacteria coated with F6b, bacteria incubated with F6b and uninfected cell extract, or bacteria incubated with infected cell extract alone. The protein kinase reaction with casein as acceptor had an optimum pH between 6 and 6.5, and declined rapidly at more acid and alkaline pH values (Fig. 3). It was dependent on the presence of M$+ ions (optimum concentration of 5 mlM), and excess divalent cations inhibited the kinase activity (Fig. 4). The reaction was CAMP independent, exhibiting a relatively high affinity for ATP as phosphate donor (KkTp at pH 6.5 = 45 p&f) (Fig. 5). Two minutes of heating F6b-p68 complexes at 100“ before the reaction eliminated all subsequent transfer.

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In vitro Phosphmylation Protein A-bound immunoprecipitate of ~68 from Ad-169 HCMV-infected cells was capable of transferring phosphate to itself and, to a lesser extent, to the light chains of F6b. When incorporated into the protein kinase reaction mixture, casein (1 mg/ml) and phosvitin (1.5 mg/ml), but not histones

263

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I

I

6.5

7.0

I

7.6

I

6.0

PH FIG. 4. Effect of pH F6b immunoprecipitates conditions and reaction Materials and Methods, of MgB” was fixed at buffer was varied from as picomoles minute-’ itated cell extract.

on p68 protein kinase activity. were run under the same mixture as described under except that the concentration 5 m&f and the pH of the KP, 5.5 to 8.0. Results are expressed milligram-’ of immunoprecip-

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Protein kinase activity was detected as early as 15 hr after infection and increased rapidly to attain a plateau between ‘72 and 96 hr pi. (Fig. lb). Increase in kinase activity roughly paralleled that of the accumulation of ~68 in infected cell nuclei (Fig. la), with which transferase activity was predominantly associated throughout infection. The enzyme could be conserved in the form of S. aureu-s-bound immunoprecipitates for as long as 6 weeks at 4” with only slight loss of activity. The only amino acid residues phosphorylated, as determined by electrophoresis of autophosphorylated p68 on thin-layer cellulose plates after acid hydrolysis, were threonine and serine (Fig. 6). Antigen Induction in Werent System The Ad-169 strain of infected cells was used for immunization of the mouse which gave rise to the hybridoma secreting F6b. We therefore examined the capacity of other human and two monkey strains of CMV to induce this protein. For this purpose, cells were infected at a m.o.i. of 1

AND

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with human strains Towne, Davis, and Ad-169, and simian strains Colburn and SGC. When cytopathic effects gained 90% of the culture, cells were pulse-labeled for 2 hr with [?S]methionine (10 pCi/ml) and extracted without separating nucleus from cytoplasm. As can be seen in Fig. 7a, all human strains tested induced the ~68. A questionable induction of p68 occurred with the Colburn strain, but none was found in cells infected with SGC monkey virus. When unlabeled immunoprecipitates from the above systems were incubated in the protein kinase reaction mixture without casein, ~68 from cells infected with human CMV became phosphorylated, as did proteins having the same molecular weight as the light chains of F6b (Fig. 7B). No phosphotransferase activity was demonstrated in immunoprecipitates of either Colburn- or SGC-infected cells. To try to determine whether the protein kinase recognized by F6b was the same as that found in the virion by Mar et al. (19’78), we performed two experiments. First, purified [35S]methionine-labeled virus was dissociated in 1% SDS, diluted to a final

6-

- IO2 V 4-

I (Inki’) CATa FIG. 5. Dependence of protein kinase activity on ATP concentration. The reaction was carried out at pH 6.5 with 5 n&f a6pp”. ATP concentrations were varied from 21 to 922 a The reciprocal of velocity (pmol min-’ mg-’ of immunoprecipitated cell extract) was plotted against the reciprocal of the ATP concentration according to Lineweaver-Burk.

HUMAN

CYTOMEGALOVIRUS-INDUCED

PROTEIN

KINASE

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DISCUSSION

0 FIG. 6. Autoradiograms of q-labeled phosphoamino acids separated by thin-layer electrophoresis at pH 3.5. Hydrolysate was combined with 2 mM of each marker phosphoamino acid before electrophoresis. Markers were located by ninhydrin staining. 9, freephosphate; Ser, serini; Thr, threonine; Tyr, tyrosine; 0, origin.

concentration of 0.1% SDS, and immunoprecipitated with ‘F6b. As a control of the integrity of ~68 following SDS treatment, infected cell extract was similarly treated and immunoprecipitated. Second, FGb-reacted, unlabeled material from SDStreated purified virus was run in a protein kinase reaction. Figures 8a and b show that McAb F6b failed to immunoprecipitate ~68 from purified virus although it continued to precipitated ~68 from SDS-treated, infected cell extracts. In addition, and consistent with the latter lack of reactivity, no protein kinase activity was detected in immunoprecipitated virus preparations, whereas SDS-treated ~68 continued to transfer phosphate.

HCMV monoclonal antibody F6b reacts with an HCMV (Davis, Towne, or Ad-169)infected, cell-derived polypeptide of 68 kDa (~68) which has protein kinase activity. Simian CMVs do not induce ~68. Electrophoresis of ~68, as well as its behavior in sucrose gradients, suggests a dimeric structure for the protein kinase. Preliminary experiments showed that the enzyme could easily be released from immunoprecipitates by 6 1M urea, and its enzymatic activity could be almost completely recovered after removal of the urea. ~68 protein kinase activity remains associated with nuclear ~68 throughout infection, despite the fact that ~68 can be detected by immunofluorescence (Amadei et I& 1983) and immunoprecipitation in the cytoplasm from 48 hr p.i. onwards. Lack of enzymatic activity in the cytoplasm is not due to binding of regulatory and catalytic subunits, since CAMP had no effect. The conformation of ~68 in the cytoplasm might result in steric hindrance of enzyme activity when F6b binds to the polypeptide. There may also be a dissociation of the enzyme from the F6b antigenic determinant-bearing polypeptide in the cytoplasm. Mar et al. (1981) have described a protein kinase activity associated with NP-40-disrupted, purified virions. We performed both immunoprecipitation and protein kinase experiments with purified virions in an attempt to ascertain whether FGb-reactive kinase and that of the virion were the same. Both experiments appeared negative. In addition, several parameters of the phosphotransferase reaction using ~68 differ from those described by Mar et al, (1979). ~68 does not transfer phosphate at high pH nor does it function in the presence of manganese. Nelson et aL (1982) have recently described a transforming DNA fragment within the HCMV genome. To date, transforming gene products of both RNA (Collett et al, 1978) and DNA viruses (Griffin et al, 1979; Smith et al, 1979) appear to be protein kinases. During the early phase of lytic infection, HCMV effectively induces a series of transient, “transformation-like”

MICHELSON,

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TARDY-PATIT,

a

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b

FIG. 7. Induction of p68-associated protein kinase activity in different systems. (a) F6b (lane 1) or anti-polio McAb (lane 2) immunoprecipitates of [86S]methionine-labeled, uninfected cells (0) or cells infected with CMV strains Colburn (C), Ad-169 (A), Davis (D), and Towne (T), and with vervet CMV SGC (S). Lanes 2 are immunoprecipitated of the same materials with an anti-polio McAb. (b) SDS-PAGE analysis of proteins phosphorylated using F6b immunoprecipitates from cells infected with various strains of human and simian CMV. Human strains (A) Ad-169, (M) Mira, (D) Davis, (T) Towne; and simian strains (C) Colburn and (S) Cercopithecus. Casein was omitted from the protein kinaae reaction mixture. The positions of metabolically radiolabeled F6b IgG light chains (LC) are indicated.

3%

v

a

changes in the host cell, such as loss of contact inhibition, disorganization of the cytoskeleton (L&se et d, 1982; Ihara et al, 1982; Albrecht et a& 1983), and stimulation of host cell nuclear and mitochondrial DNA synthesis (St. Jeor et d, 19’74; Furukawa et aL, 1976). These changes are in part attributable to proteins appearing within the first 5 hr after infection (Tanaka et C& 1975;

32P

b

FIG. 8. (a) Immunoprecipitation of purified HCMV with F6b and analysis on SDS-PAGE. Whole [85S]methionine-labeled virus (V), was dissociated in

1% SDS and then diluted ten times with NETS (Material and Methods) and immunoprecipitated with F6b. Whole purified virus (9) was dissociated in highpH Tris buffer with 1% SDS, diluted to 0.1% SDS, immunoprecipitated with F6b, and used in the protein kinase reaction. Only trace amounts of [S6S]methionine-labeled ~69 were precipitated and showed marginal phosphate transfer position of ~68. (b) Ninety six-hour, [86S]methionine-labeled, infected cell extract was treated with 1% SDS, diluted to 0.1% SDS, and immunoprecipitated with F6b. The resulting precipitate (SDS) was resolved in 10% SDS-PAGE along with untreated infected cell immunoprecipitated with F6b (0).

HUMAN

CYTOMEGALOVIRUS-INDUCED

Kamata et al, 1978; Garnett et cc& 1979; Furukawa et a& 1973), some of which are coded for in the region of the CMV-transforming DNA fragment. An in vitro translation product of messenger RNA hybridizing in this region demonstrates protein kinase activity and is immunoprecipitated by a McAb directed against one of the immediate early proteins (J. Geelen, personal communication). Numerous other herpesviruses possess protein kinases (Lemaster and Roizman, 1980; Blue and Stobbs, 1981; Kamata et ak, 1981; Rubenstein et &, 1972), and share many properties in common: Me dependence, CAMP independence (except for EBNA-associated kinase activity), preferentially autophosphorylating or transferring phosphate to viral proteins, and affinity for acidic exogenous protein substrates. Herpesvirus protein kinases, like those of other DNA viruses, phosphorylate only at threonine and serine residues. This property seems to set DNA virus protein kinases apart from RNA viral protein kinases which also phosphorylate tyrosine. In conclusion, this is first characterization and attribution of a functional role to an HCMV-induced protein using a monoclonal antibody. ACKNOWLEDGMENTS We wish to thank Dr. Axe1 Garapin, Dr. Luc Montagnier, and Dr. Michele Bouloy for their material assistance; Dr. Florian Horaud, Dr. Radu Crainic, and Dr. Bruno Blonde1 for helpful discussions; Nicole Perrin for her secretarial skills; and Nathalie Fousse for her photographic expertise. This work was supported by Grants CLR 801015, 124016, and 127018 accorded by the Institut National de la Sante et de la Recherche MBdicale, and Grant 82.L.1316 accorded by the Minis&e de l’Indust.rie et de la Recherche. REFERENCES ALBRECHT, T., SPEELMAN, D. J., and STEINSLAND, 0. S. (1983). Similarities between Cytomegalovirus induced cell rounding and contraction of smooth muscle. Life Sci 32,2273-2278. AMADEI, C., TARDY-PANIT, M., COUILLIN, P., COULON, M., CABAU, N., BouB, A., and MICHELSON, S. (1983). Kinetic study of the development and localization of Human Cytomegalovirus (HCMV)-induced antigens using monoclonal antibodies. Ann ViroL (Inst. Pasteur) E 134, 165-180.

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