Human Cytomegalovirus-associated DNA Polymerase and Protein ...
J. gen. ViroL ( 198 t), 57, 149-156. Printed in Great Britain
149
Key words: human cytomegalovirus/DNA polymerase/protein kinase
Human Cytomegalovirus-associated DNA Polymerase and Protein Kinase Activities By E N G - C H U N M A R , * P R A V I N C. P A T E L AND ENG-SHANG HUANG
Department of Medicine and Cancer Research Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A. (Accepted 13 July 1981) SUMMARY
Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgC12. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)~2_ls and poly(dC).oligo(dG)12_18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KC1 from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 #g/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)~2_~s was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0-3 M with either activated calf thymus DNA or poly(dA).oligo(dT)~2_~8 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgC12, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32p]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion. INTRODUCTION
The association of enzyme activities with purified virions, such as DNA-dependent RNA polymerase, DNA-dependent DNA polymerase, reverse transcriptase, protein kinase and so forth has been frequently demonstrated in various virus systems (McAuslan, 1974; Tan, 1975). Among the herpes group viruses, an endogenous DNA polymerization activity was found with Epstein-Barr virus (Goodman et al, 1978), and protein kinase activities were detected with equine herpesvirus (Randall et aL, 1972) and herpes simplex virus (LeMaster & Roizman, 1980; Rubenstein et al., 1974). We report here that DNA polymerase and protein kinase activities are also detected in the purified virions of human cytomegalovirus (HCMV). METHODS
Cell line and virus. The WI-38 strain of human fibroblast (Hayflick; obtained from American Tissue Culture Collection, CCL-75, Passage 24-28) was used for the entire study. The Towne Strain of HCMV was adapted and propagated by infection of WI-38 roller bottle 0033-1317/81/0000-4625 $02.00© 1981SGM
150
E.C.
MAR,
P. C. P A T E L
AND
E.
S. H U A N G
cultures at a multiplicity of 1 to 2 p.f.u./cell. Virus was harvested from the extracellular fluid as described previously (Huang et al., 1973). Labelling and purification of virus. To label virus proteins, monolayers were washed twice with Hanks' balanced salt solution 20 h after infection, and 30 ml low-phosphate medium (Hanks' balanced salt solution containing amino acids, glucose, 4 % foetal calf serum, 10 mM-glutamine, 2% NaHCO3, 100 units/ml penicillin, 100 gg/ml streptomycin sulphate and 10 -5 M of phosphate) containing 30 gCi/ml [32p]orthophosphate were added. For non-labelled virions, the infected cells were grown in minimum essential medium containing 4 % foetal calf serum, supplemented with 100 units/ml penicillin, 100 gg/ml streptomycin sulphate and I0 mM-glutamine. The extracellular virus released from infected cell cultures was collected every 4 days after infected cells showed 100 % c.p.e. The extracellular fluid was then subjected to centrifugation at 6000 rev/min in a Sorvall GSA rotor for 20 min at 2 °C. The cell-free virus particles in the supernatant were pelleted in a Spinco T-19 rotor at 18 000 rev/min for 90 min. The virus pellet was suspended in ice-cold TBS (0.05 tris-HC1 pH 7.4 0-15 M-NaC1) and dispersed by vortexing until no particulate material was visible. The suspension was layered on to a gradient of 10 to 50 % sucrose and centrifuged at 26 000 rev/min in a Beckman SW27 rotor for 1 h at 2 °C. The opaque virus band was collected and diluted with 1 vol. TBS. The virus suspension was then layered on to a preformed CsC1 gradient (1.16 to 1.37 g/ml) in TBS containing 5 mM-EDTA, and centrifuged again at 26000 rev/min for 1 h at 4 °C. The virus particles banded at a density of 1.22 to 1-24 g/ml. The purified virus was then extensively dialysed against cold TBS, and pelleted by centrifugation at 27 000 rev/min for 60 min. The freshly purified non-labelled virus was used immediately for the study of the two enzymic activities.
Preparation of enzyrne extracts DNA polymerase study. For this study the purified virus was suspended and solubilized in a hypotonic detergent buffer A [10 mM-tris-HC1 pH 7.8, 1 mM-MgCI 2, 1 mM-DTT, 0-5 % Nonidet P40 (NP40), 0.4 mM-phenylmethylsulphonyl fluoride (PMSF)] and kept on ice for 30 min. To the mixture was added an equal volume of 3.8 M-NaC1 in buffer B (20 mM-tris-HC1 pH 7.8, 10 mM-MgC12, 8 mM-DTT, 0.4 mM-PMSF) to give a final salt concentration of 1.9 M-NaCI in order to further dissociate the enzyme component from other virion complexes. This high salt mixture was centrifuged at 25 000 rev/min for 2 h at 2 °C. The clarified supernatant extract was collected and dialysed against buffer C (50 mM-tris-HC1 pH 7-8, 1 mM-EDTA, 1 mM-DTT, 0.4 mM-PMSF, 5% glycerol) to a salt concentration less than 0.01 M-NaC1, as determined by a salt conductivity meter. The dialysed material was loaded on to a phosphocellulose column (1.6 x 10 cm) and eluted with a gradient of 0.1 to 0.7 M-KC1 in buffer C. Protein kinase activity study. For this study the purified virions were dissociated in a buffer containing 50 mM-tris-HC1 pH 7.8, 100 mM-KC1, 1 mM-DTT, 0.5 % NP40 and 5 % glycerol, with the aid of vortexing for 30 s. The dissociated virions were subjected to centrifugation at 15 000 rev/min for 20 min at 2 °C and the pellet was reextracted with the same buffer three times. The supernatants were combined and used for the protein kinase activity study. DNA polymerase assay. Three systems were used in the DNA polymerase activity assay, as described elsewhere (Huang, 1975; Mar et al., 1978; Mar & Huang, 1979). In system A, activated calf thymus DNA was used as the template primer. A 0.1 ml amount of activated calf thymus DNA (200/~g/ml) was mixed with 0.1 ml of stock reaction mixture containing 0.1 M-tris-HCl pH 7.8, 25 mM-MgCI 2, 1 mM-DTT, 125 mg/ml bovine serum albumin (BSA), 0.25 mM each of dATP, dCTP, dGTP, and 1/.tCi [3HldTTP (48 Ci/mmol) together
HCMV-assoeiated enzyme activities
151
with 50 /A of enzyme fraction. The reaction was carried out at 37 °C for 1 h and was terminated by chilling in ice and adding 2 ml 20 % ice-cold trichloroacetic acid (TCA). The acid-precipitable radiolabelled material was collected by suction filtration on glass fibre paper (GF/C) or a nitrocellulose filter. It was then washed with 5% TCA. After drying, the radioactivity on the filters was determined using a liquid scintillation counter. In system B the reaction conditions employed were similar to those in system A, except that cold dATP, dCTP and dGTP were omitted, and the synthetic template primers, poly(dA), oligo(dT)12_lS, poly(rA).oligo(dT)12_lS or oligo(dT)12_~8 (2 #g for each reaction), were used instead of activated calf thymus DNA. In system C, 2 pg poly(dC).oligo(dG)~2_18 or poly(rC)-oligo(dG)~2 ~s, 0-01 mM-dGTP and 1 /,tCi [3H]dGTP (42 Ci/mmol) were used in the reaction mixture. Protein kinase assay. The standard reaction for the assay of protein kinase activity was conducted by incubating 10 pg of enzyme extract with 10 pCi [7-a2p]ATP (sp. act. 4200 Ci/mmol) in the presence or absence of histone, caesin, BSA or protamine sulphate (500 /ag/ml) in a total vol. of 0.2 ml containing 50 mM-tris-HC1 pH 7-8, 100 mM-KCI, 20 mM-MgC12, 1 mM-DTT, 0.5 % NP40 and 5 % glycerol. The reaction was carried out at 37 °C for 30 min and terminated by adding 2 ml 20% ice-cold TCA. The precipitated materials were treated in a similar manner as employed in the DNA polymerase assay. In other experiments the reaction condition was modified to determine the effect of pH; Mg z+, Mn 2+, Ca 2+, Z n 2+ and Co 2+ concentrations. Polyaerylamide gel eleetrophoresis. The 32p-labelled proteins were identified by SDSPAGE through 9 % polyacrylamide gels in a tris buffer system as described by Laemmli (1970). All samples were electrophoresed under reducing conditions. The mol. wt. of the polypeptides were determined by electrophoresing marker proteins with known mol. wt. (Bio-Rad) on the same gels and comparison of their relative migrations was as described elsewhere (Weber & Osborn, 1969). The gels were stained with 0-05 % Coomassie Brilliant Blue in an aqueous mixture containing 20% methanol and I0% acetic acid. The gels were destained in the same solvent system with occasional shaking. The gels were then vacuum-dried on to 3MM Whatman chromatography paper and exposed to X-ray film (Kodak RP1R2) for autoradiographs. Protein determination. The protein concentration was determined by the fluorescence assay of Bohlen et al. (1973). BSA was employed as a standard for protein determination. RESULTS
HCMV-assoeiated DNA polymerase As shown in Fig. 1 (a), DNA polymerase activity derived from NP40-disrupted HCMV virions was sharply eluted at 0.18 to 0.2 M-KC1 in phosphocellulose chromatography. The partially purified DNA polymerase could use the synthetic templates poly(dA), oligo(dT)lz-18, poly(dC).oligo(dG)12_18 as well as activated calf thymus DNA as the template primers (Table 1). However, the virus-associated DNA polymerase was unable to use poly(rA).oligo(dT)12_~s, poly(rC).oligo(dG)12_ls or oligo(dT)12_18 as a template primer (Table 1). This implied that the virus-associated DNA polymerase was an enzyme with a character distinct from those of reverse transcriptase (Baltimore, 1970), RNA-dependent DNA polymerase from lymphoblastoid cells (Lewis et al., 1974) and terminal deoxynucleotidyl transferase (Chang, 1971; Chang & Bollum, 1970). Divalent cations are essential for the DNA polymerase activity, as they are for the herpesvirus-induced DNA polymerase and host cell enzymes (a and fl polymerases) (Mar & Huang, 1979). Only part of the enzyme activity was maintained when Mg 2+ was replaced by Mn 2+. Around 5 to 10 mM-Mg 2÷ was needed for maximum DNA polymerization.
152
E . C. M A R , P . C. P A T E L
I
A N D E . S. H U A N G
I
I
(b) 1 2
0"5--
0 X
3
4
172--
0.4-
126-,_ 100--
0.3-
89 f
0.2-
68~ 67/59 f
0. l -
50-45--40~ 38~ 35 /
n
29-24-20
40
60
80
100
Fraction number Fig. 1. (a) Fractionation of virion-associated DNA polymerase in phosphocellulose chromatography. The purified virions from HCMV-infected WI-38 cells were dissociated with buffer A (10 mra-tris-HC1 pH 7.8, 1 mM-MgCI2, 1 mM-DTT, 0.5% NP40, 0.4 mM-PMSF) and kept on ice for 30 min, and then an equal volume of 3.8 M-NaCI in buffer B (20 mM-tris-HCl pH 7.8, 10 mM-MgC12, 8 mM-DTT, 0.4 mM-PMSF) was added. The resulting mixture was collected and dialysed extensively against buffer C (50 mM-tris-HCl pH 7-8, 1 rnM-EDTA, 0.4 mra-PMSF, 1 m~I-DTT, 5 % glycerol). The dialysate was loaded on to a phosphocellulose column (1.6 x 10 cm), which had been previously washed with acid and alkali and equilibrated with buffer C. After the enzyme was absorbed, a gradient of 0.1 to 0.7 M-KC1 in buffer C (100 ml: 100 ml) was applied. Fractions of 2 ml were collected and every other fraction was assayed for DNA polymerase activity using activated calf thymus DNA as the template palmer. The enzyme activity was eluted as a sharp peak at 0-18 to 0-2 M-KC1. O, DNA polymerase activity; II, salt concentration. (b) Autoradiography of phosphorylated polypeptides analysed in 9% SDS-PAGE. The in vitro phosphorylation of proteins was conducted by incubating 10 #g of specified proteins (determined according to the procedure of Bohlen et aL, 1973) with 10/~Ci [7-32p]ATP (sp. act. 4200 Ci/mmol) in a total vol. of 0.2 ml containing 50 mM-tris-HCl pH 7.8, 100 mM-KC1, 20 mM-MgCI2, 1 mM-DTT, 0.5% NP40 and 5% glycerol at 37°C for 30 rain. The reaction was terminated by adding 2 ml cold 20 % TCA. Precipitated materials were centrifuged at 8000 rev/min for 10 min and washed with 95% ethanol. After drying, 0-2 rnl buffer (62.5 mM-tris-HC1 pH 6.7, 5 mM-fl-mercaptoethanol, 2 % SDS, 15 % glycerol) was added to each reaction tube, and boiled for 2 min before gel electrophoresis. Each sample loaded on the gel contained 200000 ct/min. Lane 1, NP40-treated WI-38 labelled with [7-32p]ATP. Lane 2, Residual protein in the pellet from the 15000 rev/min centrifugation of NP40-treated virions labelled with [7-32P]ATP, as described in the text. Lane 3, Supernatant fraction of NP40-treated virions (centrifuged at 15000 rev/min) labelled with [y-32p]ATP. Lane 4, Phosphorylated virus labelled with [32p]orthophosphate in vivo as described elsewhere (Huang et al., 1973). Numbers indicate mol. wt. x 10-3. The blurred background above the region of 172K was due to the presence of 32p-labelled polynucleotides (DNA and RNA), which do not enter the 9 % acrylamide gel.
The virus-associated D N A p o l y m e r a s e was similar to ct-polymerase as j u d g e d b y salt elution in p h o s p h o c e l l u l o s e c h r o m a t o g r a p h y (about 0 . 2 M-KC1), resistance to P A A , and sensitivity to a m m o n i u m sulphate. H o w e v e r , the virus-associated e n z y m e was m o r e sensitive t h a n ct-polymerase to P A A w h e n poly(dA).oligo(dT)12_18 was used as the t e m p l a t e primer. The f o r m e r e n z y m e activity, but not the latter, was inhibited by 50 % at a c o n c e n t r a t i o n o f
HCMV-associated enzyme activities 100
~\
I
~.~
I
153
I I I 1'80' l~~ I I I I (b)| (a)
FL
/, :\
.N_ E
50
/I
-
100 /:~ "~
©
< zr~
~,
, ,,,~,
x\
",o
\\
20
40
60
80
100
PAA (/ag/ml)
lO 30
60
100
200 2 s o 3oo
(NH4)2SO4 (mM)
Fig. 2. Effects of (a) phosphonoacetic acid (PAA) and (b) ammonium sulphate concentration on virus-associated (0, x) and virus-induced (&, O) DNA polymerase activity. The assays were carried out as described in the text, except that PAA was added to the reaction mixture at a final concentration of 20 to 100 Bg/ml and (NH4)2SO 4 at 10 to 300 mM. Solid lines indicate that activated calf thymus DNA was used as the template primer for enzyme assays; dotted lines indicate that poly(dA).oligo(dT)12_ls was used. Activity is expressed relative to that obtained in the absence of (NH4)2SO 4 or PAA as 100 % control.
T a b l e 1. Template primer specificities of human cytomegalovirus-associated DNA polymerase
Assay system A* A A Bt B B C1: C
Template primer Activated calf thymus DNA Native calf thymus DNA Denatured calf thymus DNA Poly(dA). oligo(dT),2_18 Poly(rA). oligo(dT)12_la Oligo(dT)x2_ls Poly(dC).oligo(dG)tz_ls Poly(rC). oligo(dG)l 2_Is
Substrate [3H]dTTP, cold dNTP [3H]dTTP, cold dNTP [3H]dTTP, cold dNTP [ 3H]dTTP, cold dTTP [ aH]dTTP, cold dTTP [ 3H]dTTP, cold dTTP [3H]dGTP, cold dGTP [ 3H]dGTP, cold dGTP
DNA polymerase activity (pmol) 264 4 9 210 1 0.7 189 1.5
* System A. Activated calf thymus DNA (ACTD) was used as the template primer. A 0.1 ml amount of ACTD (200 #g/ml) was mixed with 0.1 ml of stock reaction mixture containing 0.1 M-tris-HC1 pH 7.8, 25 mM-MgCI2, 1 mM-DTT, 125 mg/ml BSA, 0.25 mM each of dATP, dCTP, dGTP, and 1 #Ci [3H]dTTP (48 Ci/mmol), together with 50 #1 of enzyme fraction. Incubation was at 37 °C for 1 h and was terminated by TCA preparation as described previously (Mar & Huang, 1979). i" System B. Conditions were similar to those in system A except that cold dATP, dCTP and dGTP were omitted, and poly(dA), oligo(dT)l~_18 (2 ltg for each reaction) was used instead of ACTD. :~ System C. Two #g poly(dC).oligo(dG),2_xs, 0.01 mM-dGTP and 1 ltCi [3H]dGTP (42 Ci/mmol) were used in the reaction mixture. 50 p g / m l P A A . In addition, the H C M V - a s s o c i a t e d D N A p o l y m e r a s e was characteristically distinct f r o m v i r u s - i n d u c e d e n z y m e . A s s h o w n in Fig. 2 (a), the v i r u s - a s s o c i a t e d e n z y m e was, in general, relatively resistant to P A A and sensitive to a m m o n i u m sulphate. O n the other hand, H C M V - i n d u c e d D N A p o l y m e r a s e was sensitive to P A A and its e n z y m e activity c o u l d be stimulated at 60 m M - ( N H 4 ) 2 S O 4. F u r t h e r m o r e , the v i r u s - i n d u c e d e n z y m e eluted at 0 . 3 2 M in p h o s p h o c e l l u l o s e c o l u m n s ( M a r & H u a n g , 1979) while the virus-associated e n z y m e eluted at 0 . 1 8 to 0 . 2 M-KC1 (Fig. 1 a).
154
E.C.
MAR,
P . C. P A T E L
AND
E . S. H U A N G
HCMV-associated protein kinase
In addition to a DNA polymerase activity, a protein kinase activity was also found to be associated with the virion of HCMV. The protein kinase required divalent cation for its enzyme activity. Phosphorylation was high in the presence of Mg 2+ but relatively weak in the presence of M n 2+, and negligible with Co 2+, Z n 2+ o r C a 2+. The optimum assay condition for the protein kinase activity in our system was 10 to 20 mM-Mg 2+ at a pH range of 6.5 to 8.5. Activity was not stimulated by cyclic nucleoside monophosphates such as cyclic-AMP and cyclic-GMP at concentrations of 0.0001 to 1 mM (data not shown). Interestingly, the protein kinase preferred its own virion proteins as phospho-acceptor to protamine sulphate. In addition, historic, caesin and BSA were found to be poor substrates (data not shown). Comparison of in vivo and in vitro phosphorylated proteins in S D S - P A GE
A comparison of phosphorylated proteins derived from virions labelled with 32p in vivo and virion proteins labelled with [?-32p]ATP in vitro was performed. The results revealed that in SDS-PAGE, 10 phosphorylated polypeptides (172K, 150K, 100K, 89K, 67K, 53K, 50K, 39K, 38K and 24K) were detectable. On the other hand, 14 polypeptides (172K, 126K, 100K, 89K, 68K, 67K, 59K, 50K, 45K, 40K, 38K, 35K, 29K and 24K) were found to be phosphorylated by the labelling in vitro with the endogenous virus-associated protein kinase. Although the polypeptide patterns obtained from these two different 32p-labelling systems were not identical, seven proteins co-migrated as shown in Fig. 1 (b) (indicated by arrows). These proteins are polypeptides with mol. wt. of 172K, 100K, 89K, 67K, 50K, 38K and 24K. The procedures for extracting the endogenous protein kinase from virions still left some protein kinase activity in the pellet. Nevertheless, the in situ [~32p]ATP-labelled proteins of the supernatant and the pellet fraction revealed a similar polypeptide pattern as shown in lanes 2 and 3 of Fig. 1 (b). DISCUSSION
There are several characteristic similarities between virion-associated DNA polymerase and host cell a-polymerase such as chromatographic behaviour in phosphocellulose chromatography, ammonium sulphate inhibition and partial resistance to PAA. This raises the question of whether the purified virions were contaminated with the host cell enzymes. However, we do not believe this to be the case since we could not detect any DNA-polymerizing activity using the intact whole virion without dissociation with NP40, while virion-associated DNA polymerase activity was inhibited by 50% at 50 ~tg/ml PAA with poly(dA), oligo(dT)12-18 as the template primer, while host cell a- and fl-polymerase were resistant to PAA under the same condition (Mar et al., 1978). Finally, the host cell fl-polymerase, which elutes at 0.45 M-KC1 in phosphocellulose chromatography, was not detected at all. Assay of the protein kinase in the in vivo 32P-labelling system failed to reveal any detectable phosphorylated host cell proteins, nor were any polypeptides corresponding to virion phosphorylated proteins found in mock-infected cells although equal amounts (200 000 ct/min) of TCA-precipitable materials were applied to the gel as shown in Fig. l(b) (lane 1, host cell; lanes 2 and 3, viral). Lastly, after several cycles of purification including isokinetic and isopycnic centrifugation, the purified virus was found to be free of host cell contamination as judged by electron microscopic and immunological methods. Taken together, these results ruled out the possibility of host cell contamination in the purified virions. Another possibility is that the enzyme activities are due to contamination by mycoplasma. Miller & Rapp (1976) reported that mycoplasma-specified DNA polymerase activity could be enhanced to 130 to 180% by the presence of 25 mM-(NH4)2SO 4 but was resistant to PAA at
H C M V - a s s o c i a t e d e n z y m e activities
155
a concentration of 100 gg/ml. In contrast, our data showed that the virus-associated DNA polymerase activity was inhibited by ammonium sulphate over a range of concentrations between 0.01 M and 0.3 M (Fig. 2b), and could be suppressed to 50% by PAA at 50 pg/ml with poly(dA), oligo(dT)12_18 as the template primer. The virus-associated DNA polymerase was relatively resistant to PAA inhibition using activated calf thymus DNA as the template primer, but this characteristic of HCMV-associated DNA polymerase is similar to that of Epstein-Barr virus (EBV)-associated DNA polymerase (Goodman et El., 1978). Furthermore, the culture and purified virus preparations were negative for mycoplasma contamination as determined by the cytofluorescence technique. Taken together, these data indicate that the DNA polymerase we are detecting in the virions is not due to mycoplasma contamination. It is a well-known fact that a protein kinase activity exists in several completely unrelated viruses (Tan, 1975). The experiments described in the paper demonstrated the presence of an endogenous protein kinase activity in highly purified preparations of human cytomegalovirus. The characteristics of the HCMV-associated protein kinase activity are similar to those reported for other herpes viruses (LeMaster & Roizman, 1980; Randall et al., 1972; Rubenstein et al., 1974) in that the enzyme is preferentially capable of phosphorylating HCMV structural proteins rather than exogenous protein substrates, it is not enhanced by the presence of cyclic AMP or cyclic GMP, and a divalent metal ion is essential for enzymic activity. The procedures to extract the endogenous protein kinase using non-ionic detergent suggest that the enzyme resides in the virus core. It is not clear from the present data whether the protein kinase is virus-coded or of host origin. The biological roles of the virus-associated protein kinase and DNA polymerase are unknown. Further studies including the topographic localization of these two virus-associated enzymes are essential to understand the biological role played by these two enzymic activities associated with virions. Part of this work was presented at the Fifth Herpes Virus Cold Spring Meeting on August 26-31, 1980. This study was supported by the National Institute of Allergy and Infectious Diseases (AD12717 and K04 A 10029) and the National Cancer Institute (CA 21773). We are grateful to Barbara Leonard for typing the manuscript. P.C.P. is a postdoctoral fellow at the Conseil de la recherche et sant6 du Quebec. REFERENCES BALTIMORE, O. (1970). Viral RNA-dependent D N A polymerase. Nature, London 226, 1209-1211. BOHLEN, P., STEIN, S., PAIRMAN, W. & UDENERIEND, S. (1973). Amino acid analysis with fluorescamine at the picomole level. Archives of Biochemistry and Biophysics 155, 202-219. CHANG, L. M. S. (1971). Development of terminal deoxynucleotidyl transferase activity in embryonic calf t h y m u s gland. Biochemical and Biophysical Research Communications 44, 123-131. CHANG, L. M. S. & BOLLUM, F. J. (1970). Deoxynucleotide-polymerizing enzymes of calf t h y m u s gland. IV. Inhibition of terminal deoxynucleotidyl transferase by metal ligands. Proceedings of the National Academy of Sciences of the United States of America 65, 1041-1048. GOODMAN, S. a., PREZYNA, C. & BENZ, W. C. (1978). Two Epstein-Barr virus-associated D N A polymerase activities. Journal of Biological Chemistry 253, 8617-8628. nUANG, E. S. (1975). H u m a n cytomegalovirus. Ili. Virus-induced D N A polymerase. Journal of Virology 16, 298-310. HUANG, E. S., CHEN, S. T. & PAGANO, J. S. (1973). H u m a n cytomegalovirus. I. Purification and characterization of viral D N A . Journal of Virology 12, 1473-1481. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680--685. LEMASTER, S. & ROIZMAN, B. (1980). Herpes simplex virus phosphoproteins II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. Journal of Virology 35, 798-811. LEWIS, B. J., ABRELL, J. W., GRAHAM SMITH, R. & GALLO, R. L. (1974). H u m a n D N A polymerase III ( R - D N A polymerase): distinction from D N A polymerase I and reverse transcriptase. Science 183, 867-869. McAUSLAN,a. R. (1974). Minireview: virus-associated enzymes. Life Sciences 14, 2085-2097.
156
E.C.
MAR,
P.
C.
PATEL
AND
E.
S. HUANG
MAR, E. C. & HUANG, E. S. (1979). Comparative study of herpes group virus-induced D N A polymerases. Intervirology 12, 73-83. MAR, E. C., HUANG, Y. S. & HUANG~E. S. (1978). Purification and characterization of varicella zoster virus-induced D N A polymerase. Journal of Virology 26, 249-256. MILLER, R. L. & RAPP, F. (1976). Distinguishing cytomegalovirus, mycoplasma, and cellular D N A polymerase. Journal of Virology 20, 564-569. RANDALL, C. C., RODGERS, H. W., DOWNER, O. N. & GENTRY, G. A. (1972). Protein kinase activity in equine herpesvirus. Journal of Virology 9, 216-222. RUBENSTEIN, A. S., GRAVELL, M. & DARLINGTON, a. (1974). Protein kinase in enveloped herpes simplex virions. Virology 50, 287-290. TAN, K. B. (1975). Comparative study of the protein kinase associated with animal viruses. Virology 19, 180-186. WEBER, K. & OSBORN, M. (1969). The reliability of molecular determination by dodecylsulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244, 4406--4412,
(Reeeived 26 February 1981)
149
Key words: human cytomegalovirus/DNA polymerase/protein kinase
Human Cytomegalovirus-associated DNA Polymerase and Protein Kinase Activities By E N G - C H U N M A R , * P R A V I N C. P A T E L AND ENG-SHANG HUANG
Department of Medicine and Cancer Research Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A. (Accepted 13 July 1981) SUMMARY
Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgC12. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)~2_ls and poly(dC).oligo(dG)12_18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KC1 from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 #g/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)~2_~s was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0-3 M with either activated calf thymus DNA or poly(dA).oligo(dT)~2_~8 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgC12, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32p]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion. INTRODUCTION
The association of enzyme activities with purified virions, such as DNA-dependent RNA polymerase, DNA-dependent DNA polymerase, reverse transcriptase, protein kinase and so forth has been frequently demonstrated in various virus systems (McAuslan, 1974; Tan, 1975). Among the herpes group viruses, an endogenous DNA polymerization activity was found with Epstein-Barr virus (Goodman et al, 1978), and protein kinase activities were detected with equine herpesvirus (Randall et aL, 1972) and herpes simplex virus (LeMaster & Roizman, 1980; Rubenstein et al., 1974). We report here that DNA polymerase and protein kinase activities are also detected in the purified virions of human cytomegalovirus (HCMV). METHODS
Cell line and virus. The WI-38 strain of human fibroblast (Hayflick; obtained from American Tissue Culture Collection, CCL-75, Passage 24-28) was used for the entire study. The Towne Strain of HCMV was adapted and propagated by infection of WI-38 roller bottle 0033-1317/81/0000-4625 $02.00© 1981SGM
150
E.C.
MAR,
P. C. P A T E L
AND
E.
S. H U A N G
cultures at a multiplicity of 1 to 2 p.f.u./cell. Virus was harvested from the extracellular fluid as described previously (Huang et al., 1973). Labelling and purification of virus. To label virus proteins, monolayers were washed twice with Hanks' balanced salt solution 20 h after infection, and 30 ml low-phosphate medium (Hanks' balanced salt solution containing amino acids, glucose, 4 % foetal calf serum, 10 mM-glutamine, 2% NaHCO3, 100 units/ml penicillin, 100 gg/ml streptomycin sulphate and 10 -5 M of phosphate) containing 30 gCi/ml [32p]orthophosphate were added. For non-labelled virions, the infected cells were grown in minimum essential medium containing 4 % foetal calf serum, supplemented with 100 units/ml penicillin, 100 gg/ml streptomycin sulphate and I0 mM-glutamine. The extracellular virus released from infected cell cultures was collected every 4 days after infected cells showed 100 % c.p.e. The extracellular fluid was then subjected to centrifugation at 6000 rev/min in a Sorvall GSA rotor for 20 min at 2 °C. The cell-free virus particles in the supernatant were pelleted in a Spinco T-19 rotor at 18 000 rev/min for 90 min. The virus pellet was suspended in ice-cold TBS (0.05 tris-HC1 pH 7.4 0-15 M-NaC1) and dispersed by vortexing until no particulate material was visible. The suspension was layered on to a gradient of 10 to 50 % sucrose and centrifuged at 26 000 rev/min in a Beckman SW27 rotor for 1 h at 2 °C. The opaque virus band was collected and diluted with 1 vol. TBS. The virus suspension was then layered on to a preformed CsC1 gradient (1.16 to 1.37 g/ml) in TBS containing 5 mM-EDTA, and centrifuged again at 26000 rev/min for 1 h at 4 °C. The virus particles banded at a density of 1.22 to 1-24 g/ml. The purified virus was then extensively dialysed against cold TBS, and pelleted by centrifugation at 27 000 rev/min for 60 min. The freshly purified non-labelled virus was used immediately for the study of the two enzymic activities.
Preparation of enzyrne extracts DNA polymerase study. For this study the purified virus was suspended and solubilized in a hypotonic detergent buffer A [10 mM-tris-HC1 pH 7.8, 1 mM-MgCI 2, 1 mM-DTT, 0-5 % Nonidet P40 (NP40), 0.4 mM-phenylmethylsulphonyl fluoride (PMSF)] and kept on ice for 30 min. To the mixture was added an equal volume of 3.8 M-NaC1 in buffer B (20 mM-tris-HC1 pH 7.8, 10 mM-MgC12, 8 mM-DTT, 0.4 mM-PMSF) to give a final salt concentration of 1.9 M-NaCI in order to further dissociate the enzyme component from other virion complexes. This high salt mixture was centrifuged at 25 000 rev/min for 2 h at 2 °C. The clarified supernatant extract was collected and dialysed against buffer C (50 mM-tris-HC1 pH 7-8, 1 mM-EDTA, 1 mM-DTT, 0.4 mM-PMSF, 5% glycerol) to a salt concentration less than 0.01 M-NaC1, as determined by a salt conductivity meter. The dialysed material was loaded on to a phosphocellulose column (1.6 x 10 cm) and eluted with a gradient of 0.1 to 0.7 M-KC1 in buffer C. Protein kinase activity study. For this study the purified virions were dissociated in a buffer containing 50 mM-tris-HC1 pH 7.8, 100 mM-KC1, 1 mM-DTT, 0.5 % NP40 and 5 % glycerol, with the aid of vortexing for 30 s. The dissociated virions were subjected to centrifugation at 15 000 rev/min for 20 min at 2 °C and the pellet was reextracted with the same buffer three times. The supernatants were combined and used for the protein kinase activity study. DNA polymerase assay. Three systems were used in the DNA polymerase activity assay, as described elsewhere (Huang, 1975; Mar et al., 1978; Mar & Huang, 1979). In system A, activated calf thymus DNA was used as the template primer. A 0.1 ml amount of activated calf thymus DNA (200/~g/ml) was mixed with 0.1 ml of stock reaction mixture containing 0.1 M-tris-HCl pH 7.8, 25 mM-MgCI 2, 1 mM-DTT, 125 mg/ml bovine serum albumin (BSA), 0.25 mM each of dATP, dCTP, dGTP, and 1/.tCi [3HldTTP (48 Ci/mmol) together
HCMV-assoeiated enzyme activities
151
with 50 /A of enzyme fraction. The reaction was carried out at 37 °C for 1 h and was terminated by chilling in ice and adding 2 ml 20 % ice-cold trichloroacetic acid (TCA). The acid-precipitable radiolabelled material was collected by suction filtration on glass fibre paper (GF/C) or a nitrocellulose filter. It was then washed with 5% TCA. After drying, the radioactivity on the filters was determined using a liquid scintillation counter. In system B the reaction conditions employed were similar to those in system A, except that cold dATP, dCTP and dGTP were omitted, and the synthetic template primers, poly(dA), oligo(dT)12_lS, poly(rA).oligo(dT)12_lS or oligo(dT)12_~8 (2 #g for each reaction), were used instead of activated calf thymus DNA. In system C, 2 pg poly(dC).oligo(dG)~2_18 or poly(rC)-oligo(dG)~2 ~s, 0-01 mM-dGTP and 1 /,tCi [3H]dGTP (42 Ci/mmol) were used in the reaction mixture. Protein kinase assay. The standard reaction for the assay of protein kinase activity was conducted by incubating 10 pg of enzyme extract with 10 pCi [7-a2p]ATP (sp. act. 4200 Ci/mmol) in the presence or absence of histone, caesin, BSA or protamine sulphate (500 /ag/ml) in a total vol. of 0.2 ml containing 50 mM-tris-HC1 pH 7-8, 100 mM-KCI, 20 mM-MgC12, 1 mM-DTT, 0.5 % NP40 and 5 % glycerol. The reaction was carried out at 37 °C for 30 min and terminated by adding 2 ml 20% ice-cold TCA. The precipitated materials were treated in a similar manner as employed in the DNA polymerase assay. In other experiments the reaction condition was modified to determine the effect of pH; Mg z+, Mn 2+, Ca 2+, Z n 2+ and Co 2+ concentrations. Polyaerylamide gel eleetrophoresis. The 32p-labelled proteins were identified by SDSPAGE through 9 % polyacrylamide gels in a tris buffer system as described by Laemmli (1970). All samples were electrophoresed under reducing conditions. The mol. wt. of the polypeptides were determined by electrophoresing marker proteins with known mol. wt. (Bio-Rad) on the same gels and comparison of their relative migrations was as described elsewhere (Weber & Osborn, 1969). The gels were stained with 0-05 % Coomassie Brilliant Blue in an aqueous mixture containing 20% methanol and I0% acetic acid. The gels were destained in the same solvent system with occasional shaking. The gels were then vacuum-dried on to 3MM Whatman chromatography paper and exposed to X-ray film (Kodak RP1R2) for autoradiographs. Protein determination. The protein concentration was determined by the fluorescence assay of Bohlen et al. (1973). BSA was employed as a standard for protein determination. RESULTS
HCMV-assoeiated DNA polymerase As shown in Fig. 1 (a), DNA polymerase activity derived from NP40-disrupted HCMV virions was sharply eluted at 0.18 to 0.2 M-KC1 in phosphocellulose chromatography. The partially purified DNA polymerase could use the synthetic templates poly(dA), oligo(dT)lz-18, poly(dC).oligo(dG)12_18 as well as activated calf thymus DNA as the template primers (Table 1). However, the virus-associated DNA polymerase was unable to use poly(rA).oligo(dT)12_~s, poly(rC).oligo(dG)12_ls or oligo(dT)12_18 as a template primer (Table 1). This implied that the virus-associated DNA polymerase was an enzyme with a character distinct from those of reverse transcriptase (Baltimore, 1970), RNA-dependent DNA polymerase from lymphoblastoid cells (Lewis et al., 1974) and terminal deoxynucleotidyl transferase (Chang, 1971; Chang & Bollum, 1970). Divalent cations are essential for the DNA polymerase activity, as they are for the herpesvirus-induced DNA polymerase and host cell enzymes (a and fl polymerases) (Mar & Huang, 1979). Only part of the enzyme activity was maintained when Mg 2+ was replaced by Mn 2+. Around 5 to 10 mM-Mg 2÷ was needed for maximum DNA polymerization.
152
E . C. M A R , P . C. P A T E L
I
A N D E . S. H U A N G
I
I
(b) 1 2
0"5--
0 X
3
4
172--
0.4-
126-,_ 100--
0.3-
89 f
0.2-
68~ 67/59 f
0. l -
50-45--40~ 38~ 35 /
n
29-24-20
40
60
80
100
Fraction number Fig. 1. (a) Fractionation of virion-associated DNA polymerase in phosphocellulose chromatography. The purified virions from HCMV-infected WI-38 cells were dissociated with buffer A (10 mra-tris-HC1 pH 7.8, 1 mM-MgCI2, 1 mM-DTT, 0.5% NP40, 0.4 mM-PMSF) and kept on ice for 30 min, and then an equal volume of 3.8 M-NaCI in buffer B (20 mM-tris-HCl pH 7.8, 10 mM-MgC12, 8 mM-DTT, 0.4 mM-PMSF) was added. The resulting mixture was collected and dialysed extensively against buffer C (50 mM-tris-HCl pH 7-8, 1 rnM-EDTA, 0.4 mra-PMSF, 1 m~I-DTT, 5 % glycerol). The dialysate was loaded on to a phosphocellulose column (1.6 x 10 cm), which had been previously washed with acid and alkali and equilibrated with buffer C. After the enzyme was absorbed, a gradient of 0.1 to 0.7 M-KC1 in buffer C (100 ml: 100 ml) was applied. Fractions of 2 ml were collected and every other fraction was assayed for DNA polymerase activity using activated calf thymus DNA as the template palmer. The enzyme activity was eluted as a sharp peak at 0-18 to 0-2 M-KC1. O, DNA polymerase activity; II, salt concentration. (b) Autoradiography of phosphorylated polypeptides analysed in 9% SDS-PAGE. The in vitro phosphorylation of proteins was conducted by incubating 10 #g of specified proteins (determined according to the procedure of Bohlen et aL, 1973) with 10/~Ci [7-32p]ATP (sp. act. 4200 Ci/mmol) in a total vol. of 0.2 ml containing 50 mM-tris-HCl pH 7.8, 100 mM-KC1, 20 mM-MgCI2, 1 mM-DTT, 0.5% NP40 and 5% glycerol at 37°C for 30 rain. The reaction was terminated by adding 2 ml cold 20 % TCA. Precipitated materials were centrifuged at 8000 rev/min for 10 min and washed with 95% ethanol. After drying, 0-2 rnl buffer (62.5 mM-tris-HC1 pH 6.7, 5 mM-fl-mercaptoethanol, 2 % SDS, 15 % glycerol) was added to each reaction tube, and boiled for 2 min before gel electrophoresis. Each sample loaded on the gel contained 200000 ct/min. Lane 1, NP40-treated WI-38 labelled with [7-32p]ATP. Lane 2, Residual protein in the pellet from the 15000 rev/min centrifugation of NP40-treated virions labelled with [7-32P]ATP, as described in the text. Lane 3, Supernatant fraction of NP40-treated virions (centrifuged at 15000 rev/min) labelled with [y-32p]ATP. Lane 4, Phosphorylated virus labelled with [32p]orthophosphate in vivo as described elsewhere (Huang et al., 1973). Numbers indicate mol. wt. x 10-3. The blurred background above the region of 172K was due to the presence of 32p-labelled polynucleotides (DNA and RNA), which do not enter the 9 % acrylamide gel.
The virus-associated D N A p o l y m e r a s e was similar to ct-polymerase as j u d g e d b y salt elution in p h o s p h o c e l l u l o s e c h r o m a t o g r a p h y (about 0 . 2 M-KC1), resistance to P A A , and sensitivity to a m m o n i u m sulphate. H o w e v e r , the virus-associated e n z y m e was m o r e sensitive t h a n ct-polymerase to P A A w h e n poly(dA).oligo(dT)12_18 was used as the t e m p l a t e primer. The f o r m e r e n z y m e activity, but not the latter, was inhibited by 50 % at a c o n c e n t r a t i o n o f
HCMV-associated enzyme activities 100
~\
I
~.~
I
153
I I I 1'80' l~~ I I I I (b)| (a)
FL
/, :\
.N_ E
50
/I
-
100 /:~ "~
©
< zr~
~,
, ,,,~,
x\
",o
\\
20
40
60
80
100
PAA (/ag/ml)
lO 30
60
100
200 2 s o 3oo
(NH4)2SO4 (mM)
Fig. 2. Effects of (a) phosphonoacetic acid (PAA) and (b) ammonium sulphate concentration on virus-associated (0, x) and virus-induced (&, O) DNA polymerase activity. The assays were carried out as described in the text, except that PAA was added to the reaction mixture at a final concentration of 20 to 100 Bg/ml and (NH4)2SO 4 at 10 to 300 mM. Solid lines indicate that activated calf thymus DNA was used as the template primer for enzyme assays; dotted lines indicate that poly(dA).oligo(dT)12_ls was used. Activity is expressed relative to that obtained in the absence of (NH4)2SO 4 or PAA as 100 % control.
T a b l e 1. Template primer specificities of human cytomegalovirus-associated DNA polymerase
Assay system A* A A Bt B B C1: C
Template primer Activated calf thymus DNA Native calf thymus DNA Denatured calf thymus DNA Poly(dA). oligo(dT),2_18 Poly(rA). oligo(dT)12_la Oligo(dT)x2_ls Poly(dC).oligo(dG)tz_ls Poly(rC). oligo(dG)l 2_Is
Substrate [3H]dTTP, cold dNTP [3H]dTTP, cold dNTP [3H]dTTP, cold dNTP [ 3H]dTTP, cold dTTP [ aH]dTTP, cold dTTP [ 3H]dTTP, cold dTTP [3H]dGTP, cold dGTP [ 3H]dGTP, cold dGTP
DNA polymerase activity (pmol) 264 4 9 210 1 0.7 189 1.5
* System A. Activated calf thymus DNA (ACTD) was used as the template primer. A 0.1 ml amount of ACTD (200 #g/ml) was mixed with 0.1 ml of stock reaction mixture containing 0.1 M-tris-HC1 pH 7.8, 25 mM-MgCI2, 1 mM-DTT, 125 mg/ml BSA, 0.25 mM each of dATP, dCTP, dGTP, and 1 #Ci [3H]dTTP (48 Ci/mmol), together with 50 #1 of enzyme fraction. Incubation was at 37 °C for 1 h and was terminated by TCA preparation as described previously (Mar & Huang, 1979). i" System B. Conditions were similar to those in system A except that cold dATP, dCTP and dGTP were omitted, and poly(dA), oligo(dT)l~_18 (2 ltg for each reaction) was used instead of ACTD. :~ System C. Two #g poly(dC).oligo(dG),2_xs, 0.01 mM-dGTP and 1 ltCi [3H]dGTP (42 Ci/mmol) were used in the reaction mixture. 50 p g / m l P A A . In addition, the H C M V - a s s o c i a t e d D N A p o l y m e r a s e was characteristically distinct f r o m v i r u s - i n d u c e d e n z y m e . A s s h o w n in Fig. 2 (a), the v i r u s - a s s o c i a t e d e n z y m e was, in general, relatively resistant to P A A and sensitive to a m m o n i u m sulphate. O n the other hand, H C M V - i n d u c e d D N A p o l y m e r a s e was sensitive to P A A and its e n z y m e activity c o u l d be stimulated at 60 m M - ( N H 4 ) 2 S O 4. F u r t h e r m o r e , the v i r u s - i n d u c e d e n z y m e eluted at 0 . 3 2 M in p h o s p h o c e l l u l o s e c o l u m n s ( M a r & H u a n g , 1979) while the virus-associated e n z y m e eluted at 0 . 1 8 to 0 . 2 M-KC1 (Fig. 1 a).
154
E.C.
MAR,
P . C. P A T E L
AND
E . S. H U A N G
HCMV-associated protein kinase
In addition to a DNA polymerase activity, a protein kinase activity was also found to be associated with the virion of HCMV. The protein kinase required divalent cation for its enzyme activity. Phosphorylation was high in the presence of Mg 2+ but relatively weak in the presence of M n 2+, and negligible with Co 2+, Z n 2+ o r C a 2+. The optimum assay condition for the protein kinase activity in our system was 10 to 20 mM-Mg 2+ at a pH range of 6.5 to 8.5. Activity was not stimulated by cyclic nucleoside monophosphates such as cyclic-AMP and cyclic-GMP at concentrations of 0.0001 to 1 mM (data not shown). Interestingly, the protein kinase preferred its own virion proteins as phospho-acceptor to protamine sulphate. In addition, historic, caesin and BSA were found to be poor substrates (data not shown). Comparison of in vivo and in vitro phosphorylated proteins in S D S - P A GE
A comparison of phosphorylated proteins derived from virions labelled with 32p in vivo and virion proteins labelled with [?-32p]ATP in vitro was performed. The results revealed that in SDS-PAGE, 10 phosphorylated polypeptides (172K, 150K, 100K, 89K, 67K, 53K, 50K, 39K, 38K and 24K) were detectable. On the other hand, 14 polypeptides (172K, 126K, 100K, 89K, 68K, 67K, 59K, 50K, 45K, 40K, 38K, 35K, 29K and 24K) were found to be phosphorylated by the labelling in vitro with the endogenous virus-associated protein kinase. Although the polypeptide patterns obtained from these two different 32p-labelling systems were not identical, seven proteins co-migrated as shown in Fig. 1 (b) (indicated by arrows). These proteins are polypeptides with mol. wt. of 172K, 100K, 89K, 67K, 50K, 38K and 24K. The procedures for extracting the endogenous protein kinase from virions still left some protein kinase activity in the pellet. Nevertheless, the in situ [~32p]ATP-labelled proteins of the supernatant and the pellet fraction revealed a similar polypeptide pattern as shown in lanes 2 and 3 of Fig. 1 (b). DISCUSSION
There are several characteristic similarities between virion-associated DNA polymerase and host cell a-polymerase such as chromatographic behaviour in phosphocellulose chromatography, ammonium sulphate inhibition and partial resistance to PAA. This raises the question of whether the purified virions were contaminated with the host cell enzymes. However, we do not believe this to be the case since we could not detect any DNA-polymerizing activity using the intact whole virion without dissociation with NP40, while virion-associated DNA polymerase activity was inhibited by 50% at 50 ~tg/ml PAA with poly(dA), oligo(dT)12-18 as the template primer, while host cell a- and fl-polymerase were resistant to PAA under the same condition (Mar et al., 1978). Finally, the host cell fl-polymerase, which elutes at 0.45 M-KC1 in phosphocellulose chromatography, was not detected at all. Assay of the protein kinase in the in vivo 32P-labelling system failed to reveal any detectable phosphorylated host cell proteins, nor were any polypeptides corresponding to virion phosphorylated proteins found in mock-infected cells although equal amounts (200 000 ct/min) of TCA-precipitable materials were applied to the gel as shown in Fig. l(b) (lane 1, host cell; lanes 2 and 3, viral). Lastly, after several cycles of purification including isokinetic and isopycnic centrifugation, the purified virus was found to be free of host cell contamination as judged by electron microscopic and immunological methods. Taken together, these results ruled out the possibility of host cell contamination in the purified virions. Another possibility is that the enzyme activities are due to contamination by mycoplasma. Miller & Rapp (1976) reported that mycoplasma-specified DNA polymerase activity could be enhanced to 130 to 180% by the presence of 25 mM-(NH4)2SO 4 but was resistant to PAA at
H C M V - a s s o c i a t e d e n z y m e activities
155
a concentration of 100 gg/ml. In contrast, our data showed that the virus-associated DNA polymerase activity was inhibited by ammonium sulphate over a range of concentrations between 0.01 M and 0.3 M (Fig. 2b), and could be suppressed to 50% by PAA at 50 pg/ml with poly(dA), oligo(dT)12_18 as the template primer. The virus-associated DNA polymerase was relatively resistant to PAA inhibition using activated calf thymus DNA as the template primer, but this characteristic of HCMV-associated DNA polymerase is similar to that of Epstein-Barr virus (EBV)-associated DNA polymerase (Goodman et El., 1978). Furthermore, the culture and purified virus preparations were negative for mycoplasma contamination as determined by the cytofluorescence technique. Taken together, these data indicate that the DNA polymerase we are detecting in the virions is not due to mycoplasma contamination. It is a well-known fact that a protein kinase activity exists in several completely unrelated viruses (Tan, 1975). The experiments described in the paper demonstrated the presence of an endogenous protein kinase activity in highly purified preparations of human cytomegalovirus. The characteristics of the HCMV-associated protein kinase activity are similar to those reported for other herpes viruses (LeMaster & Roizman, 1980; Randall et al., 1972; Rubenstein et al., 1974) in that the enzyme is preferentially capable of phosphorylating HCMV structural proteins rather than exogenous protein substrates, it is not enhanced by the presence of cyclic AMP or cyclic GMP, and a divalent metal ion is essential for enzymic activity. The procedures to extract the endogenous protein kinase using non-ionic detergent suggest that the enzyme resides in the virus core. It is not clear from the present data whether the protein kinase is virus-coded or of host origin. The biological roles of the virus-associated protein kinase and DNA polymerase are unknown. Further studies including the topographic localization of these two virus-associated enzymes are essential to understand the biological role played by these two enzymic activities associated with virions. Part of this work was presented at the Fifth Herpes Virus Cold Spring Meeting on August 26-31, 1980. This study was supported by the National Institute of Allergy and Infectious Diseases (AD12717 and K04 A 10029) and the National Cancer Institute (CA 21773). We are grateful to Barbara Leonard for typing the manuscript. P.C.P. is a postdoctoral fellow at the Conseil de la recherche et sant6 du Quebec. REFERENCES BALTIMORE, O. (1970). Viral RNA-dependent D N A polymerase. Nature, London 226, 1209-1211. BOHLEN, P., STEIN, S., PAIRMAN, W. & UDENERIEND, S. (1973). Amino acid analysis with fluorescamine at the picomole level. Archives of Biochemistry and Biophysics 155, 202-219. CHANG, L. M. S. (1971). Development of terminal deoxynucleotidyl transferase activity in embryonic calf t h y m u s gland. Biochemical and Biophysical Research Communications 44, 123-131. CHANG, L. M. S. & BOLLUM, F. J. (1970). Deoxynucleotide-polymerizing enzymes of calf t h y m u s gland. IV. Inhibition of terminal deoxynucleotidyl transferase by metal ligands. Proceedings of the National Academy of Sciences of the United States of America 65, 1041-1048. GOODMAN, S. a., PREZYNA, C. & BENZ, W. C. (1978). Two Epstein-Barr virus-associated D N A polymerase activities. Journal of Biological Chemistry 253, 8617-8628. nUANG, E. S. (1975). H u m a n cytomegalovirus. Ili. Virus-induced D N A polymerase. Journal of Virology 16, 298-310. HUANG, E. S., CHEN, S. T. & PAGANO, J. S. (1973). H u m a n cytomegalovirus. I. Purification and characterization of viral D N A . Journal of Virology 12, 1473-1481. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680--685. LEMASTER, S. & ROIZMAN, B. (1980). Herpes simplex virus phosphoproteins II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. Journal of Virology 35, 798-811. LEWIS, B. J., ABRELL, J. W., GRAHAM SMITH, R. & GALLO, R. L. (1974). H u m a n D N A polymerase III ( R - D N A polymerase): distinction from D N A polymerase I and reverse transcriptase. Science 183, 867-869. McAUSLAN,a. R. (1974). Minireview: virus-associated enzymes. Life Sciences 14, 2085-2097.
156
E.C.
MAR,
P.
C.
PATEL
AND
E.
S. HUANG
MAR, E. C. & HUANG, E. S. (1979). Comparative study of herpes group virus-induced D N A polymerases. Intervirology 12, 73-83. MAR, E. C., HUANG, Y. S. & HUANG~E. S. (1978). Purification and characterization of varicella zoster virus-induced D N A polymerase. Journal of Virology 26, 249-256. MILLER, R. L. & RAPP, F. (1976). Distinguishing cytomegalovirus, mycoplasma, and cellular D N A polymerase. Journal of Virology 20, 564-569. RANDALL, C. C., RODGERS, H. W., DOWNER, O. N. & GENTRY, G. A. (1972). Protein kinase activity in equine herpesvirus. Journal of Virology 9, 216-222. RUBENSTEIN, A. S., GRAVELL, M. & DARLINGTON, a. (1974). Protein kinase in enveloped herpes simplex virions. Virology 50, 287-290. TAN, K. B. (1975). Comparative study of the protein kinase associated with animal viruses. Virology 19, 180-186. WEBER, K. & OSBORN, M. (1969). The reliability of molecular determination by dodecylsulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244, 4406--4412,
(Reeeived 26 February 1981)
