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DNA-dependent RNA polymerase in African swine fever virus
VIROLOGY
101,
169-175
(1980)
DNA-Dependent JUAN
RNA Polymerase in African Swine Fever Virus
KUZNAR,’
Centro de Biologia Molecular
MARIA (CSIC-UAM),
L. SALAS,
AND ELADIO
Universidad Authoma,
VINUELA
Canto Blunco, Madrid-.&
Spain
Accepted November 7,1979 African swine fever (ASF) virus has a DNA-dependent RNA polymerase activity which does not require the addition of exogenous DNA. The polymerase activity copurifies with ASF virus and reaches a maximum specific activity value of 5-15 pmol UMP/pg of virus protein, when the virus particles are pretreated with NP-40. In vitro RNA synthesis by ASF virus requires higher concentrations of ATP than of GTP or CTP. The RNA product has a sedimentation rate of 6-14 S and anneals specifically with virus DNA. INTRODUCTION
tion of Eagle’s minimum essential medium (DMEM), supplemented with 10% calf serum. The origin of the ASF virus adapted to grow in Vero cells, as well as the conditions for plaque assay, have been reported by Enjuanes et aE. (1976a). ~~~~~ution of ASF virus. Sub~on~uent Vero cell cultures were infected with ASF virus at a multiplicity of infection of 1 PFU/cell. After an adsorption period of 2 hr at 3’1” the cells were washed with phosphate-buffered saline (PBS) and incubated 44-48 hr at 37” in DMEM with 2% calf serum. At the end of the infection period, the culture medium was collected and the cells and cell debris were removed by centrifugation at 3000 g for 5 min and 4”. The extracellular virus in the supernatant was concentrated by precipitation with 5% polyethylene glycol (Enjuanes et at., 1976b). The pellet was resuspended in PBS and treated with a hydroxyapatite slurry in 1.0 M phosphate buffer pH 6.8 to give a final concentration of 0.2 M phosphate. After 15 min in an ice bath, the suspension was centrifuged for 5 min at 3000 g. The supernatant was layered onto a linear density gradient from 15 to 30% (w/v) potassium tartrate in PBS and centrifuged for 1.5 hr at 35,000 rpm and 4” in the SW 40 rotor. The virus, which banded at a density of 1.16 g/cm3, was collected, diluted in PBS, and centrifuged 1 hr as above. The virus pellet was resuspended in PBS and stored in portions at -70”.
The genome of African swine fever (ASF) virus is a crosslinked double-stranded DNA (Ortin et al,, 1979) with a molar mass of 100 x IO6 g mol-’ (Enjuanes et at., 19’76a). Although ASF virus multiplies in the cytoplasm of the infected cell (Breese and de Boer, 1966>, virus-infected enucleated cells do not produce infectious progeny virus (Ortin and Viiiuela, 1977). The role of the cell nucleus in the infection is unknown, but even if the nucleus of the infected cell were a site of virus DNA and/or RNA synthesis, as in the case of frog virus 3 (Goorha et aZ., 1977, 1978), this possibility does not preclude the presence in ASF virus particles of an RNA polymerase activity similar to that present in poxviruses (Kates and McAuslan, 1967; Munyon et al., 1967; Schwartz and Dales, 1971). In this report we show that ASF virus contains an RNA polymerase which synthesizes in vitro RNA complementary to virus DNA and describe some properties of the enzyme activity. MATERIALS
AND METHODS
Cells and virus. Vera cells (CCL 31) were obtained from the American Type Culture Collection and grown in Dulbecco’s modifica1 Present address: Departamento de Quimica, Casilla 13OV, Universidad de Chile, Valparaiso, Chile. * To whom reprint requests should be addressed. 169
0~2-682~80~03016~07$02.~~0 Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
KUZNAR,SALAS,ANDVImUELA
170
In some experiments, after the hydroxyapatite treatment, the virus was subjected to one of the following purification steps. The sample was layered onto a discontinuous gradient of 50, 35, and 25% (w/v) sucrose layers in PBS and centrifuged for 1.5 hr at 25,000 rpm and 4” in the SW 40 rotor. Alternatively, the virus suspension was chromatographed in controlled-pore glass, which had been previously treated with 2 vol of 1% (w/v> polyethylene glycol 20,000 in PBS and equilibrated with degassified PBS. The virus was eluted from the column with PBS. The volume of the sample was about l/lOth that of the column. RNA polymerase assay. Enzyme assays were based on the incorporation of 13H]UTP (1 &i per assay; specific activity, 0.1 Ci/ mmol) into acid-insoluble material. Immediately before the reaction, the virus samples were treated 20 min at room temperature with 7.5 x 10p3% NP-40. The standard assay mixture contained in a final volume of 250 ~1: 40 m&f Tris- HCI (pH 7.9), 8 n&f MgC&, 1 n-J! MnCl,, 25 n&f (NH&SO,, 0.05 mM dithiothreitol, 2 mJ4 ATP, 0.2 mil4 CTP, 0.2 n-J4 GTP, 0.04 mM UTP, and sample pretreated with NP40. The reaction mixture was incubated 60 min at 37” and the TCA-insoluble material was collected on GF/C filters and counted in a scintillation spectrometer. Sedimentation sized in vitro.
analysis
of RNA
synthe-
RNA synthesis was terminated by addition of SDS and EDTA to final concentrations of 0.5% and 20 mM, respectively, and the solution was heated 2 min in a bath of boiling water and cooled in ice. RNA was sedimented on 5 to 20% sucrose gradient in 10 n-&l sodium acetate (pH 6.0)-100 mM NaCl-0.1% SDS in the SW 40 rotor at 35,000 rpm for 5.2 hr at 18”. Fractions of 0.4 ml were collected from the bottom, precipitated with TCA, and filtered on GF/C glass-fiber filters. [5,6-3H]Uridine-labeled Vero,ribosomal and transfer RNAs were centrifuged in a paral: lel gradient as sedimentation standards. Purification
of RNA
synthesized
in vitro.
To isolate radioactive RNA, 160 pg of virus protein was used in a standard reaction mixture of 1.25 ml containing 50 #Zi of
[“H]UTP. Following the incubation time, the reaction mixture received 20 pg Q/3 RNA, SDS to a final concentration of O.l%, and 100 pg/ml proteinase K. After 30 min at room temperature, the sample was adjusted to 0.1 A4 sodium acetate (pH 5.2)-5 mJ4 EDTA (acetate-EDTA buffer) and extracted twice for 5 min at 65” with an equal volume of phenol saturated with distilled water. The RNA was precipitated with 2 vol of ethanol and resuspended in 150 ~1 of acetate-EDTA buffer. After chromatography on Sephadex G-50, the RNA was precipitated from the excluded volume with ethanol. The pellet was dissolved in 50 ~1 of 20 mM Tris-HCl (pH 8.0)-5 m&f MgCl* and the solution incubated 20 min at 30” with 10 pg/ml RNase-free DNase I and then 30 min with 50 pg/ml proteinase K. The RNA solution, adjusted to 0.1 M NaCI, was precipitated with ethanol and the pellet resuspended in 1.2 M phosphate buffer, pH 6.8. Preparation
of virus
and host cell DNA.
The purified ASF virus pellet obtained as described before was resuspended in a buffer containing 10 mM Tris-HCI (pH S.O), 10 m&f EDTA, 10 mM NaCl, and 500 pg/ml proteinase K. After 20 min at 4”, Sarkosyl was added to a final concentration of 0.5% and the mixture incubated overnight at 4”. Following the Sarkosylproteinase K treatment, the sample was extracted three times with 1 vol of phenol equilibrated with 50 mJ4 Tris-HCl (pH 7.8), 100 m&Z NaCl, and 10 mM EDTA. The DNA was dialyzed against 500 vol of 0.1 x SSC (1 x SSC is 0.15 A4 sodium chloride-O.015 M sodium citrate, pH 7.0). To isolate host cell DNA, confluent Vero cells were resuspended in a buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 10 mM NaCI, and 0.5% Sarkosyl (10 x lo6 cell/ml). The lysate was treated with 50 pg/ml proteinase K during 30 min at 30” and then extracted three times with phenol saturated with 50 n-J4 Tris-HCl (pH 7.8), 100 mM NaCl, and 10 n&f EDTA. The DNA was precipitated from the aqueous phase with 2 vol of ethanol, resuspended in 0.1 x SSC and incubated with 50 pglml RNase A during 30 min at 37”. The DNA preparation was again treated with Sarkosyl
ASF VIRUS RNA POLYMERASE
171
TABLE 1 RNA POLYMERASE
ACTIVITY
AND VIRUS
INFECTIVITY
DURING PURIFICATION OF ASF VIRUS~
Purification step
Proteinb (mg)
Virus infectivity (PFU wg-’ x lo-*)
RNA polymerase activity (pmol h-’ @g-l)
1. Polyethylene glycol 2. Hydroxyapatite 3. Tartrate gradient
46.2 33.3 2.4
6.7 7.0 16.7
0.55 0.71 5.6
a The culture medium of Vero cells infected with ASF virus (m.o.i. = 1) was collected at 48 hr postinfection
and processed as indicated under Materials and Methods. b Protein was assayed according to Lowry et al. (1951).
and proteinase K and then phenolyzed and precipitated with ethanol as indicated before. DNA-RNA hybridization and ribonuclease treatment. The conditions for the hybridization of RNA to DNA in aqueous solution were those described by Vogelstein and Gillespie (1977). The hybridization mixture contained radioactive RNA, ASF virus or Vero cell DNA, 0.36 M potassium phosphate (pH 6.8), and 70% formamide. Portions of 3 ~1 were flamesealed in 5-~1 capillary pipets, heated 10 min in a bath with boiling water, and then incubated 4 days at 46”. After hybridization, the content of each capillary tube was diluted to 750 ~1 with 3 x SSC and divided in two portions. One was incubated 30 min at 37” with 50 pug/ml RNase A and 0.2 pg/ml RNase Tl and the second portion was incubated without enzymes. The acid-precipitable radioactive material in the first portion, relative to that in the second one, was used to calculate the percentage of RNA resistant to the RNases. Materials. Ribonucleoside triphosphates were from Sigma Chemical Company; [3H]UTP and [5,6-3H]uridine from the Radiochemical Centre, Amersham; pancreatic RNase and DNase (ribonuclease free) and RNase Tl from Worthington Biochemical Corporation; proteinase K from Merck; hydroxyapatite, DNA grade, from Bio-Rad; controlled-pore glass, CPG10, 3125 A mean pore diameter, 80/120 mesh size, from Serva Feinbiochemica. All other chemicals were reagent grade. Qp RNA was a gift from E. Domingo.
6 1.0
0.5
” N
P
FRACTION
NUMBER
FIG. 1. Copurification of an RNA polymerase activity with ASF virus. ASF virus released to the culture medium from virus-infected Vero cells was concentrated with polyethylene glycol, treated with hydroxyapatite, followed by centrifugation in potassium tartrate (A) or in sucrose (B) gradient or chromatography on controlled-pore glass (C). RNA polymerase activity and virus titer were measured in the fractions collected from the gradients or column. Before enzyme assay, the tartrate gradient fractions were dialyzed against PBS.
172
KUZNAR.
SALAS, AND ~I~UELA
E NP-40
CONCEMRATION
RIBONUCLEOSIDE
TRIPYOSPHATE CONCENTRATtON,mM
x IO’, %
FIG. 2. Effect of NP-40 on ASF virus RNA polymerase. RNA polymerase activity was measured under standard conditions, except that the concentration of NP-40 w&s varied as indicated in the graph. The zero value of NP-40 concentration indicates the incorporation of pH]UTP by an untreated ASF virus sample. RESULTS
Copuri$cation of an RNA Activity and ASF Virus
PoLymeruse
Culture medium from ASF virus-infected Vero cells contains an RNA polymerase whose specific activity increases l&fold TABLE
2
PROPERTIESOFTHE REA~ION~ATA~Y~E~BY ASF VIRUS RNA POLYMERASE Reaction conditions Standard system -ATP -GTP -CTP +Denatured calf thymus DNA, 80 pg/ml +Actinomyein D, 50 CLgiml +cz-Amanitin, 40 pg/ml + Rifampicin, 50 E*&/ml +Rifampicin, 100 FgIml +Pancreatic DNaae, 15 pg/ml +Pancreatic RNase, 15 pg/ml” +O.l M KOHb
Enzyme activity (pmol h-l &g-l) 6.3 co.2 0.2 1.2 6.3 co.2 6.2 5.5 3.2 4.4 co.2 co.2
o Following the incubation time in the standard system, the mixture was heated for 10 min in a bath of boiling water, cooled in ice, and treated for 30 min at 37” in the presence of RNaae. a After incubation, KOH was added to a final concentration of 0.1 M and the mixture was heated for 10 min in a bath of boiling water.
FIG. 3. Effect of ribonucleoside triphosphate concentration on ASF virus RNA polymerase activity. Standard assay conditions were used. (A) The concentration of ribonucleoside triphosphates was varied as shown in the graph. (0) ATP; (0) GTP, (A) CTP. (B) After 60 min reaetion, the mixture was supplemented with either 2 m&f ATP or dATP. (e) No addition; (0) addition of ATP, (A) addition of dATP.
after virus purification (Table 1). The enzymatic activity sediments with the virus after potassium tartrate (Fig. 1A) or sucrose (Fig. 1B) gradient centrifugation and elutes in the exclusion volume, with the infective particles, after controlled-pore glass chromato~aphy (Fig. 1C). Other preparations of ASF virus showed a good overlapping of the enzymatic activity and the infectivity after column chromatography. Figure 2 shows that when the virus samples were incubated with NP-40 before the assay, the RNA polymerase activity increased. Pwperties of the RNA P~l~rn~rase Aesoc~ted to ASF Vi?-ue
A~t~~‘ty
Table 2 shows some ch~acte~stics of the reaction catalyzed by ASF virus RNA polymerase. The enzyme requires ail four ribonucleoside triphosphates and is not
ASF VIRUS RNA POLYMERASE
stimulated by the addition of exogenous DNA. The reaction is completely inhibited by actinomycin D, and only partially by rifampicin and pancreatic DNase. a-Amanitin has no effect on the reaction. RNase or KOH treatment hydrolyzes the acidinsoluble reaction product to acid-soluble material. Figure 3A shows that ASF virus RNA polymerase requires for maximal activity an ATP concentration higher than that of GTP or CTP. To test the possibility of a rapid depletion of ATP during the incubation, the reaction mixture was supplemented with either ATP or dATP when the rate of RNA synthesis began to decrease. Figure 3B shows that the addition of those nucleotides has no effect on the rate of RNA synthesis. Under standard assay conditions, the RNA polymerase activity is proportional to virus protein concentration in a range from 2 to at least 30 pg (Fig. 4) and is linear with time for 60 min at 37” (Fig. 3B). The optimal concentrations of MgCl,, MnCl,, and (NH&SO, for the reaction were 5, 0.75, and 20 mM, respectively, and the optimal pH value was 8.0 (Fig. 5). Size and Complementary Sequences of the RNA Synthesized in Vitro by ASF Virus RNA Polymerase Figure 6 shows that under standard assay conditions, most of the RNA synthesized in vitro by the ASF virus RNA
ASF
VIRUS
PROTEIN,
173
4 5 i
n
s_
16
I
40
u” +
20
L
12 mN
60
B E % k
5
6 CONCENTRATION.
I
20 40 60 (NH,12 SO, CONCENTRATION,
60 mM
FIG. 5. Effect of divalent cations, ammonium sulfate, and pH on ASF virus RNA polymerase activity. The standard reaction mixtures were used except that the concentrations of MgCl, or MnCl, (A) and ammonium sulfate (B) or the pH (C) were varied as indicated in the graph.
polymerase has a sedimentation coefficient of 6-14 S. To test whether the RNA synthesized by ASF virus is complementary to virus DNA, a fixed amount of [3H]RNA was hybridized to increasing concentrations of either ASF virus or Vero cell DNA. At least 70% of the RNA annealed with virus DNA and essentially none with host cell DNA (Fig. 7). As a hybridization control, Vero cell DNA hybridized with radioactive ribosomal and transfer RNAs from the same cells (data not shown).
pg
FIG. 4. Effect of ASF virus protein concentration on RNA polymerase activity. Assay conditions were as described under Materials and Methods. The amount of ASF virus protein was varied as indicated in the graph.
DISCUSSION
The enzymatic activity described in this paper has the properties of a DNA-dependent RNA polymerase. RNA synthesis re-
174
KUZNAR,
FRACTION
SALAS, AND VIiWELA
NUMBER
FIG. 6. Sucrose gradient sedimentation of RNA synthesized in vitro by ASF virus RNA polymerase. The arrows indicate the position of the $H-labeled rRNA and tRNA markers from Vero cells centrifuged in a parallel gradient.
quired all four ribonucleoside triphosphates, was completely inhibited by aetinomycin D, and the product was susceptible to pancreatic RNase and to alkaline hydrolysis. Since the polymerase activity which copurifies with ASF virus is not sensitive to cr-amanitin (an inhibitor of cellular RNA polymerases II and III) and requires previous treatment of purified virions with NP-40 for full activity, it seems that the enzyme is an integral viral component rather than a contamination. Evidence supporting this idea comes from the fact that ASF virus RNA polymerase activity does not require an exogeneous DNA template and that the RNA product synthesized in vitro anneals specifically with viral DNA. Furthermore, the in vitro RNA synthesis is only 30% inhibited when DNase I is added to the reaction mixture. It seems unlikely that the RNA polymerase activity is a contaminant enzyme which uses the inner ASF virus DNA and not an added DNA template. The weak effect of rifampicin on the RNA polymerase activity rules out the possibility that the measured reaction were due to bacterial contamination. ASF virus RNA polymerase shares a number of properties with a similar enzymatic activity present in poxviruses. The specific activities of both enzymes when assayed in partially dis~pt~ virions are comparable (Kates and McAuslan, 1967), they require higher concentrations of ATP than of the other ~~nucleoside triphos-
phates (Kates and Beeson, 1970), and the patterns of enzyme inhibition by a number of semisynthetic derivatives of rifampicin are similar (Szilagyi and Pennington, 1971; unpublished results). Furthermore, we will show elsewhere that the RNA synthesized in vitro by ASF virus is polyadenylated and methylated and that the virus contains other enzymatic activities, among them the protein kinase described by Polatnick et al. (1974), also present in poxviruses (Moss, 1974). These properties, together with the existence of crosslinks in the genomes of vaccinia (Berns and Silverman, 1970; Geshelin and Berns, 1974) and ASF virus (Ortin et al., 1979) suggest that both virus, independently of their mo~hology and host range, use a similar, if not identical, biochemical strategy for the early phase of gene expression. Together with the iridescent viruses from insects and frog virus 3, ASF virus has been classified as an Iridovirus (Fenner, 1976). It is not known whether or not all the members of the family contain crosslinked DNA, but there are reports that suggest that iridescent viruses types 2 and 6 (Kelly and Tinsley, 1973) and frog virus 3 (Gravel1 and ~romeans, 1971; Gaby and Kucera, 1974) contain an RNA polymerase, as well as other enzymatic activities similar to those present in poxviruses (Kelly and Robertson, 1973). It has been reported that the DNA of ASF virus is infective (Adldinger et al., 1966). This result, however, should be re-
O-0
,o 0.6
, 1.2
I.8
9 2.4
ONA, clg
FIG. 7. Hybridization of RNA synthesized in vitro to DNA. The hyb~di~tion mixture contained 2.4 ng of PH]RNA (1400 cpm) and the amounts of ASF virus (e) or Vero cell (0) DNA indicated in the graph.
ASF VIRUS RNA POLYMERASE
examined since it seems incompatible with the presence in ASF virus of an RNA polymerase. Experiments to test whether this activity is involved in the transcription of early genes are in progress. ACKNOWLEDGMENTS This work was supported by grants from Comision Asesora para el Desarrollo de la Investigacicin Cientifica and Comision Administradora de1 Descuento Complementario. REFERENCES ADLDINGER, H. K., STONE, S. S., HESS, W. R., and BACHRACH, H. L. (1966). Extraction of infectious deoxyribonucleic acid from African swine fever virus. Virology 30, 750-752. BERNS, K. I., and SILYER~A~, C. (1970). Natural occurrence of cross-linked vaccinia virus deoxyribonucleic acid. J. Viral. 5, 299-304. BREESE, S. S., JR., and DEBOER, C. J. (1966). Electron microscope observations of African swine fever virus in tissue culture cells. Virology 28, 420-428.
ENJUANES, L., CARRASCOSA,A. L., MORENO, M. A., and VI~UELA, E. (1976a). Titration of African swine fever (ASF) virus. J. Gen. Viral. 32, 471477.
ENJUANES, L., CARRASCOSA,A. L., and VIRUELA, E. (197613). Isolation and properties of the DNA of African swine fever (ASF) virus. J. Gen. Viral. 32,479-&E.
FENNER, F. (1976). “Classification and Nomenclature of Viruses,” Second Report of the International Committee on Taxonomy of Viruses. Karger, Basel. GABY, N. S., and KUCERA, L. S. (1974). DNAdependent RNA polymerase associated with subviral particles of polyhedral cytoplasmic deoxyribovirus. J. Vi&. 14, 231-233. GESHELIN, P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. S&785-796.
GRAVELL, M., and CROMEANS, T. L. (1971). Mechanisms involved in nongenetic reactivation of frog polyhedral cytoplasmic deoxyribovirus: Evidence for an RNA polymerase in the virion. Virology 46,39-49.
KATES, J. R., and MCAUS~N, B. R. flS67). Poxvirus DNA-dependent RNA polymerase. Proc. Nat. Acad. Sci. USA 58, 134-141. KATES, J., and BEESON, J. (1970). Ribonucleic acid synthesis in vaccinia virus. I. The mechanism of synthesis and release in vaccinia cores. J. Mol. Biol.
50, l-18.
KELLY, D. C., and TINSLEY, T, W. (1973). Ribonucleic acid polymerase activity associated with particles of iridescent virus types 2 and 6. J. Invertebr. Pathol. 22, 199-202. KELLY, D. C., and ROBERTSON, J. S. (1973). Icosahedral cytoplasmie deoxyriboviruses. J. Gen. viroz. 20, 17-41. LOWRY, 0. H., ROSEBROU~H, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. C&n. 193,265-275. Moss, B. (1974). Reproduction of poxviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 3, pp. 405-474. Plenum, New York. MUNYON, W., PAOLETTI, E., and GRACE, J. T., Ja. (1967). RNA polymerase activity in purified infectious vaccinia virus. Proc. Nat. Acad. Sci. USA 58,2280-2287.
ORTIN, J., ENJUANES, L., and VIRUELA, E. (1979). Cross-links in the DNA of African swine fever virus. J. Viral. 31, 579-583. ORTIN, J., and VINUELA, E. (lS77). Requirement of cell nucleus for African swine fever virus replication in Vero cells. J. Viral. 21, 902-905. POLATNICK, J., PAU, I. C., and GRAVELL, M. (1974). Protein kinase activity in African swine fever virus. Arch. ~esamte V~~fo~~h. 44, 156-159. SCHWARTZ, J., and DALES, S. (1971). Biogenesis of poxviruses: Identification of four enzyme activities within purified Yaba tumor virus. Virology 45, 797-801.
GOORHA, R., MURTI, G., GRANOFF, A., ~~~TIREY, R. (1918). Macromole~~~ synthesis in cells infected by frog virus 3. VIII. The nucleus is a site of frog virus 3 DNA and RNA synthesis. Virology 84,32-50.
GOORHA, R., WILLIS, D. B., Macromolecular synthesis virus 3. VI. Frog virus ent on the cell nucleus. J.
175
and GRANOFF, A. (1977). in cells infected by frog 3 replication is dependViral. 21, 302-305.
SZILAGYI, J. F., and PENNINGTON, T. H. (1971). Effect of rifamyrins and related antibiotics on the deoxyr~bonueleie acid-dependent ribonucleic acid polymerase of vaceinia virus particles. J. ViroE. 8, 133-141. VOGELSTEIN, B., and GILLESPIE, D. (1977). RNADNA hybridization in solution without DNA reannealing. Biochem. Biophys. Res. Commun. 75, 1127-1132.
101,
169-175
(1980)
DNA-Dependent JUAN
RNA Polymerase in African Swine Fever Virus
KUZNAR,’
Centro de Biologia Molecular
MARIA (CSIC-UAM),
L. SALAS,
AND ELADIO
Universidad Authoma,
VINUELA
Canto Blunco, Madrid-.&
Spain
Accepted November 7,1979 African swine fever (ASF) virus has a DNA-dependent RNA polymerase activity which does not require the addition of exogenous DNA. The polymerase activity copurifies with ASF virus and reaches a maximum specific activity value of 5-15 pmol UMP/pg of virus protein, when the virus particles are pretreated with NP-40. In vitro RNA synthesis by ASF virus requires higher concentrations of ATP than of GTP or CTP. The RNA product has a sedimentation rate of 6-14 S and anneals specifically with virus DNA. INTRODUCTION
tion of Eagle’s minimum essential medium (DMEM), supplemented with 10% calf serum. The origin of the ASF virus adapted to grow in Vero cells, as well as the conditions for plaque assay, have been reported by Enjuanes et aE. (1976a). ~~~~~ution of ASF virus. Sub~on~uent Vero cell cultures were infected with ASF virus at a multiplicity of infection of 1 PFU/cell. After an adsorption period of 2 hr at 3’1” the cells were washed with phosphate-buffered saline (PBS) and incubated 44-48 hr at 37” in DMEM with 2% calf serum. At the end of the infection period, the culture medium was collected and the cells and cell debris were removed by centrifugation at 3000 g for 5 min and 4”. The extracellular virus in the supernatant was concentrated by precipitation with 5% polyethylene glycol (Enjuanes et at., 1976b). The pellet was resuspended in PBS and treated with a hydroxyapatite slurry in 1.0 M phosphate buffer pH 6.8 to give a final concentration of 0.2 M phosphate. After 15 min in an ice bath, the suspension was centrifuged for 5 min at 3000 g. The supernatant was layered onto a linear density gradient from 15 to 30% (w/v) potassium tartrate in PBS and centrifuged for 1.5 hr at 35,000 rpm and 4” in the SW 40 rotor. The virus, which banded at a density of 1.16 g/cm3, was collected, diluted in PBS, and centrifuged 1 hr as above. The virus pellet was resuspended in PBS and stored in portions at -70”.
The genome of African swine fever (ASF) virus is a crosslinked double-stranded DNA (Ortin et al,, 1979) with a molar mass of 100 x IO6 g mol-’ (Enjuanes et at., 19’76a). Although ASF virus multiplies in the cytoplasm of the infected cell (Breese and de Boer, 1966>, virus-infected enucleated cells do not produce infectious progeny virus (Ortin and Viiiuela, 1977). The role of the cell nucleus in the infection is unknown, but even if the nucleus of the infected cell were a site of virus DNA and/or RNA synthesis, as in the case of frog virus 3 (Goorha et aZ., 1977, 1978), this possibility does not preclude the presence in ASF virus particles of an RNA polymerase activity similar to that present in poxviruses (Kates and McAuslan, 1967; Munyon et al., 1967; Schwartz and Dales, 1971). In this report we show that ASF virus contains an RNA polymerase which synthesizes in vitro RNA complementary to virus DNA and describe some properties of the enzyme activity. MATERIALS
AND METHODS
Cells and virus. Vera cells (CCL 31) were obtained from the American Type Culture Collection and grown in Dulbecco’s modifica1 Present address: Departamento de Quimica, Casilla 13OV, Universidad de Chile, Valparaiso, Chile. * To whom reprint requests should be addressed. 169
0~2-682~80~03016~07$02.~~0 Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
KUZNAR,SALAS,ANDVImUELA
170
In some experiments, after the hydroxyapatite treatment, the virus was subjected to one of the following purification steps. The sample was layered onto a discontinuous gradient of 50, 35, and 25% (w/v) sucrose layers in PBS and centrifuged for 1.5 hr at 25,000 rpm and 4” in the SW 40 rotor. Alternatively, the virus suspension was chromatographed in controlled-pore glass, which had been previously treated with 2 vol of 1% (w/v> polyethylene glycol 20,000 in PBS and equilibrated with degassified PBS. The virus was eluted from the column with PBS. The volume of the sample was about l/lOth that of the column. RNA polymerase assay. Enzyme assays were based on the incorporation of 13H]UTP (1 &i per assay; specific activity, 0.1 Ci/ mmol) into acid-insoluble material. Immediately before the reaction, the virus samples were treated 20 min at room temperature with 7.5 x 10p3% NP-40. The standard assay mixture contained in a final volume of 250 ~1: 40 m&f Tris- HCI (pH 7.9), 8 n&f MgC&, 1 n-J! MnCl,, 25 n&f (NH&SO,, 0.05 mM dithiothreitol, 2 mJ4 ATP, 0.2 mil4 CTP, 0.2 n-J4 GTP, 0.04 mM UTP, and sample pretreated with NP40. The reaction mixture was incubated 60 min at 37” and the TCA-insoluble material was collected on GF/C filters and counted in a scintillation spectrometer. Sedimentation sized in vitro.
analysis
of RNA
synthe-
RNA synthesis was terminated by addition of SDS and EDTA to final concentrations of 0.5% and 20 mM, respectively, and the solution was heated 2 min in a bath of boiling water and cooled in ice. RNA was sedimented on 5 to 20% sucrose gradient in 10 n-&l sodium acetate (pH 6.0)-100 mM NaCl-0.1% SDS in the SW 40 rotor at 35,000 rpm for 5.2 hr at 18”. Fractions of 0.4 ml were collected from the bottom, precipitated with TCA, and filtered on GF/C glass-fiber filters. [5,6-3H]Uridine-labeled Vero,ribosomal and transfer RNAs were centrifuged in a paral: lel gradient as sedimentation standards. Purification
of RNA
synthesized
in vitro.
To isolate radioactive RNA, 160 pg of virus protein was used in a standard reaction mixture of 1.25 ml containing 50 #Zi of
[“H]UTP. Following the incubation time, the reaction mixture received 20 pg Q/3 RNA, SDS to a final concentration of O.l%, and 100 pg/ml proteinase K. After 30 min at room temperature, the sample was adjusted to 0.1 A4 sodium acetate (pH 5.2)-5 mJ4 EDTA (acetate-EDTA buffer) and extracted twice for 5 min at 65” with an equal volume of phenol saturated with distilled water. The RNA was precipitated with 2 vol of ethanol and resuspended in 150 ~1 of acetate-EDTA buffer. After chromatography on Sephadex G-50, the RNA was precipitated from the excluded volume with ethanol. The pellet was dissolved in 50 ~1 of 20 mM Tris-HCl (pH 8.0)-5 m&f MgCl* and the solution incubated 20 min at 30” with 10 pg/ml RNase-free DNase I and then 30 min with 50 pg/ml proteinase K. The RNA solution, adjusted to 0.1 M NaCI, was precipitated with ethanol and the pellet resuspended in 1.2 M phosphate buffer, pH 6.8. Preparation
of virus
and host cell DNA.
The purified ASF virus pellet obtained as described before was resuspended in a buffer containing 10 mM Tris-HCI (pH S.O), 10 m&f EDTA, 10 mM NaCl, and 500 pg/ml proteinase K. After 20 min at 4”, Sarkosyl was added to a final concentration of 0.5% and the mixture incubated overnight at 4”. Following the Sarkosylproteinase K treatment, the sample was extracted three times with 1 vol of phenol equilibrated with 50 mJ4 Tris-HCl (pH 7.8), 100 m&Z NaCl, and 10 mM EDTA. The DNA was dialyzed against 500 vol of 0.1 x SSC (1 x SSC is 0.15 A4 sodium chloride-O.015 M sodium citrate, pH 7.0). To isolate host cell DNA, confluent Vero cells were resuspended in a buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 10 mM NaCI, and 0.5% Sarkosyl (10 x lo6 cell/ml). The lysate was treated with 50 pg/ml proteinase K during 30 min at 30” and then extracted three times with phenol saturated with 50 n-J4 Tris-HCl (pH 7.8), 100 mM NaCl, and 10 n&f EDTA. The DNA was precipitated from the aqueous phase with 2 vol of ethanol, resuspended in 0.1 x SSC and incubated with 50 pglml RNase A during 30 min at 37”. The DNA preparation was again treated with Sarkosyl
ASF VIRUS RNA POLYMERASE
171
TABLE 1 RNA POLYMERASE
ACTIVITY
AND VIRUS
INFECTIVITY
DURING PURIFICATION OF ASF VIRUS~
Purification step
Proteinb (mg)
Virus infectivity (PFU wg-’ x lo-*)
RNA polymerase activity (pmol h-’ @g-l)
1. Polyethylene glycol 2. Hydroxyapatite 3. Tartrate gradient
46.2 33.3 2.4
6.7 7.0 16.7
0.55 0.71 5.6
a The culture medium of Vero cells infected with ASF virus (m.o.i. = 1) was collected at 48 hr postinfection
and processed as indicated under Materials and Methods. b Protein was assayed according to Lowry et al. (1951).
and proteinase K and then phenolyzed and precipitated with ethanol as indicated before. DNA-RNA hybridization and ribonuclease treatment. The conditions for the hybridization of RNA to DNA in aqueous solution were those described by Vogelstein and Gillespie (1977). The hybridization mixture contained radioactive RNA, ASF virus or Vero cell DNA, 0.36 M potassium phosphate (pH 6.8), and 70% formamide. Portions of 3 ~1 were flamesealed in 5-~1 capillary pipets, heated 10 min in a bath with boiling water, and then incubated 4 days at 46”. After hybridization, the content of each capillary tube was diluted to 750 ~1 with 3 x SSC and divided in two portions. One was incubated 30 min at 37” with 50 pug/ml RNase A and 0.2 pg/ml RNase Tl and the second portion was incubated without enzymes. The acid-precipitable radioactive material in the first portion, relative to that in the second one, was used to calculate the percentage of RNA resistant to the RNases. Materials. Ribonucleoside triphosphates were from Sigma Chemical Company; [3H]UTP and [5,6-3H]uridine from the Radiochemical Centre, Amersham; pancreatic RNase and DNase (ribonuclease free) and RNase Tl from Worthington Biochemical Corporation; proteinase K from Merck; hydroxyapatite, DNA grade, from Bio-Rad; controlled-pore glass, CPG10, 3125 A mean pore diameter, 80/120 mesh size, from Serva Feinbiochemica. All other chemicals were reagent grade. Qp RNA was a gift from E. Domingo.
6 1.0
0.5
” N
P
FRACTION
NUMBER
FIG. 1. Copurification of an RNA polymerase activity with ASF virus. ASF virus released to the culture medium from virus-infected Vero cells was concentrated with polyethylene glycol, treated with hydroxyapatite, followed by centrifugation in potassium tartrate (A) or in sucrose (B) gradient or chromatography on controlled-pore glass (C). RNA polymerase activity and virus titer were measured in the fractions collected from the gradients or column. Before enzyme assay, the tartrate gradient fractions were dialyzed against PBS.
172
KUZNAR.
SALAS, AND ~I~UELA
E NP-40
CONCEMRATION
RIBONUCLEOSIDE
TRIPYOSPHATE CONCENTRATtON,mM
x IO’, %
FIG. 2. Effect of NP-40 on ASF virus RNA polymerase. RNA polymerase activity was measured under standard conditions, except that the concentration of NP-40 w&s varied as indicated in the graph. The zero value of NP-40 concentration indicates the incorporation of pH]UTP by an untreated ASF virus sample. RESULTS
Copuri$cation of an RNA Activity and ASF Virus
PoLymeruse
Culture medium from ASF virus-infected Vero cells contains an RNA polymerase whose specific activity increases l&fold TABLE
2
PROPERTIESOFTHE REA~ION~ATA~Y~E~BY ASF VIRUS RNA POLYMERASE Reaction conditions Standard system -ATP -GTP -CTP +Denatured calf thymus DNA, 80 pg/ml +Actinomyein D, 50 CLgiml +cz-Amanitin, 40 pg/ml + Rifampicin, 50 E*&/ml +Rifampicin, 100 FgIml +Pancreatic DNaae, 15 pg/ml +Pancreatic RNase, 15 pg/ml” +O.l M KOHb
Enzyme activity (pmol h-l &g-l) 6.3 co.2 0.2 1.2 6.3 co.2 6.2 5.5 3.2 4.4 co.2 co.2
o Following the incubation time in the standard system, the mixture was heated for 10 min in a bath of boiling water, cooled in ice, and treated for 30 min at 37” in the presence of RNaae. a After incubation, KOH was added to a final concentration of 0.1 M and the mixture was heated for 10 min in a bath of boiling water.
FIG. 3. Effect of ribonucleoside triphosphate concentration on ASF virus RNA polymerase activity. Standard assay conditions were used. (A) The concentration of ribonucleoside triphosphates was varied as shown in the graph. (0) ATP; (0) GTP, (A) CTP. (B) After 60 min reaetion, the mixture was supplemented with either 2 m&f ATP or dATP. (e) No addition; (0) addition of ATP, (A) addition of dATP.
after virus purification (Table 1). The enzymatic activity sediments with the virus after potassium tartrate (Fig. 1A) or sucrose (Fig. 1B) gradient centrifugation and elutes in the exclusion volume, with the infective particles, after controlled-pore glass chromato~aphy (Fig. 1C). Other preparations of ASF virus showed a good overlapping of the enzymatic activity and the infectivity after column chromatography. Figure 2 shows that when the virus samples were incubated with NP-40 before the assay, the RNA polymerase activity increased. Pwperties of the RNA P~l~rn~rase Aesoc~ted to ASF Vi?-ue
A~t~~‘ty
Table 2 shows some ch~acte~stics of the reaction catalyzed by ASF virus RNA polymerase. The enzyme requires ail four ribonucleoside triphosphates and is not
ASF VIRUS RNA POLYMERASE
stimulated by the addition of exogenous DNA. The reaction is completely inhibited by actinomycin D, and only partially by rifampicin and pancreatic DNase. a-Amanitin has no effect on the reaction. RNase or KOH treatment hydrolyzes the acidinsoluble reaction product to acid-soluble material. Figure 3A shows that ASF virus RNA polymerase requires for maximal activity an ATP concentration higher than that of GTP or CTP. To test the possibility of a rapid depletion of ATP during the incubation, the reaction mixture was supplemented with either ATP or dATP when the rate of RNA synthesis began to decrease. Figure 3B shows that the addition of those nucleotides has no effect on the rate of RNA synthesis. Under standard assay conditions, the RNA polymerase activity is proportional to virus protein concentration in a range from 2 to at least 30 pg (Fig. 4) and is linear with time for 60 min at 37” (Fig. 3B). The optimal concentrations of MgCl,, MnCl,, and (NH&SO, for the reaction were 5, 0.75, and 20 mM, respectively, and the optimal pH value was 8.0 (Fig. 5). Size and Complementary Sequences of the RNA Synthesized in Vitro by ASF Virus RNA Polymerase Figure 6 shows that under standard assay conditions, most of the RNA synthesized in vitro by the ASF virus RNA
ASF
VIRUS
PROTEIN,
173
4 5 i
n
s_
16
I
40
u” +
20
L
12 mN
60
B E % k
5
6 CONCENTRATION.
I
20 40 60 (NH,12 SO, CONCENTRATION,
60 mM
FIG. 5. Effect of divalent cations, ammonium sulfate, and pH on ASF virus RNA polymerase activity. The standard reaction mixtures were used except that the concentrations of MgCl, or MnCl, (A) and ammonium sulfate (B) or the pH (C) were varied as indicated in the graph.
polymerase has a sedimentation coefficient of 6-14 S. To test whether the RNA synthesized by ASF virus is complementary to virus DNA, a fixed amount of [3H]RNA was hybridized to increasing concentrations of either ASF virus or Vero cell DNA. At least 70% of the RNA annealed with virus DNA and essentially none with host cell DNA (Fig. 7). As a hybridization control, Vero cell DNA hybridized with radioactive ribosomal and transfer RNAs from the same cells (data not shown).
pg
FIG. 4. Effect of ASF virus protein concentration on RNA polymerase activity. Assay conditions were as described under Materials and Methods. The amount of ASF virus protein was varied as indicated in the graph.
DISCUSSION
The enzymatic activity described in this paper has the properties of a DNA-dependent RNA polymerase. RNA synthesis re-
174
KUZNAR,
FRACTION
SALAS, AND VIiWELA
NUMBER
FIG. 6. Sucrose gradient sedimentation of RNA synthesized in vitro by ASF virus RNA polymerase. The arrows indicate the position of the $H-labeled rRNA and tRNA markers from Vero cells centrifuged in a parallel gradient.
quired all four ribonucleoside triphosphates, was completely inhibited by aetinomycin D, and the product was susceptible to pancreatic RNase and to alkaline hydrolysis. Since the polymerase activity which copurifies with ASF virus is not sensitive to cr-amanitin (an inhibitor of cellular RNA polymerases II and III) and requires previous treatment of purified virions with NP-40 for full activity, it seems that the enzyme is an integral viral component rather than a contamination. Evidence supporting this idea comes from the fact that ASF virus RNA polymerase activity does not require an exogeneous DNA template and that the RNA product synthesized in vitro anneals specifically with viral DNA. Furthermore, the in vitro RNA synthesis is only 30% inhibited when DNase I is added to the reaction mixture. It seems unlikely that the RNA polymerase activity is a contaminant enzyme which uses the inner ASF virus DNA and not an added DNA template. The weak effect of rifampicin on the RNA polymerase activity rules out the possibility that the measured reaction were due to bacterial contamination. ASF virus RNA polymerase shares a number of properties with a similar enzymatic activity present in poxviruses. The specific activities of both enzymes when assayed in partially dis~pt~ virions are comparable (Kates and McAuslan, 1967), they require higher concentrations of ATP than of the other ~~nucleoside triphos-
phates (Kates and Beeson, 1970), and the patterns of enzyme inhibition by a number of semisynthetic derivatives of rifampicin are similar (Szilagyi and Pennington, 1971; unpublished results). Furthermore, we will show elsewhere that the RNA synthesized in vitro by ASF virus is polyadenylated and methylated and that the virus contains other enzymatic activities, among them the protein kinase described by Polatnick et al. (1974), also present in poxviruses (Moss, 1974). These properties, together with the existence of crosslinks in the genomes of vaccinia (Berns and Silverman, 1970; Geshelin and Berns, 1974) and ASF virus (Ortin et al., 1979) suggest that both virus, independently of their mo~hology and host range, use a similar, if not identical, biochemical strategy for the early phase of gene expression. Together with the iridescent viruses from insects and frog virus 3, ASF virus has been classified as an Iridovirus (Fenner, 1976). It is not known whether or not all the members of the family contain crosslinked DNA, but there are reports that suggest that iridescent viruses types 2 and 6 (Kelly and Tinsley, 1973) and frog virus 3 (Gravel1 and ~romeans, 1971; Gaby and Kucera, 1974) contain an RNA polymerase, as well as other enzymatic activities similar to those present in poxviruses (Kelly and Robertson, 1973). It has been reported that the DNA of ASF virus is infective (Adldinger et al., 1966). This result, however, should be re-
O-0
,o 0.6
, 1.2
I.8
9 2.4
ONA, clg
FIG. 7. Hybridization of RNA synthesized in vitro to DNA. The hyb~di~tion mixture contained 2.4 ng of PH]RNA (1400 cpm) and the amounts of ASF virus (e) or Vero cell (0) DNA indicated in the graph.
ASF VIRUS RNA POLYMERASE
examined since it seems incompatible with the presence in ASF virus of an RNA polymerase. Experiments to test whether this activity is involved in the transcription of early genes are in progress. ACKNOWLEDGMENTS This work was supported by grants from Comision Asesora para el Desarrollo de la Investigacicin Cientifica and Comision Administradora de1 Descuento Complementario. REFERENCES ADLDINGER, H. K., STONE, S. S., HESS, W. R., and BACHRACH, H. L. (1966). Extraction of infectious deoxyribonucleic acid from African swine fever virus. Virology 30, 750-752. BERNS, K. I., and SILYER~A~, C. (1970). Natural occurrence of cross-linked vaccinia virus deoxyribonucleic acid. J. Viral. 5, 299-304. BREESE, S. S., JR., and DEBOER, C. J. (1966). Electron microscope observations of African swine fever virus in tissue culture cells. Virology 28, 420-428.
ENJUANES, L., CARRASCOSA,A. L., MORENO, M. A., and VI~UELA, E. (1976a). Titration of African swine fever (ASF) virus. J. Gen. Viral. 32, 471477.
ENJUANES, L., CARRASCOSA,A. L., and VIRUELA, E. (197613). Isolation and properties of the DNA of African swine fever (ASF) virus. J. Gen. Viral. 32,479-&E.
FENNER, F. (1976). “Classification and Nomenclature of Viruses,” Second Report of the International Committee on Taxonomy of Viruses. Karger, Basel. GABY, N. S., and KUCERA, L. S. (1974). DNAdependent RNA polymerase associated with subviral particles of polyhedral cytoplasmic deoxyribovirus. J. Vi&. 14, 231-233. GESHELIN, P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. S&785-796.
GRAVELL, M., and CROMEANS, T. L. (1971). Mechanisms involved in nongenetic reactivation of frog polyhedral cytoplasmic deoxyribovirus: Evidence for an RNA polymerase in the virion. Virology 46,39-49.
KATES, J. R., and MCAUS~N, B. R. flS67). Poxvirus DNA-dependent RNA polymerase. Proc. Nat. Acad. Sci. USA 58, 134-141. KATES, J., and BEESON, J. (1970). Ribonucleic acid synthesis in vaccinia virus. I. The mechanism of synthesis and release in vaccinia cores. J. Mol. Biol.
50, l-18.
KELLY, D. C., and TINSLEY, T, W. (1973). Ribonucleic acid polymerase activity associated with particles of iridescent virus types 2 and 6. J. Invertebr. Pathol. 22, 199-202. KELLY, D. C., and ROBERTSON, J. S. (1973). Icosahedral cytoplasmie deoxyriboviruses. J. Gen. viroz. 20, 17-41. LOWRY, 0. H., ROSEBROU~H, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. C&n. 193,265-275. Moss, B. (1974). Reproduction of poxviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 3, pp. 405-474. Plenum, New York. MUNYON, W., PAOLETTI, E., and GRACE, J. T., Ja. (1967). RNA polymerase activity in purified infectious vaccinia virus. Proc. Nat. Acad. Sci. USA 58,2280-2287.
ORTIN, J., ENJUANES, L., and VIRUELA, E. (1979). Cross-links in the DNA of African swine fever virus. J. Viral. 31, 579-583. ORTIN, J., and VINUELA, E. (lS77). Requirement of cell nucleus for African swine fever virus replication in Vero cells. J. Viral. 21, 902-905. POLATNICK, J., PAU, I. C., and GRAVELL, M. (1974). Protein kinase activity in African swine fever virus. Arch. ~esamte V~~fo~~h. 44, 156-159. SCHWARTZ, J., and DALES, S. (1971). Biogenesis of poxviruses: Identification of four enzyme activities within purified Yaba tumor virus. Virology 45, 797-801.
GOORHA, R., MURTI, G., GRANOFF, A., ~~~TIREY, R. (1918). Macromole~~~ synthesis in cells infected by frog virus 3. VIII. The nucleus is a site of frog virus 3 DNA and RNA synthesis. Virology 84,32-50.
GOORHA, R., WILLIS, D. B., Macromolecular synthesis virus 3. VI. Frog virus ent on the cell nucleus. J.
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and GRANOFF, A. (1977). in cells infected by frog 3 replication is dependViral. 21, 302-305.
SZILAGYI, J. F., and PENNINGTON, T. H. (1971). Effect of rifamyrins and related antibiotics on the deoxyr~bonueleie acid-dependent ribonucleic acid polymerase of vaceinia virus particles. J. ViroE. 8, 133-141. VOGELSTEIN, B., and GILLESPIE, D. (1977). RNADNA hybridization in solution without DNA reannealing. Biochem. Biophys. Res. Commun. 75, 1127-1132.