VIROLOGY
92,
271-277 (1979)
Inhibition of Activity of Encephalomyocarditis Virus-induced RNA Polymerase by Antibodies against Cellular Components T. M. DMITRIEVA,
M. V. SHCHEGLOVA,
AND
V. I. AGOL’
A. N. Belozersky Laboratoy of Molecular Biology and Bioorganic Chemistry and Department Moscow State University, and Institute of Poliomyelitis and Viral Encephalitides, the U. S. S. R. Academy of Medical Sciences, Moscow, U. S. S. R.
of Virology,
Accepted September II, 1978
Antibodies raised against a fraction of homogenate of uninfected Krebs II cells are able to inhibit the RNA polymerase activity of replication complexes induced upon infection of these cells with encephalomyocarditis virus. The inhibitory activity of these antibodies can be absorbed by the cellular fraction used for immunization. When tested with separated replication complexes synthesizing preferentially either single- or double-stranded virus-specific RNA, the anti-host antibodies inhibit the formation of single-stranded RNA totally and the synthesis of double-stranded RNA partially. The results are consistent with a notion that a host cell component(s) is a functionally important part of viral replication complexes. INTRODUCTION
The polypeptide composition of RNA polymerases induced upon infection with picornaviruses is at present rather poorly known. There is some evidence that this enzyme contains a virus-specific polypeptide, NCVP4 or E, of a molecular weight of 56 to 58 kilodaltons (Lundquist et al., 1974; Loesch and Arlinghaus, 1975; Traub et al., 1976). Partially purified preparations of mengovirus replication complex also contain a host cell protein of 40 kilodaltons (Loesch and Arlinghaus, 19’75), but its functional participation in the viral RNA replication machinery has not been documented. On the other hand, the structure and function of an RNA phage-induced RNA-dependent RNA polymerase, the Q@ replicase, has been elucidated in great detail (Eoyang and August, 1974). This multicomponent enzyme consists of one virusspecific polypeptide and three cellular proteins. In addition, at least one more host protein is required for the phage RNA replication. It has been reported that anti1 Author to whom requests for reprints should be sent. 271
bodies raised against the host cell components of Q/3 replicase are able to inhibit the activity of this enzyme in vitro (Carmichael et al., 1976). In an attempt to gain some information on the possible participation of host components in the process of picornavirus RNA replication, we decided to test the effect of antibodies against host cell proteins on the activity of encephalomyocarditis (EMC) virus-induced RNA polymerase. We report here that such antibodies do inhibit the synthesis of single- and double-stranded EMC virus RNA in vitro. Preliminary results of this study have been published elsewhere (Dmitrieva et al., 1977). MATERIALS
AND METHODS
Isolation of replication complexes. Template-containing replication complexes capable of synthesis of viral RNA in vitro were isolated from EMC virus-infected Krebs II cells after 5 hr of infection by a previously described procedure (Dmitrieva and Agol, 1974) which included the treatment of cell homogenate pellet (40,000 g, 15 min) with deoxycholate and dextran sulfate and ethanol precipitation. The membrane-free 0042~6822/79/020271-07$02.00/0 Copyright 0 1979by AcademicPress, Inc. All rights of reproduction in any form reserved.
272
DMITRIEVA,
SHCHEGLOVA,
crude preparations of replicative complexes obtained at this step were subjected to sucrose density gradient centrifugation to obtain partially purified complexes preferentially synthesizing either single- or double-stranded EMC virus-specific RNA. The RNA polymerase activity of these complexes was assayed by measuring the incorporation of label from [14C]GTP into the acid-insoluble material (Dmitrieva and Agol, 1974). The values of incorporation in the absence of three unlabeled ribonucleoside triphosphates (comprising usually about 2 to 4%) were subtracted from those obtained in the complete incubation mixture.
AND AGOL
Absorption of immune y-globulins. Preparations of immune y-globulins were mixed with the PdOfraction, such as those used for the immunization, in a ratio of 1 to 2 with respect to protein. After overnight stirring at 4” the suspension was centrifuged at 40,000 g for 20 min and the supernatant obtained was subjected to the same procedure of absorption with a fresh portion of P,,. The y-globulins obtained after the second absorption were concentrated by dialysis against dry Sephadex G-200. Treatment y-globulins.
of replication
complexes with
Preparations of replication complexes in a solution containing 0.0011M MgC&, 0.01&f Tris-HCI, pH 7.5 were Analysis of products formed by replication complexes. The RNA products formed mixed with preparations of either immune by replication complexes were deproteinized or control y-globulins in 0.01 M Na phoswith phenol, precipitated with ethanol, phate, pH 7.5 and were incubated at 37 and subjected to centrifugation in a 5 to for 15 min. Then components for the assay 20% sucrose concentration gradient pre- of RNA polymerase activity (Dmitrieva pared in a solution containing 0.11M NaCl, and Agol, 1974) were added and the incuba0.01 M Tris-HCl, pH 7.5, and 0.001 it4 tion was continued for another 30 min. EDTA at 45,000 rpm for 150 min at 4” in a Then the samples were treated either to determine radioactivity in the acid-insoluble Beckman SW-50 rotor. After centrifugation, each fraction (ca. 0.25 ml) was collected material or to analyze the RNA products in 1 ml of a solution containing 0.15 1M formed. NaCl, 0.015 1M sodium citrate, pH 7.5 and the radioactivity of the acid-insoluble RESULTS material was determined before and after treatment with pancreatic RNase (10 General Characterization of the Inhibitory pg/ml, 30 min, 30”). Action of Anti-host Antibodies on the Immunization procedure and isolation of Activity of EMC Virus-Induced RNA y-globulins. The homogenates of noninPol ymerase fected Krebs II cells (Dmitrieva and Agol, 1974) were sedimented at 40,000 g for 15 The RNA polymerase activity in crude min and the pellet (P4& was used for the immunization of rabbits. For the first unfractionated preparations of EMC virusimmunization, two portions each of 4 to 5 induced replication complexes was measmg of the protein of this fraction in com- ured in the presence of different concenplete Freund’s adjuvant were injected into a trations of immune y-globulins. The results presented in Fig. 1 show that the enzyme popliteal lymph node and intramuscularly. Intramuscular reimmunization with the activity is inhibited in the presence of antigen was repeated several times at 40- antibodies, a considerable, at least IO-fold, day intervals. Preparations of the immune weight excess of y-globulins being required y-globulins were isolated from the sera for a nearly maximal inhibition. A ratio of complex protein taken on the seventh to ninth days after y-globulins:replication reimmunization by a previously described equal to 10 has been chosen for further method (Baumstark et al., 1964) with minor experiments. At this ratio, the inhibition of modifications. Control preparations of y- RNA polymerase activity usually amounted globulins were similarly isolated from pre- to not less than 80%. Sera taken from a number of immunized rabbits possessed immunization sera.
INHIBITION
OF EMC VIRUS-INDUCED
g-globulir@epluation amplexes protein FIG. 1. Effect of different concentrations of immune y-globulins on the RNA polymerase activity of crude replication complexes. Each sample (0.4 ml) contained 180 pg of protein of crude replication complexes and different amounts of a preparation of immune globulins. After incubation at 37” for 15 min, the components required for the RNA polymerase activity assay were added and the incubation was continued for an additional 30 min. Radioactivity in the acid-insoluble material was determined. The incorporation of label in the samples without y-globulins (5600 cpm) represented the control activity of RNA polymerase.
such activity. Preparations of control preimmunization y-globulins exerted only a slight inhibitory effect, not exceeding 20% (Table 1). Absorption of the immune y-globulins with the PdOfraction of homogenate from uninfected cells resulted in a significant, albeit apparently not complete, loss of their inhibitory activity (Table 1, Expt 3). An absorption experiment has also been carried out in a different way. A preparation of immune y-globulins was incubated at 37” for 15 min with increasing amounts of the P,, from uninfected cells. A preparation of unfractionated EMC virus-induced replication complexes as well as components
POLYMERASE
273
ACTIVITY
for the RNA polymerase activity assay were then added and the incubation was continued for another 30 min. The results presented in Fig. 2 indicate that such an in situ absorption was also highly efficient in eliminating the inhibitory effect of immune y-globulins on the polymerase activity. It might be suggested that the inhibitory antibodies were directed not against cellular proteins but rather against doublestranded RNA possibly present in the material used for immunization. To test this hypothesis, an experiment with in situ absorption of immune y-globulins by poly(r1) *poly(rC) was performed. The results presented in Fig. 3 show that the preincubation of immune y-globulins with this doublestranded polyribonucleotide failed to influence the inhibitory activity of the antibodies. Thus, this activity could not be explained on the basis of interaction of immune y - globulins with virus - specific double stranded RNA present in replication complexes. TABLE 1 THE EFFECT OF PREPARATIONS OF Y-GLOBULINS ON THE RNA POLYMERASE ACTIVITY OF CRUDE REPLICATION COMPLEXES OF EMC VIRUS
Incorporation of label from [W]GTP
Expt
Preparation of globulins
c@mg of protein of replication complex
%
1
None Control Immune
39,060 34,550 6,950
100 88 18
2
None Control Immune
49,240 42,346 9,840
100 86 20
3
None Control Immune Immune, absorbed
65,410 55,250 7,930 45,690
100 84 12 69
274
DMITRIEVA,
SHCHEGLOVA,
AND AGOL
of the gradient was assayed before and after incubation with immune y-globulins. The polymerase activity of every fraction was found to be inhibited by the antibodies. In the present work, a more detailed study of this point has been carried out. The replication complexes synthesizing preferentially either single- or doublestranded RNA were isolated and separately incubated with either immune or control y-globulins. Then the products formed by these globulin-treated complexes in the 1 I RNA polymerase reaction were extracted loo 200 and characterized by means of sucrose PqO Protein, pq density gradient centrifugation and determination of sensitivity to pancreatic FIG. 2. Effect of preincubation of immune yglobulins with P, on their capacity to inhibit the RNase treatment. Figure 4 shows the reRNA polymerase activity of crude replication com- sults of assays of RNA polymerase activity plexes. Samples, eat;. containing 300 pg of a in each fraction obtained after sedimentation of a preparation of crude replication preparation of immune y-globulins, were incubated at 37” for 15 min with amounts of P4,, protein indicomplexes in a sucrose gradient. Two peaks cated on the abscissa. Then 60 pg of protein of crude replication complexes and components for the assay of RNA polymerase activity were added to each fraction. After an additional 30 min of incubation, radioactivity in the acid-insoluble material was determined (solid line). The broken line indicates the extent of incorporation of the label in samples containing neither y-globulin nor P,,.
The Effect of Immune y-Globulins on the Polymerase Activity of Replication Complexes Preferentially Synthesizing Either Single- or Double-Stranded Viral RNA The crude replication complexes isolated from cells infected with picornaviruses (Arlinghaus and Polatnick, 1969; Arlinghaus et al., 1972; Caliguiri, 1974), including EMC virus (Dmitrieva and Agol, 1974), can be separated in at least two fractions preferentially synthesizing either singleor double-stranded viral RNA. It seemed to be of interest to investigate the effect of immune y-globulins on the RNA polymerase activity of each of these fractions. In a preliminary experiment (Dmitrieva et al., 1977), the crude replication complexes were subjected to sedimentation in a sucrose concentration gradient and the RNA polymerase activity in each fraction
x1Q4
St-
4r---5’ E 34 --X--
-*----mm____
* ______m-
------
*-
!
FIG. 3. Effect of preincubation of immune yglobulins with poly(rI).poly(rC) on their capacity to inhibit the RNA polymerase activity of crude replication complexes. Samples, each containing 500 pg of a preparation of immune y-globulins, were incubated at 37” for 15 min with the amounts of poly(r1). poly(rC) indicated on the abscissa. Then crude replication complexes (50 pg of protein) were added to each sample and incubation was continued for 15 min. Next, components for the assay of RNA polymerase activity were added and after 30 min of additional incubation, radioactivity in the acidinsoluble material was determined (broken line). Control sample (solid line) contained no antibodies.
INHIBITION
0
OF EMC VIRUS-INDUCED
POLYMERASE
RNA material was still formed (Fig. 5b). The light replication complex treated with control y-globulins, on the other hand, synthesized almost exclusively RNase-resistant double-stranded replicative form RNA (Fig. 6a). The synthesis of this product was greatly, although not completely, inhibited after the treatment of the light complex with immune y-globulins (Fig. 6b).
80s c
6
2001
,
0
I
2
I
I
4
I
I
6
I
I
8
I
275
ACTIVITY
I
I
I
I
I
c
I
10 12 14
x 28s
l&S
nrI
a
fraction number
FIG. 4. Sedimentation profile of RNA polymerase activity of replication complexes. A preparation of crude replication complexes (1.2 mg of protein) was subjected to centrifugation in a 10 to 50% sucrose concentration gradient prepared in 0.004 M MgCl,, 0.01 M Tris, pH 7.5 (Beckman SW-27 rotor, 17 hr, 15,000 rpm, 2”). Components for the assay of RNA polymerase activity were added to aliquots (100~1) of each fraction and radioactivity in the acid-insoluble material was determined. 80
of activity are evident. Peak fractions 2 and 11 were taken as representatives of “heavy” and “light” replication complexes, respectively. The aliquots of these fractions were incubated with either immune or control -y-globulins, the components required for RNA synthesis were added, and the products synthesized were subjected to centrifugation in sucrose gradients. The radioactivity of acid-insoluble material in each gradient fraction was measured before and after the treatment with pancreatic RNase. The products formed by the heavy replication complex treated with control globulins contained predominantly pancreatic RNase-sensitive, that is single-stranded, RNA species, among which a genome size, 37 S, RNA was present in a considerable amount (Fig. 5a). After pretreatment with immune y-globulins, this complex synthesized practically no single-stranded RNA although a small quantity of double-stranded
B
t
40
fwtiot
number
FIG. 5. RNA products formed by the “heavy” replication complex pretreated with control (a) or immune (b) y-globulins. Aliquots (1 ml) of fraction 2 of the gradient presented in Fig. 4 were incubated with either control or immune y-globulins (550 pg of protein) at 37” for 15 min and then components for the RNA polymerase activity assay were added. After 30 min of incubation, the samples were deproteinized by phenol extraction and RNA was precipitated with ethanol and then subjected to centrifugation in 5 to 20% sucrose concentration gradients (Beckman SW-50 rotor, 150 min, 45,000 rpm, 4”). Radioactivity in the acid-insoluble material was determined before (solid lines) and after (broken lines) treatment with pancreatic RNase. Arrows show the position of ribosomal RNAs from the Krebs II cells centrifuged in a parallel gradient.
276
DMITRIEVA,
SHCHEGLOVA,
AND AGOL
The inhibitory effect of our preparations of immune y-globulins cannot be explained a x by the presence of RNase activity. First, neither similarly prepared y-globulins from preimmunization sera nor absorbed immune ly\ y-globulins possess a comparable inhibitory I/’ activity. Second, a sensitive assay for RNase activity, using EMC virus RNA as 100 1I the substrate and its sedimentation be2s * \ 18s havior as the criterion of nuclease activity, gave essentially negative results with yii i1 globulins (data not shown). I i I I Alternatively, it may be suggested that the inhibition of RNA polymerase is due to the interaction of antibodies with some host cell proteins which contaminated the preparations of replicative complexes but are not functionally important components of the enzyme. The nonspecific precipitate formed as a result of such interaction might somehow inactivate the polymerase. It is not possible at present to disprove such an assumption rigorously but several considerations seem to make it unlikely. First, there is no evident reason for believing that unspecific precipitation would result in a dramatic drop of the enzyme activity. Moreover, the in situ absorption experiment (Fig. 2) indicates that the precipitate itself, if formed, can hardly inacti0 vate the polymerase. Second, the inhibition by immune y-globulins of the enzyme FIG. 6. RNA products formed by the “light” replicaactivity in any sedimentation fraction of tion complex pretreated with control (a) or immune replication complexes (Dmitrieva et al., (b) y-globulins. The experiment was performed as 1977) necessitates the additional assumpdescribed in the legend to Fig. 5 but fraction 11 of the gradient presented in Fig. 4 was used instead of tion that the contaminating proteins responsible for the inhibition are distributed fraction 2. along the entire sucrose gradient. Third, an unequal inhibition by immune y-globDISCUSSION ulins of the synthesis of single- and doubleThe results presented in this paper indi- stranded RNA catalyzed by the same heavy cate that antibodies directed against cellu- replication complex (Fig. 5) also contralar components, most likely proteins, are dicts a simple contaminant hypothesis. able to exert an inhibitory effect on the Therefore, we prefer at present to explain RNA polymerase activity of EMC virus- the inhibitory action of immune y-globulins specific replication complexes. The simplest on the RNA polymerase activity of replicaand most likely explanation of this effect tion complexes by their interaction with consists in the assumption that a host cell cellular proteins which are essential comprotein(s) is the intrinsic component of ponents of these complexes. Unequivocal virus-induced RNA polymerase. Neverthe- proof of this notion requires, however, less, other explanations of the effect additional experiments. The second point which merits discussion should be considered. 150
t
INHIBITION
OF EMC VIRUS-INDUCED
is the inhibition by cellular antibodies of the polymerase activity of both replication complexes synthesizing preferentially either single- or double-stranded virusspecific RNA. This fact appears to indicate that host cell proteins participate in both reactions. The results presented in Figs. 5 and 6 show that the synthesis of singlestranded RNA is completely inhibited while the synthesis of double-stranded RNA is impaired only partially. This difference suggests that the host cell component(s) is more important for the formation of singlestranded RNA. The present experiments, however, do not provide any information allowing us to decide whether the same or different host proteins are required for the synthesis of both species of virusspecific RNA. REFERENCES
ARLINGHAUS, R. B., and POLATNICK, J. (1969). The isolation of two enzyme-ribonucleic acid complexes involved in the synthesis of foot-and-mouth disease virus ribonucleic acid. Proc. Nat. Acad. Sci. USA 62, 821-828. ARLINGHAUS, R. B., SYREWICZ,J. J., and LOESCH, W. T. (1972). RNA polymerase complexes from mengovirus infected cells. Arch. Ges. Vimsforsch. 38, 17-28. BAUMSTARK, I. S., DAFFIN, R. F., and BARDAWIE, W. A. (1964). A preparative method for the
POLYMERASE
ACTIVITY
277
separation of 7s’ y-globulin from human serum. Arch. Biochem. Biophys. 108, 514-522. CALIGUIRI, L. A. (1974). Analysis of RNA associated with the poliovirus RNA replication complexes. Virology
58, 526-535.
CARMICHAEL, G. G., LANDERS, T. A., and WEBER, K. (1976). Immunochemical analysis of the functions of the subunits of phage Qp ribonucleic acid replicase. J. Biol. Chew 251, 2744-2753. DMITRIEVA, T. M., and AGOL, V. I. (1974). Selective inhibition of the synthesis of single-stranded RNA of encephalomyocarditis virus by 2-(a-hydroxybenzyl)-bensimidazole in cell-free systems. Arch. Ges. Vimsforsch.
45, 17-26.
DMITRIEVA, T. M., SENKEVICH,T. G., KOMISSAROVA, E. V., and AGOL, V. I. (1977). Immunological elucidation of cellular component of RNA polymerase of encephalomyocarditis virus. Dokl. A.N. SSSR 232, 949-952.
EOYANG, L., and AUGUST, J. T. (1974). Reproduction of RNA bacteriophages. In “Comprehensive Virology,” (Fraenkel-Conrat, H., and Wagner, R. R., eds.) Vol. 2, pp. l-59. Plenum Press, New York. LOESCH,W. T., and ARLINGHAUS, R. B. (1975). Stable polipeptide associated with the 250s mengovirusinduced RNA polymerase structure. Arch. Viral. 47, 201-209. LUNDQUIST, R. E., EHRENFELD, E., and MAIZEL, J. V. (1974). Isolation of a viral polypeptide associated with poliovirus RNA polymerase. Proc. Nat. Acad. Sci. USA 71, 4773-4777.
TRAUB, A., DISKIN, B., ROSENBERG, R., and KALMAR, N. S. (1976). Isolation and properties of the replicase of encephalomyocarditis virus. J. Viral.
18, 375-386.
92,
271-277 (1979)
Inhibition of Activity of Encephalomyocarditis Virus-induced RNA Polymerase by Antibodies against Cellular Components T. M. DMITRIEVA,
M. V. SHCHEGLOVA,
AND
V. I. AGOL’
A. N. Belozersky Laboratoy of Molecular Biology and Bioorganic Chemistry and Department Moscow State University, and Institute of Poliomyelitis and Viral Encephalitides, the U. S. S. R. Academy of Medical Sciences, Moscow, U. S. S. R.
of Virology,
Accepted September II, 1978
Antibodies raised against a fraction of homogenate of uninfected Krebs II cells are able to inhibit the RNA polymerase activity of replication complexes induced upon infection of these cells with encephalomyocarditis virus. The inhibitory activity of these antibodies can be absorbed by the cellular fraction used for immunization. When tested with separated replication complexes synthesizing preferentially either single- or double-stranded virus-specific RNA, the anti-host antibodies inhibit the formation of single-stranded RNA totally and the synthesis of double-stranded RNA partially. The results are consistent with a notion that a host cell component(s) is a functionally important part of viral replication complexes. INTRODUCTION
The polypeptide composition of RNA polymerases induced upon infection with picornaviruses is at present rather poorly known. There is some evidence that this enzyme contains a virus-specific polypeptide, NCVP4 or E, of a molecular weight of 56 to 58 kilodaltons (Lundquist et al., 1974; Loesch and Arlinghaus, 1975; Traub et al., 1976). Partially purified preparations of mengovirus replication complex also contain a host cell protein of 40 kilodaltons (Loesch and Arlinghaus, 19’75), but its functional participation in the viral RNA replication machinery has not been documented. On the other hand, the structure and function of an RNA phage-induced RNA-dependent RNA polymerase, the Q@ replicase, has been elucidated in great detail (Eoyang and August, 1974). This multicomponent enzyme consists of one virusspecific polypeptide and three cellular proteins. In addition, at least one more host protein is required for the phage RNA replication. It has been reported that anti1 Author to whom requests for reprints should be sent. 271
bodies raised against the host cell components of Q/3 replicase are able to inhibit the activity of this enzyme in vitro (Carmichael et al., 1976). In an attempt to gain some information on the possible participation of host components in the process of picornavirus RNA replication, we decided to test the effect of antibodies against host cell proteins on the activity of encephalomyocarditis (EMC) virus-induced RNA polymerase. We report here that such antibodies do inhibit the synthesis of single- and double-stranded EMC virus RNA in vitro. Preliminary results of this study have been published elsewhere (Dmitrieva et al., 1977). MATERIALS
AND METHODS
Isolation of replication complexes. Template-containing replication complexes capable of synthesis of viral RNA in vitro were isolated from EMC virus-infected Krebs II cells after 5 hr of infection by a previously described procedure (Dmitrieva and Agol, 1974) which included the treatment of cell homogenate pellet (40,000 g, 15 min) with deoxycholate and dextran sulfate and ethanol precipitation. The membrane-free 0042~6822/79/020271-07$02.00/0 Copyright 0 1979by AcademicPress, Inc. All rights of reproduction in any form reserved.
272
DMITRIEVA,
SHCHEGLOVA,
crude preparations of replicative complexes obtained at this step were subjected to sucrose density gradient centrifugation to obtain partially purified complexes preferentially synthesizing either single- or double-stranded EMC virus-specific RNA. The RNA polymerase activity of these complexes was assayed by measuring the incorporation of label from [14C]GTP into the acid-insoluble material (Dmitrieva and Agol, 1974). The values of incorporation in the absence of three unlabeled ribonucleoside triphosphates (comprising usually about 2 to 4%) were subtracted from those obtained in the complete incubation mixture.
AND AGOL
Absorption of immune y-globulins. Preparations of immune y-globulins were mixed with the PdOfraction, such as those used for the immunization, in a ratio of 1 to 2 with respect to protein. After overnight stirring at 4” the suspension was centrifuged at 40,000 g for 20 min and the supernatant obtained was subjected to the same procedure of absorption with a fresh portion of P,,. The y-globulins obtained after the second absorption were concentrated by dialysis against dry Sephadex G-200. Treatment y-globulins.
of replication
complexes with
Preparations of replication complexes in a solution containing 0.0011M MgC&, 0.01&f Tris-HCI, pH 7.5 were Analysis of products formed by replication complexes. The RNA products formed mixed with preparations of either immune by replication complexes were deproteinized or control y-globulins in 0.01 M Na phoswith phenol, precipitated with ethanol, phate, pH 7.5 and were incubated at 37 and subjected to centrifugation in a 5 to for 15 min. Then components for the assay 20% sucrose concentration gradient pre- of RNA polymerase activity (Dmitrieva pared in a solution containing 0.11M NaCl, and Agol, 1974) were added and the incuba0.01 M Tris-HCl, pH 7.5, and 0.001 it4 tion was continued for another 30 min. EDTA at 45,000 rpm for 150 min at 4” in a Then the samples were treated either to determine radioactivity in the acid-insoluble Beckman SW-50 rotor. After centrifugation, each fraction (ca. 0.25 ml) was collected material or to analyze the RNA products in 1 ml of a solution containing 0.15 1M formed. NaCl, 0.015 1M sodium citrate, pH 7.5 and the radioactivity of the acid-insoluble RESULTS material was determined before and after treatment with pancreatic RNase (10 General Characterization of the Inhibitory pg/ml, 30 min, 30”). Action of Anti-host Antibodies on the Immunization procedure and isolation of Activity of EMC Virus-Induced RNA y-globulins. The homogenates of noninPol ymerase fected Krebs II cells (Dmitrieva and Agol, 1974) were sedimented at 40,000 g for 15 The RNA polymerase activity in crude min and the pellet (P4& was used for the immunization of rabbits. For the first unfractionated preparations of EMC virusimmunization, two portions each of 4 to 5 induced replication complexes was measmg of the protein of this fraction in com- ured in the presence of different concenplete Freund’s adjuvant were injected into a trations of immune y-globulins. The results presented in Fig. 1 show that the enzyme popliteal lymph node and intramuscularly. Intramuscular reimmunization with the activity is inhibited in the presence of antigen was repeated several times at 40- antibodies, a considerable, at least IO-fold, day intervals. Preparations of the immune weight excess of y-globulins being required y-globulins were isolated from the sera for a nearly maximal inhibition. A ratio of complex protein taken on the seventh to ninth days after y-globulins:replication reimmunization by a previously described equal to 10 has been chosen for further method (Baumstark et al., 1964) with minor experiments. At this ratio, the inhibition of modifications. Control preparations of y- RNA polymerase activity usually amounted globulins were similarly isolated from pre- to not less than 80%. Sera taken from a number of immunized rabbits possessed immunization sera.
INHIBITION
OF EMC VIRUS-INDUCED
g-globulir@epluation amplexes protein FIG. 1. Effect of different concentrations of immune y-globulins on the RNA polymerase activity of crude replication complexes. Each sample (0.4 ml) contained 180 pg of protein of crude replication complexes and different amounts of a preparation of immune globulins. After incubation at 37” for 15 min, the components required for the RNA polymerase activity assay were added and the incubation was continued for an additional 30 min. Radioactivity in the acid-insoluble material was determined. The incorporation of label in the samples without y-globulins (5600 cpm) represented the control activity of RNA polymerase.
such activity. Preparations of control preimmunization y-globulins exerted only a slight inhibitory effect, not exceeding 20% (Table 1). Absorption of the immune y-globulins with the PdOfraction of homogenate from uninfected cells resulted in a significant, albeit apparently not complete, loss of their inhibitory activity (Table 1, Expt 3). An absorption experiment has also been carried out in a different way. A preparation of immune y-globulins was incubated at 37” for 15 min with increasing amounts of the P,, from uninfected cells. A preparation of unfractionated EMC virus-induced replication complexes as well as components
POLYMERASE
273
ACTIVITY
for the RNA polymerase activity assay were then added and the incubation was continued for another 30 min. The results presented in Fig. 2 indicate that such an in situ absorption was also highly efficient in eliminating the inhibitory effect of immune y-globulins on the polymerase activity. It might be suggested that the inhibitory antibodies were directed not against cellular proteins but rather against doublestranded RNA possibly present in the material used for immunization. To test this hypothesis, an experiment with in situ absorption of immune y-globulins by poly(r1) *poly(rC) was performed. The results presented in Fig. 3 show that the preincubation of immune y-globulins with this doublestranded polyribonucleotide failed to influence the inhibitory activity of the antibodies. Thus, this activity could not be explained on the basis of interaction of immune y - globulins with virus - specific double stranded RNA present in replication complexes. TABLE 1 THE EFFECT OF PREPARATIONS OF Y-GLOBULINS ON THE RNA POLYMERASE ACTIVITY OF CRUDE REPLICATION COMPLEXES OF EMC VIRUS
Incorporation of label from [W]GTP
Expt
Preparation of globulins
c@mg of protein of replication complex
%
1
None Control Immune
39,060 34,550 6,950
100 88 18
2
None Control Immune
49,240 42,346 9,840
100 86 20
3
None Control Immune Immune, absorbed
65,410 55,250 7,930 45,690
100 84 12 69
274
DMITRIEVA,
SHCHEGLOVA,
AND AGOL
of the gradient was assayed before and after incubation with immune y-globulins. The polymerase activity of every fraction was found to be inhibited by the antibodies. In the present work, a more detailed study of this point has been carried out. The replication complexes synthesizing preferentially either single- or doublestranded RNA were isolated and separately incubated with either immune or control y-globulins. Then the products formed by these globulin-treated complexes in the 1 I RNA polymerase reaction were extracted loo 200 and characterized by means of sucrose PqO Protein, pq density gradient centrifugation and determination of sensitivity to pancreatic FIG. 2. Effect of preincubation of immune yglobulins with P, on their capacity to inhibit the RNase treatment. Figure 4 shows the reRNA polymerase activity of crude replication com- sults of assays of RNA polymerase activity plexes. Samples, eat;. containing 300 pg of a in each fraction obtained after sedimentation of a preparation of crude replication preparation of immune y-globulins, were incubated at 37” for 15 min with amounts of P4,, protein indicomplexes in a sucrose gradient. Two peaks cated on the abscissa. Then 60 pg of protein of crude replication complexes and components for the assay of RNA polymerase activity were added to each fraction. After an additional 30 min of incubation, radioactivity in the acid-insoluble material was determined (solid line). The broken line indicates the extent of incorporation of the label in samples containing neither y-globulin nor P,,.
The Effect of Immune y-Globulins on the Polymerase Activity of Replication Complexes Preferentially Synthesizing Either Single- or Double-Stranded Viral RNA The crude replication complexes isolated from cells infected with picornaviruses (Arlinghaus and Polatnick, 1969; Arlinghaus et al., 1972; Caliguiri, 1974), including EMC virus (Dmitrieva and Agol, 1974), can be separated in at least two fractions preferentially synthesizing either singleor double-stranded viral RNA. It seemed to be of interest to investigate the effect of immune y-globulins on the RNA polymerase activity of each of these fractions. In a preliminary experiment (Dmitrieva et al., 1977), the crude replication complexes were subjected to sedimentation in a sucrose concentration gradient and the RNA polymerase activity in each fraction
x1Q4
St-
4r---5’ E 34 --X--
-*----mm____
* ______m-
------
*-
!
FIG. 3. Effect of preincubation of immune yglobulins with poly(rI).poly(rC) on their capacity to inhibit the RNA polymerase activity of crude replication complexes. Samples, each containing 500 pg of a preparation of immune y-globulins, were incubated at 37” for 15 min with the amounts of poly(r1). poly(rC) indicated on the abscissa. Then crude replication complexes (50 pg of protein) were added to each sample and incubation was continued for 15 min. Next, components for the assay of RNA polymerase activity were added and after 30 min of additional incubation, radioactivity in the acidinsoluble material was determined (broken line). Control sample (solid line) contained no antibodies.
INHIBITION
0
OF EMC VIRUS-INDUCED
POLYMERASE
RNA material was still formed (Fig. 5b). The light replication complex treated with control y-globulins, on the other hand, synthesized almost exclusively RNase-resistant double-stranded replicative form RNA (Fig. 6a). The synthesis of this product was greatly, although not completely, inhibited after the treatment of the light complex with immune y-globulins (Fig. 6b).
80s c
6
2001
,
0
I
2
I
I
4
I
I
6
I
I
8
I
275
ACTIVITY
I
I
I
I
I
c
I
10 12 14
x 28s
l&S
nrI
a
fraction number
FIG. 4. Sedimentation profile of RNA polymerase activity of replication complexes. A preparation of crude replication complexes (1.2 mg of protein) was subjected to centrifugation in a 10 to 50% sucrose concentration gradient prepared in 0.004 M MgCl,, 0.01 M Tris, pH 7.5 (Beckman SW-27 rotor, 17 hr, 15,000 rpm, 2”). Components for the assay of RNA polymerase activity were added to aliquots (100~1) of each fraction and radioactivity in the acid-insoluble material was determined. 80
of activity are evident. Peak fractions 2 and 11 were taken as representatives of “heavy” and “light” replication complexes, respectively. The aliquots of these fractions were incubated with either immune or control -y-globulins, the components required for RNA synthesis were added, and the products synthesized were subjected to centrifugation in sucrose gradients. The radioactivity of acid-insoluble material in each gradient fraction was measured before and after the treatment with pancreatic RNase. The products formed by the heavy replication complex treated with control globulins contained predominantly pancreatic RNase-sensitive, that is single-stranded, RNA species, among which a genome size, 37 S, RNA was present in a considerable amount (Fig. 5a). After pretreatment with immune y-globulins, this complex synthesized practically no single-stranded RNA although a small quantity of double-stranded
B
t
40
fwtiot
number
FIG. 5. RNA products formed by the “heavy” replication complex pretreated with control (a) or immune (b) y-globulins. Aliquots (1 ml) of fraction 2 of the gradient presented in Fig. 4 were incubated with either control or immune y-globulins (550 pg of protein) at 37” for 15 min and then components for the RNA polymerase activity assay were added. After 30 min of incubation, the samples were deproteinized by phenol extraction and RNA was precipitated with ethanol and then subjected to centrifugation in 5 to 20% sucrose concentration gradients (Beckman SW-50 rotor, 150 min, 45,000 rpm, 4”). Radioactivity in the acid-insoluble material was determined before (solid lines) and after (broken lines) treatment with pancreatic RNase. Arrows show the position of ribosomal RNAs from the Krebs II cells centrifuged in a parallel gradient.
276
DMITRIEVA,
SHCHEGLOVA,
AND AGOL
The inhibitory effect of our preparations of immune y-globulins cannot be explained a x by the presence of RNase activity. First, neither similarly prepared y-globulins from preimmunization sera nor absorbed immune ly\ y-globulins possess a comparable inhibitory I/’ activity. Second, a sensitive assay for RNase activity, using EMC virus RNA as 100 1I the substrate and its sedimentation be2s * \ 18s havior as the criterion of nuclease activity, gave essentially negative results with yii i1 globulins (data not shown). I i I I Alternatively, it may be suggested that the inhibition of RNA polymerase is due to the interaction of antibodies with some host cell proteins which contaminated the preparations of replicative complexes but are not functionally important components of the enzyme. The nonspecific precipitate formed as a result of such interaction might somehow inactivate the polymerase. It is not possible at present to disprove such an assumption rigorously but several considerations seem to make it unlikely. First, there is no evident reason for believing that unspecific precipitation would result in a dramatic drop of the enzyme activity. Moreover, the in situ absorption experiment (Fig. 2) indicates that the precipitate itself, if formed, can hardly inacti0 vate the polymerase. Second, the inhibition by immune y-globulins of the enzyme FIG. 6. RNA products formed by the “light” replicaactivity in any sedimentation fraction of tion complex pretreated with control (a) or immune replication complexes (Dmitrieva et al., (b) y-globulins. The experiment was performed as 1977) necessitates the additional assumpdescribed in the legend to Fig. 5 but fraction 11 of the gradient presented in Fig. 4 was used instead of tion that the contaminating proteins responsible for the inhibition are distributed fraction 2. along the entire sucrose gradient. Third, an unequal inhibition by immune y-globDISCUSSION ulins of the synthesis of single- and doubleThe results presented in this paper indi- stranded RNA catalyzed by the same heavy cate that antibodies directed against cellu- replication complex (Fig. 5) also contralar components, most likely proteins, are dicts a simple contaminant hypothesis. able to exert an inhibitory effect on the Therefore, we prefer at present to explain RNA polymerase activity of EMC virus- the inhibitory action of immune y-globulins specific replication complexes. The simplest on the RNA polymerase activity of replicaand most likely explanation of this effect tion complexes by their interaction with consists in the assumption that a host cell cellular proteins which are essential comprotein(s) is the intrinsic component of ponents of these complexes. Unequivocal virus-induced RNA polymerase. Neverthe- proof of this notion requires, however, less, other explanations of the effect additional experiments. The second point which merits discussion should be considered. 150
t
INHIBITION
OF EMC VIRUS-INDUCED
is the inhibition by cellular antibodies of the polymerase activity of both replication complexes synthesizing preferentially either single- or double-stranded virusspecific RNA. This fact appears to indicate that host cell proteins participate in both reactions. The results presented in Figs. 5 and 6 show that the synthesis of singlestranded RNA is completely inhibited while the synthesis of double-stranded RNA is impaired only partially. This difference suggests that the host cell component(s) is more important for the formation of singlestranded RNA. The present experiments, however, do not provide any information allowing us to decide whether the same or different host proteins are required for the synthesis of both species of virusspecific RNA. REFERENCES
ARLINGHAUS, R. B., and POLATNICK, J. (1969). The isolation of two enzyme-ribonucleic acid complexes involved in the synthesis of foot-and-mouth disease virus ribonucleic acid. Proc. Nat. Acad. Sci. USA 62, 821-828. ARLINGHAUS, R. B., SYREWICZ,J. J., and LOESCH, W. T. (1972). RNA polymerase complexes from mengovirus infected cells. Arch. Ges. Vimsforsch. 38, 17-28. BAUMSTARK, I. S., DAFFIN, R. F., and BARDAWIE, W. A. (1964). A preparative method for the
POLYMERASE
ACTIVITY
277
separation of 7s’ y-globulin from human serum. Arch. Biochem. Biophys. 108, 514-522. CALIGUIRI, L. A. (1974). Analysis of RNA associated with the poliovirus RNA replication complexes. Virology
58, 526-535.
CARMICHAEL, G. G., LANDERS, T. A., and WEBER, K. (1976). Immunochemical analysis of the functions of the subunits of phage Qp ribonucleic acid replicase. J. Biol. Chew 251, 2744-2753. DMITRIEVA, T. M., and AGOL, V. I. (1974). Selective inhibition of the synthesis of single-stranded RNA of encephalomyocarditis virus by 2-(a-hydroxybenzyl)-bensimidazole in cell-free systems. Arch. Ges. Vimsforsch.
45, 17-26.
DMITRIEVA, T. M., SENKEVICH,T. G., KOMISSAROVA, E. V., and AGOL, V. I. (1977). Immunological elucidation of cellular component of RNA polymerase of encephalomyocarditis virus. Dokl. A.N. SSSR 232, 949-952.
EOYANG, L., and AUGUST, J. T. (1974). Reproduction of RNA bacteriophages. In “Comprehensive Virology,” (Fraenkel-Conrat, H., and Wagner, R. R., eds.) Vol. 2, pp. l-59. Plenum Press, New York. LOESCH,W. T., and ARLINGHAUS, R. B. (1975). Stable polipeptide associated with the 250s mengovirusinduced RNA polymerase structure. Arch. Viral. 47, 201-209. LUNDQUIST, R. E., EHRENFELD, E., and MAIZEL, J. V. (1974). Isolation of a viral polypeptide associated with poliovirus RNA polymerase. Proc. Nat. Acad. Sci. USA 71, 4773-4777.
TRAUB, A., DISKIN, B., ROSENBERG, R., and KALMAR, N. S. (1976). Isolation and properties of the replicase of encephalomyocarditis virus. J. Viral.
18, 375-386.
