Behavior of virus-specific activities in tissue cultures infected with myxoviruses after chemical changes of the viral ribonucleic acid

VIROLOGY

22,

169-176 (1964)

Behavior

of Virus-Specific

Myxoviruses

after

Activities

Chemical

CHRISTOPH Max-Plan&Institut

in Tissue

Changes

SCHOLTISSEK

Cultures

of the Viral AND RUDOLF

fiir Virusforschung, Biologisch-Me&inische Tiibingen, Germany Accepted

October

infected

Ribonucleic

with Acid

ROTT Abteilung,

14, 1963

The base composition of NDV-RNA has been determined. The RNA which is synthesized in NDV-infected cells in the presence of actinomycin is identical with NDV RNA. No other RNA is synthesized in significant amounts under the conditions employed. Fowl plague virus (which belongs with the influenza A viruses) and NDV are inactivated by Bayer A 139, an ethylene iminoquinone, following first-order kinetics. If partially inactivated samples of fowl plague virus were added to tissue cultures, under certain conditions several virus components like hemagglutinin, neuraminidase, S-antigen, and viral RNA were still synthesized without a corresponding yield of infeatious virus particles. This effect was not observed with NDV. INTRODUCTION

Newcastle disease virus (NDV) and fowl plague virus are both myxoviruses and are characteristic oft this group in morphology, chemical composition, and biological properties. Each belongs to a different subgroup, however, and they differ from each other as follows: 1. Fowl plague virus belongs to the influenza A subgroup and contains an inner component (S-antigen) morphologically different from that of NDV, which belongs to the parainfluenza subgroup (for a review see Schafer, 1963). 2. ‘The multiplication of fowl plague virus is inhibited by actinomycin D whereas that of NDV is not (Barry et al., 1962; Rott and Scholtissek, 1964). 3. Genetic studies have demonstrated that influenza viruses show a high degree of genetic recombination whereas with NDV, recombination has not been demonstrated (Burnet, 1951; Granoff, 1959; Hirst, 1962). 4. With influenza viruses, multiplicity reactivation has been observed (Barry, 1961; Scholtissek et al., 1962a; Schafer and Rott,

1962), but not in the case of NDV (Scholtissek et al., 1962a; Barry, 1962; Rott and Schafer, 1962). 5. A von Magnus phenomenon has been found with influenza viruses, but not with NDV (for a survey see Waterson, 1962). In most of these earlier studies intact virus preparations were employed, but in the current work we tried to study the differences between these two virus subgroups by investigating virus preparations that had been changed specifically in the virus genome: Viruses were inactivated by treatment with an ethylene iminoquinone (Bayer A 139), which is known to react mainly with the phosphate groups of the nucleic acids and to break the phosphatesugar backbone, leaving the proteins intact (Scholtissek, 1957; Scholtissek et al., 1962a). These virus preparations were added to tissue culture and tested for their ability to induce the synthesis of virusspecific products. A short note on the production of virus-specific RNA with noninfectious fowl plague virus has already been published (Scholtissek and Rott, 1963).

169

170

SCHOLTISSEK

:: N/cH2 I H2c,R 1 lxx /N d R’c”2

H3c

FIG. 1. Bayer A 139. R = residue to make the compound water soluble. MATERIBI,

Irirus.

The

AND METHODS

“Restock” strain of fowl plague virus and the “Italien” strain of NDV were used throughout. The viruses were passaged in embryonated eggs, isolated by centrifugation, and taken up in tris-maleinate buffer pH 6.7. Tissue cultures and their infection. Chick embryo cells were prepared from ll-dayold embryos according to Kraus and Schafer (1963). The cell layers (about lo7 cells per culture in petri dishes of 9 cm diameter, 24 hours old) were washed with Earle’s saline containing 5 mM glucose and 2 mM glutamine without phosphate and were infected with the virus samples as described earlier (Scholtissek and Rott,, 1961a). In the case of fowl plague virus the input multiplicity was about 100 plaque-forming units (PFIJ) per cell in the noninactivated samples. After two washings with the abovementioned medium only about 10% of the virus remained on the cell layers (Breitenfeld and Schafer, 19571. About the same number of virus particles were employed, whether cultures were treated with act,ive or inactive virus. Such a high multiplicity was necessary in order to infect almost all cells synchronously (Breitenfeld and Schiifer, 1957). Assays. The infectivity was determined by the plaque test as described by Schafer et al. (1959). The hemagglutination test was performed in the usual manner with chick erythrocytes (for fowl plague virus at room temperature, for NDV at 4°C). The microcomplement fixation test (Fulton and Dumbell, 1949) as modified by Hennessen (1955) was employed. Q is the ratio of t.he amount of complement causing 50% hemolysis in the reaction mixture with antiserum to that with normal serum. For the fowl plague virus the antiserum was ob-

.4XD ROTT

tained from mice infected with influenza FM1 strain. This serum reacts only with the S antigen of fowl plague virus (Schafer, 1955). For NDV, convalescent ant,iscrum from hamsters was used (Schafer and Rott, 1959). The neuraminidase activity was determined according to Aminoff (1961) using X-acetylneuraminic acid lactose. The R!YA synthesis in NDV-infected cells, as measured by the incorporation of C’4-uridine, was determined as follows: Tissue cultures in petri dishes were infected with NDV and received at 2 hours post infection (p.i.) 5 pg actinomycin D per milliliter, which inhibits cellular RNA synthesis almost totally (Rott and Scholtissek, 1964). At 3 hours pi. 1 PC of C14-uridine (5.7 PC/ mg) was added to each culture, and 3 hours later the cultures were washed at 0”: once with NaCl-phosphate buffer pH 7.2 (0.7% NaCl, 0.02 M phosphate), 4 times with 6% trichloroacetic acid, twice with ethanol, and once with ether. The cell layers were dissolved in concentrated formic acid, transferred to aluminum disks, dried, counted; the values were corrected for self-absorption. Control cultures were either not infected or did not receive actinomycin. It has been shown by Kingsbury (1962)) that viral RNA synthesis occurs between 3 and 6 hours pi. Inactivation of the virus samples. The virus samples were inactivated in 0.1 N tris-maleinate buffer pH 6.7 with 1% of Bayer A 139 at 21°C. Bayer A 139 is an ethylene iminoquinone of the general formula shown in Fig. 1. The inactivation was stopped by dilution of an aliquot into an equal volume of ice cold tris-maleinate buffer pH 8.0 followed by dialysis against NaCl-phosphate buffer pH 7.2 at 4”. It has been shown that the Bayer A 139 reacts with nucleic acids mainly at a pH 5 7 (Scholtissek, 1957). Determination of the base composition of NDV-RNA. In order to exclude contamination by cellular RNA, viral RNA produced in the presence of actinomycin was investigated. This RNA was labeled with Ps2. To NDV-infected cultures, P32 (0.7 mC in 6 ml medium for each culture) and 5 pg actinomycin D per milliliter were added immediately after infection. At 8 hours p.i. the cell layers were washed once with cold

BEHAVIOR

OF MYXOVIRUSES

NaCl-phosphate buffer pH 7.2 and frozen. The virus was isolated by freezing and thawing three times, removing of the cell debris by centrifugation, and adsorption on and elution from chick erythrocytes (Rott et al., 1961). The RNA was extracted from the virus samples by hot phenol (about 50”) plus 0.5% dodecylsulfate in 0.01 N acetate buffer pH 5.1 (Wecker, 1959; Scherrer and Darnell, 1962). A corresponding procedure was used when RNA was extracted from infected cells. After precipitation of the extracted RNA with ethanol at -10” it was dissolved in water and reprecipitated by ice cold 0.4 N HC104. The RNA was digested by 0.3 N KOH at 37” over night. The monophosphates were separated on Dowex-1 columns in the formate form. The CMP was eluted by 0.1 N formic acid, AMP by 1 N formic acid, the UMP by 0.1 N formic acid plus 0.1 N ammonium formate, the GMP by 3 N formic acid. In this way UMP is free of contamination by orthophosphate (Hurlbert et al., 1954; Scholtissek, unpublished). The P32 was determined in each fraction. Chemicals and isotopes. The Bayer A 139 was a gift of the Bayerwerke, Leverkusen, Germany. Actinomycin D (C,) was a gift of Merck, Sharp, and Dohme, New York. P32 was obtained from the Kernreaktor Bau- und Betriebsgesellschaft mbH., Karlsruhe, Germany. The P32 was counted in a fluid counter with a counting efficiency of about 8% (Frieseke & Hoepfner GmbH, Erlangen-Bruck, Germany). C14-uridine was purchased from Schwarz BioResearch, Inc., Orangeburg, New York. The C14radioactivity was determined in a windowless flow counter model FH 516 (Frieseke & Hoepfner). RESULTS

Inactivation

Kinetics

Bayer A 139 reacts with nucleic acids at a pH of 5 7 without reacting with proteins (Scholtissek, 1957). The inactivation of fowl plague virus and NDV by this compound in general follows first-order kinetics as shown in Fig. 2. The break in the curves at about 1 hour may be ascribed to solution in the lipid shell of the viruses of the

WITH

ALTERED

RN.4

171

‘Or

“4

hrs. of inacfivalion FIG. 2. Inactivation of fowl plague virus and NDV

(0)

(X)

by Bayer A 139.

lipophilic Bayer A 139, which cannot be removed easily by dialysis or dilution. This phenomenon has been discussed more thoroughly by Scholtissek et al. (1962a). Determination NDV-RNA

anti

Characterization

of

The determination and characterization of fowl plague virus RNA within infected cells was described earlier (Scholtissek and Rott, 1961a). In the course of these experiments the synthesis of NDV-RNA in infected tissue cultures was measured by the incorporation of C14-uridine into the infected cells in the presence of actinomycin. It has been shown that actinomycin inhibits cellular RNA synthesis, but not that of several RNA-containing viruses (Reich et al., 1961; Kingsbury, 1962). Under the conditions employed (C14-uridine from 3 to 6 hours p.i.) the synthesis of RNA in infected actinomycin-inhibited cells is 10% that in noninfected and noninhibited controls. In noninfected and actinomycin-inhibited cultures the incorporation of C14-uridine into RNA was 0.3% of that in untreated uninfected controls. The base composition of the newly formed RNA of the cells infected with NDV and treated with actinomycin is identical with that of purified NDV and completely different from that of the noninfected host

172

SCHOLTISSEK

cells (Table 1). Under the conditions employed (P32 from 0 to 8 hours p.i.), RNAsynthesis in the infected cells is about 16% that in noninfected and untreated cells. One can see that no significant amounts of RNA with a base composition different from that of viral RNA is synthesized in NDV-infected cells in the presence of actinomycin. Therefore the viral RNA seems to be the only functional template available for viral protein synthesis. The base composition and the inactivation kinetics of NDV are in agreement with the concept of a singlestranded RNA. Only about 25% of the viral RNA in the infected cells is incorporated into the virus particles isolated 8 hours p.i. TABLE

1

BASE COMPOSITION OF NDV-RNA, RNA INFECTED .~ND ACTINOMYCIN-TREATED FIBROBLASTS, ‘\ND NORMAL CELLULAR

NDV-RNA

CMP AMP UMP GMP

27.0 26.1 22.0 24.9

a Values tions.

f 1.0 & 0.3 f 0.7 z!= 0.9

RNA of NDV-infected cells plus

27.2 27.0 21.7 24.1

f k zk f

0.9 0.7 0.4 0.6

OF NDVCHICK

RNA.a Cellular RNA

29.0 20.1 20.0 30.9

f f f zk

0.4 0.4 0.3 0.4

(TO) are averages of four determina-

AND

ROTT

Action of Partially Tissue Cultures

Inactivated

NDV

on

Samples of NDV were inactivated for different periods and added to tissue cultures and the various activities determined 8 hours p.i. (for RNA see Material and Methods). The results presented in Fig. 3 were obtained. During the process of inactivation of NDV with Bayer A 139, the virus simultaneously lost infectivity (PFU), ability to induce the synthesis of hemagglutmm, neuraminidase, or any virus-specific proteins detectable by the complement fixation test (CF test), and viral RNA. Action of Partially Inactivated Virus on Tissue Cultures

Fowl Plague

A completely different picture was obtained when corresponding experiments were performed with fowl plague virus (Fig. 4). In contrast to NDV, there was a pronounced delay in the loss of the capacity to synthesize the different virus components during the inactivation of fowl plague virus. In the fowl plague system, the first event after the diminution of the infectivity by gradual inactivation was a drop in the induction

of the

hemagglutinin

synthesis

followed by that of the neuraminidase synthesis. Still later there was a corresponding decrease in the complement fixation titer.

FIG. 3. Synthesis of viral components by inactivated NDV. On the left ordinate is the percentage of the yield of the viral components 8 hours after infection in the cell layers (except viral RNA) ; right ordinate & indicates total viral antigen; inactivation time is on the abscissa. a = plaque-forming units; X = hemagglutinin titer; 0 = neuraminidase; 0 = complement-fixing activity; l = viral RNA.

BEHAVIOR

OF MYXOVIRUSES

WITH

ALTERED

173

RNA 3 5 4,s 4

60

3

40 2 2G

.I ‘12

1

2

3

4

5

6

8 hrs

FIG. 4. Synthesis of viral components by inactivated fowl plague virus. The various viral activities were determined 7 hours after infection. For further explanations see Fig. 3. & shows the amount of S-antigen.

which measures the amount of the inner component, the S-antigen. In an earlier communication it has been shown that the viral RNA-synthesis goes down in parallel with the complement fixation titer (Scholtissek and Rott, 1963). DISCUSSION

It has been shown that during the stepwise chemical inactivation of NDV the drop in the infectivity is paralleled by the loss of the ability to induce the synthesis of any virus-specific product as detected by the available tests. In this respect NDV behaves differently from fowl plague virus. With fowl plague virus a marked delay in the loss of the ability to induce the synthesis of the different virus-specific components was found, although the yield of infectious (plaque forming) virus dropped very soon. The processesthat might occur during inactivation of fowl plague virus are understandable if the data of Fig. 4 are presented on a semilogarithmic scale as shown in Fig. 5. In order to get a synchronous infection of all cells it was necessary to use a relatively high multiplicity. The multihit curves in Fig. 5 are explained if one assumes an effective multiplicity of around 6. Since the

different curves extrapolate to approximately the same ordinate value, one hit per target for each virus component is enough to abolish the ability to induce the synthesis of the corresponding components. According to Fig. 5 the target size for hemagglutinin is about one-half of that for infectivity; that for the neuraminidase is about one-fourth and that for S-antigen about one-eighth or one-tenth of the target size for infectivity. It has been shown that prior to the synthesis of viral RNA “early protein(s) ” has to be synthesized (Scholtissek and Rott, 1961b, c). Since the curve for the viral RNA synthesis follows that of the CF titer (Scholtissek and Rott, 1963), the target size for the “early protein (s) ” also should be around one-tenth that for the infectivity. There are at least two possible explanations as to how the different cistrons function spatially along the genetic material. Scheme 1 shows a nonoverlapping arrangement of the different targets (except infectivity). Each cistron has to be inactivated independently in order to abolish the induction of the synthesis of a particular virus-specific component. The target size is identical with the size of the cistron.

SCHOLTISSEK

AND

ROTT

\ \

I

I

1

0

;

2

3

I

4

I

5

I

6

I

7

I

I

I

8

9

10 hours

of

inactivation

FIG. 5. Synthesis of viral components by inactivated fowl plague virus. The data of Fig. 4 and of a second experiment are plotted on a semilogarithmic scale.

scheme 7

infectivity , hemagglutinin

scheme 2

I

hemaggkffinin

nrumminidase

Scheme 2 demonstrates an overlapping arrangement of the targets, which might represent the sequence in the different functions of the subunits. This means that a hit

into ,the cistron that codes for the “early protein(s) ” will abolish also the ability to synthesize all the other virus-specific eomponents. A hit into the cistron that codes for

BEHAVIOR

OF MYXOVIRUSES

the hemagglutinin does not inhibit the synthesis of the “early protein(s) ,” S-antigen, and neuraminidase, but will abolish the ability to produce hemagglutinin and infectious virus. In this case there is no direct correspondence between the target size and the size of the cistron. We have evidence that the second scheme -at least in part-may be correct: (1) The sequence in the time course of appearance of the different viral components within the infected cell (Scholtissek et al., 1962b) corresponds to the sequence of the inhibition of the synthesis of these components after partial inactivation of the virus. (2) As long as completely functional “early protein (s) ’ is not synthesized, no other virusspecific component can be produced (Scholtissek and Rott, 1961b, c). Since scheme 2 seems to be correct, no reasonable statement on the size of the cistrons can be derived from the target size except that of t,he “early protein(s) .” As mentioned above, the target size for the “early protein(s)” is about one-tenth that for infectivity. Since the RNA per virus particle corresponds to a molecular weight of about two million (equal to about 6000 nucleotides; see Schafer, 1963), the cistron for the “early protein(s)” contains roughly 600 nucleotides corresponding to about 200 amino acids, if one assumes a triplet code. For this est,imate the assumption is made that all nucleotides of the viral RNA are necessary for infectivity; 200 amino acids is a reasonable number for a single protein. Under these circumstances it might be doubted that more than one ‘!early protein” could exist in the fowl plague system for starting the viral RNA synthesis. It may be mentioned that scheme 2 would fit better with a new hypothesis for explaining the von Magnus phenomenon, which may be the result of some kind of an autocatalytic shortening of the viral genome during undiluted passages of influenza viruses (Rott and Scholtissek, 1963). There is no explanation for the difference between NDV and fowl plague virus in response to Bayer A 139. Further investigations are needed to clarify this point. The present communication stresses again the fundamental differences between these two

WITH

ALTERED

RNA

viruses, which are morphologically in various respects.

175 similar

ACKNOWLEDGMENTS We wish tc thank Professor W. Schafer for his interest in these studies and Dr. W. Vielmetter for his very helpful discussions. We are grateful to Mrs. U. Schafer-Fuhr, Miss M. Seyffer, and Mr. 0. Harzrr for their technical assistance and to Dr. E. Eckert for helping us to translate the manuscript. These studies were supported by the “Deutsche Forschungsgemeinschaft.” REFERENCES D. (1961). Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hgdrolysates of sialomucoids. Biothem. J. 81,384-392. BARRY, R. D. (1961). The multiplication of influenza virus. Virology 14,398-405. BARRY, R. D. (1962). Failure.of Newcastle disease to undergo multiplicity reactivation. Nature 193, 96-97. BARRY, R. D., IVES, D. R., and CRUICKSHANK, J. G. (1962). Participation of deoxyribonucleic acid in the multiplication of influenza virus. Nature 194, 1139-I 140. BREITENFELD, P. M., and SCHAFER, W. (1957). The formation of fowl plague virus antigens in infected cells, as studied with fluorescent antibodies. Virology 4, 328-345. BURNET, F. M. (1951). A genetic approach to variation in influenza viruses. J. Gen. Microbial. 5, 46-66. FULTON, F., and DUMBELL, K. R. (1949). The serological comparison of strains of influenza virus. J. Gen. Microbial. 3, 97-111. GRANOFF, A. (1959). Studies on mixed infection with Newcastle disease virus. I. Isolation of Newcastle disease virus mutants and tests for genetic recombination between them. Virology 9, 636648. HENNESSEK, W. (1955). Uber eine Influenza-Komplementbindungsreaktion fiir die Praxis. 2. Hyg. Znjektionskrankh. 141, 557-564. HIRST, G. K. (1962). Genetic recombination with Newcastle disease virus, poliovirus, and influenza. Cold Spring Harbor Symp. Quant. Biol. 27, 303-309. HURLBERT, R. B., Scrrn%rrz, H., BRUMM, A. F., and POTTER, V. R. (1954). Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. J. Biol. Chem. 209,2%39. KINGSBURY, D. W. (1962). Use of actinomycin D to unmask RNil-synthesis induced by Newcastle disease virus. Biochem. Biophys. Res Commun. 9, 156-161. KRAUS, W., and SCHAFER, W. (1963). Apparatur BMINOFF,

176

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