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Relationship between viral RNA and viral protein synthesis
VIROLOGY
17, 110-117 (1962)
Relationship
between
Viral
RNA
EBERHARD The W&tar
Institute
of Anatomy
KLAUS Virus Laboratories
and
Viral
Protein
Synthesis’
WECKER
and Biology,
Philadelphia,
Pennsylvania
HUMMELER
of The Children’s Hospital of Philadelphia, and Medical of The University of Pennsylvania, Philadelphia
OTMAR Virus Laboratories
GOETZ2
of The Chillren’s Accepted
School
January
Hospital
of Philadelphia
24, 1962
Low concentrations of p-fluorophenylalanine (5-10 @g/ml) inhibit the maturation of Western equine encephalomyelitis and poliomyelitis viruses. Much higher concentrations are required (125 pg/ml) to inhibit the synthesis of the respective infectious RNA. By means of fluorescent antibodies against poliovirus, it was found that the formation of the viral antigens and the viral RNA are inhibited in a parallel manner by FPA. The implications of these findings suggesting a mutual dependency of viral protein and viral RNA synthesis are discussed. INTRODUCTION
Significant evidence has been presented to indicate that in HeLa cells infected with the Mahoney strain of poliovirus, the synthesis of viral RNA, viral proteins, and infectious virus particles are almost simultaneous events. Infectious viral RNA can be extracted from the cell only at the time of, or 30 minutes prior to, virus maturation (Darnell et al., 1961). In contrast, with Eastern and Western equine encephalomyelitis virus (EEE and WEE), encephalomyocarditis virus (EMC) , and mouse encephalomyelitis virus (ME), a considerable amount, if not all, of the in‘This work was supported in part by a contract with the United States Army Chemical Corps Biological Laboratories, Fort Detrick, Maryland, a grant from Samuel S. Fels Fund, and grant E-2405 of the National Institutes of Health, United States Public Health Service. ’ On leave of absence from Unirersit&tskinderklinik, Munich, Germany. 110
fectious viral RNA that can be extracted by means of “cold phenol” seems to be derived from an intracellular precursor which appears up to 2 hours prior to the maturation of the first new virus particles (Huppert and Sanders, 1958; Wecker, 1960; Hausen and Schgfer, 1961). Furthermore, with WEE it was shown that the amino acid analog p-fluorophenylalanine can inhibit the synthesis of infectious virus as well as that of infectious viral RNA. It was suggested that this is due to an intimate relationship between the synthesis of a nonenzyme protein and the viral RNA synthesis; e.g., if the former is inhibited by FPA, the latter will also be prevented (Wecker and Schonne, 1961). It was recently reported that when much smaller concentrations of FPA are used, HeLa cells infected with poliovirus still produce a considerable amount of infectious RNA, but no infectious virus (Levintow and Darnell, 1961). Moreover, it was
VIRAL
RNA AND VIRAL
concluded from tracer experiments that under these conditions no viral protein is being synthesized by the inhibited cells, which would signify the dissociation of the synthesis of viral RNA from that of viral protein. This view is in contrast to the notion that viral RNA synthesis may be dependent upon the concurrent synthesis of viral proteins. Such a possibility was suggest,ed by experiments with WEE and more recently with foot-and-mouth disease virus (FMD) (Wecker and Schonne, 1961; Brown et al., 1961). The highly specific and rather sensitive technique of demonstrating intracellular antigens of poliovirus by means of fluorescent antibodies (Hinuma and Hummeler, 1961) offered a possibility of investigating this problem directly. The polio protein occurs in two distinct antigenic manifestations. One, the so-called N antigen (for native) can be converted into H antigen (for heated)1 by exposure of the virus in vitro to 56” C for 30 minutes (Hummeler and Hamparian, 1958). N antigenicity is associated with infectious as well as noninfectious, but structurally complete, virus particles. H antigenicity is displayed by structurally incomplete virus particles that are noninfectious and lack RNA (Hummeler et al., 1962). It is the aim of the present study tal find out whether or not polio RNA can be synthesized in cells inhibited by FPA without the accompanying synthesis of viral proteins, and the further characterization of the antigen found. MATERIAL
AND
METHODS
l’irus. The Mahoney strain of type 1 poliomyelitis virus was used in all experiments. The virus was produced in tissue cultures of HeLa cells. Tissue culture cells. The Sa line of HeLa cells was cultivated on glass in a growth medium consisting of Eagle’s medium in Earle’s bala,nced salt solution (EE) containing 10% calf serum. Inhibition experiments. Monolayers of cells were washed with phosphate-buffered saline (PBS) and exposed for 1 hour at 37” to Mahoney virus at a multiplicity of about 250. After two washings with PBS, EE
PROTEIN
SYNTHESIS
111
lacking the amino acid phenylalanine (PAfree medium) was added containing various concentrations of nn-p-fluorophenylalanine (FPA) (Nutritional Biochemicals Corporation). The controls received PA free medium without the inhibitor. This was considered to be time zero. At various times the medium was removed and the cells were scraped off the glass by means of a rubber policeman. Following one washing in PBS, the cells were resuspended in EE and a sample was taken for virus assay. Then they were immediately frozen in a dry ice-acetone mixture and stored at -50” until extraction for RNA or virus assay. Extraction of infectious Rl\‘A. Each sample contained about log cells in 4 ml of EE. After the cells were thawed, the following mixture was added to each sample: 4 ml of 0.02% Versene; 2 ml of 0.2 M phosphate buffer pH 7.4, and 10 ml of 80% phenol. In this mixture the cells were shaken at 4” for 8 minutes and then centrifuged at 3000 rpm for 10 minutes. The aqueous phase was extracted a second time with 10 ml of 80% phenol as above. After the second extraction, the aqueous phase was mixed with two volumes of ethanol and kept in the icebath until the precipitation of nucleic acid was completed (ea. 2 hours). The centrifuged precipitate was washed once in a mixture of ethanol and 0.02 M phosphate buffer pH 7.2 in the ratio 2: 1 and redissolved in 3 ml of distilled water. To that, 7 ml of M MgSO, in 0.01 &f Tris buffer pH 7.5 were added. This was considered to be a 1:lO dilution of the RNA sample. Subsequently, fivefold dilutions were made in M MgS04 for infectivity assay according to Holland et al. (1960). Infectivity assay for RXA. Monolayers of primary monkey kidney cells were prepared in 60-mm petri dishes using EE + 10% calf serum as growth medium. The medium was removed and the layers were incubated for 15 minutes at 37” in a CO, incubator with 2 ml of physiological saline, containing 0.01 M phosphate buffer pH 7.2 and 0.02 M MgSO, (Mg saline). Then the layers were washed again with Mg saline. For inoculation, 0.2 ml of the respective
112
RECKER,
HUMMELER
RNA dilution in J!f MgS04 were dropped into the center of the dish. The plates were held at room temperature (ca. 20”) for 20 minutes and t,hen washed with EE. Thereafter, they were overlaid with 2.5 ml of 2% agar in water + 2.5 ml of ‘*alkaline overlay medium” according to Vogt et al. (1957). When the agar had solidified, the plates were incubated in a CO, incubator (ea. 4% COO) for 3 days. The cells were then stained by adding 3 ml of neutral red 1: 10 000 in PBS on top of the agar overlay. Plaques could be read after 2-3 hours of further incubation. Virus assay. The samples were thawed and homogenized for 3 minutes in a cooled sonic oscillation chamber. Tenfold dilutions were made in PBS. The monkey kidney monolayers were washed once with PBS before inoculation of 0.2 ml of virus dilution. After 1 hour of incubation, the cells were directly overlaid as described above. Also, staining and reading of the plaques were done as described above. Antisera. Antisera against the N and H antigens of poliomyelitis were prepared in guinea pigs as reported using purified virus preparation (Hummeler and Tumilowicz, 1960). Production of rabbit anti-guinea pig globulin and its conjugation with isothiocyanate have been described previously (Hinuma and Hummeler, 1961). Preparation and fixation of coverslip cultures. Cultures were washed with maintenance medium and infected at a multiplicity of 250. The maintenance medium consisted of 2% calf serum in EE. After incubation at 37” for 1 hour, the inoculum was removed and the cultures were washed twice with PBS. Following this, 2.0 ml of either FPA medium or PA-free medium were added to the tubes. The same amount of maintenance medium was added to some coverslips for control purposes. At varying time intervals after infection, as stated in Results, sets of infected coverslip cultures were harvested and washed twice in phosphate-buffered saline. The washed coverslips were transferred, while still moist, to acetone at -20” and fixed for 10 minutes according to Goetz and Hummeler (to be published).
AND
GOETZ
Antibody staining. Fixed coverslip cultures were stained by the indirect method. They were overlaid with either anti-N or anti-H guinea pig sera in a dilution of 1:8 and 1: 12, respectively, and incubated in a moist chamber at 37” for 30 minutes. They were then washed in phosphatebuffered saline, and again overlaid with the conjugated rabbit anti-guinea pig globulin in a dilution of 1:4. After furt,her incubation for 30 minutes, the coverslips were washed, dried, and mounted on glass slides, using a recently described semipermanent mounting medium (Rodriguez and Deinhardt, 1960). RESULTS
The Ejfect of Various Concentrations of FPA upon the Synthesis of Infectious RNA and Infectious Viruses In order to inhibit completely the synthesis of infectious WEE RNA, relatively high concentrations of FPA were found to be necessary (600 rg/ml) although concentrations of 10 pg/ml prevent the formation of infectious WEE virus. With poliovirus, concentrations of about 5 pg/ml were reported to inhibit the production of infectious viruses but not that of infectious RNA (Levintow and Darnell, 1961). It was, therefore, of interest to find out whether or not FPA at higher concentrations would inhibit the viral RNA synthesis of polio as has been shown for WEE. This objective was achieved by investigating the respective dose-response curves. Figure 1 shows the results with WEE. While the synthesis of infectious virus is strongly inhibited by FPA concentrations as low as 5 pg/ml, the inhibition of viral RNA synthesis requires much higher doses and follows an altogether different dose-response curve. As shown in Fig. 2, similar results were obtained with poliomyelitis virus. These findings lead to two conclusions: (1) The inhibition of the formation of infectious RNA viruses, e.g., poliomyelitis and WEE by FPA, cannot be explained simply on the basis that this amino acid analog prevents the synthesis of the respective
VIRAL
RNA AND VIRAL
viral RNA: virus synthesis is much more sensitive to FPA than is the respective viral RNA synthesis. (2) As was already known for WEE, the viral RNA synthesis of polio can also be inhibited by FPA. Both types of RNA display similar dose responses. From earlier experiments with WEE, it had been concluded that FPA inhibits the synthesis of the viral RNA by inhibiting the synthesis of a nonenzyme protein, the concurrent formation of this protein, possibly the viral protein, being essential for the simultaneous viral RNA synthesis (Wecker and Schonne, 1961). Brown et al. have recently arrived at similar conclusions from their pertinent studies with foot-andmouth disease virus (Brown et al., 1961). Owing to the availability of highly specific fluorescent antibodies against poliovirus, it now became possible to investigate this problem further: Can the synthesis of viral RNA and viral protein be dissociated by FPA? The results of these studies are described in the following section.
PROTEIN
113
SYNTHESIS
FPA pg /ml
FIG. 1. Dose response of WEE virus and RNA synthesis to FPA.
The Effect of Various Concentrations of FPA upon the Synthesis of J’irus-Specific Proteins Two types of experiments were performed. In the first, slides were harvested at different time intervals after infection while 5 pg of FPA was present in the medium. In the second experiment, infected cultures were incubated for 4 hours with varying amounts of FPA. The respective concentrations were 5, 25, and 125 rg/ml. The results of these experiments were as follows: In the first type of experiment, weak perinuclear staining could be seen at 2$$ hours after infection in the PA-free control cells, whereas the cells exposed to FPA did not show specific staining. At the 3-hour interval, the antigens were also evident in the FPA-treated cultures, although the staining was more pronounced in the PA-free controls. This difference was still obvious at 4 hours, but the intensity had increased in both types of preparations. At 5 hours, little difference in staining intensity between PA-free and FPA-treated cells could be seen (Fig. 4,A-D). In spite of the
IO 20 30 40 50 60 70 60 SO 100110 120 FP, g/m, FIG. 2. Dose response of poliovirus RNA synthesis to FPA.
and viral
difference in staining intensity in the earlier hours, it should be pointed out that in the presence of FPA, practically all cells showed synthesis of antigen. It has previously been shown that even with maximal multiplicit,y
114
WECKER,
HUMMELER
of infection obtainable, only about as% of the HeLa cells show synthesis of antigen (Hinuma and Hummeler, 1961 ‘I. A morphological difference in the localization of antigens was observed. The control cultures showed a normal development of antigen, e.g., a beginning of synthesis around the nucleus at 2’$“&-3 hours pi. and a subsequent distribution of the viral antigen throughout the cytoplasm. This distribution in the cytoplasm was delayed in the FPA-treated cells. Although the intensity of staining increased markedly with time, the antigens were confined perinuclearly for a longer time. Only at the end of the observation time, that is at the fifth hour, could antigen be detected throughout the cytoplasm. This was particularly true for the N antigen, whereas H antigen was still primarily confined to a perinuclear location. The observed delay of antigen production in the presence of 5 pg FPA/ml was reflected by a similar delay of viral RNA synthesis as demonstrated in Fig. 3. The second type of experiment was done with varying amounts of FPA and harvests 4 hours after infection. At the 5-pg concentration of FPA, results were obtained simi-
5.01
AND
GOETZ
FIG. 4. Fluorescent antibody studies of polioinfected HcLa cells using anti-N guinea pig scrum. $. Control in PA-free medium, 4 hours p.i. B. With 5 rg/ml of FPA, 4 hours pi. C. Control in PA-free medium, 5 hours p.i. D. With 5 rg/mI of FPA, 5 hours p.i. E. With 25 pg/ml of FPA, 5 hours p.i. F. With 125 fig/ml of FPA, 5 hours pi.
lar to those of the first experiment. The controls showed the expected amount of staining for N and H antigens. A considerable degree of cytopathic effect was apparent at 4 hours p.i. in the controls whereas none was observed in the presence of FPA. With 25 pg/ml of FPA the staining for both antigens showed a marked decrease in intensity, but very little if any was to be seen with 125 pg/ml (Fig. 4,DF). Within the limits of quantitation, it is thus apparent that the synthesis of viral proteins, e.g., the viral antigens and that of viral RNA, responds in a similar fashion to increasing amounts of the inhibitor. DISCUSSION
FIG. 3. Synthesis of poliovirus ence and in the absence of FPA.
RNA
in the pres-
The amino acid analog p-fluorophenylalanine (FPA) is a known inhibitor of t,he
VIRAL
RNA
AND
VIRAL
synthesis of infectious animal viruses (Ackermann and Maassab, 1955; Ackermann et al., 1954; Zimmermann and Schiifer, 1960; Wecker and Schonne, 1961; Levintow and Darnell, 1961; Brown et al., 1961; Scholtissek and Rott, 1961). Three main mechanisms of its inhibitory action suggest’ed by results with these and other systems have been discussed: 1. FPA is incorporated into protein, rendering it nonfunctional (Munier and Cohen, 1959; Zimmermann and Schafer, 1960; Scholtissek and Rott, 1961). 2. FPA inhibits the synthesis of proteins as studied in bacterial systems (Halvarson and Spiegelman, 1952) and with poliovirus (Levintow and Darnell, 1961). In the latter case low FPA concentrations (5 pg/ml) inhibit the formation of infectious virus but still permit the synthesis of infectious viral RNA. Levintow and Darnell also studied the protein constitution of mature poliovirus particles produced following reversion of the inhibition by FPA. They found that none of the proteins incorporated into such viruses had been synthesized during the inhibition, but only subsequent to the reversion. This led to the conclusion that presumably no viral proteins at all were formed in the presence of FPA. 3. FPA also inhibits the synthesis of viral RNA as demonstrated in the case of WEE, fowl plague, and foot-and-mouth disease viruses (Wecker apd Schonne, 1961; Scholtissek and Rott, 1961; Brown et al., 1961). In these experiments much higher concentrations of the inhibitor have been used (125 &ml or more). The present work was aimed at gaining additions1 evidence about the inhibitory mechanism of FPA on virus synthesis which could possibly lead to a more generalized understanding. The second possibility cited above offered the easiest access to further experistudies. Fluorescent antibodies mental against poliovirus demonstrated that at low FPA concentrations, which inhibit the formation of infectious virus but still permit the synthesis of a substantial amount of infectious RNA, there is also a consid-
PROTEIN
SYNTHESIS
115
erable amount of viral antigens produced. The cells displayed a bright fluorescence, using antisera to both the N and the H antigens. The demonstration of N antigenicity is of particular significance. N antigens are regularly associated with polioviruses which are infectious or at least structurally complete (Hummeler et al., 1962). Furthermore, N antigens undoubtedly represent a native viral protein. The presence of H antigens might be explained on the basis of conversion from N antigens during the fixation of the cells. This was observed before an improved technique became available whereby the still-moist cells are fixed in acetone at -20” (Goetz and Hummeler, 1961). On the other hand, crude poliovirus preparations also contain both types of antigens without undergoing any manipulation (Roizman et al., 1958; Hummeler and Hamparian, 1958). At any rate, it seems that FPA at low concentrations (about 5 pg/ml) does not inhibit the viral protein synthesis completely. Rather, the proteins formed, although antigenically competent, do not appear to be acceptable for subsequent incorporation into virus, perhaps because they contain some FPA. This interpretation of the experiments brings them into agreement with those mentioned in point (1’1. Greater difficulties are presented by attempting to integrate the results that FPA at higher concentrations (125 pg/ml or more) ran inhibit the synthesis of viral RNA as well. In the case of WEE, this inhibition could not be attributed to an effect of FPA on the synthesis of novel enzymes which might be necessary for the synthesis of the novel type RNA, i.e., the viral RNA. Rather, it was suggested that the set of normal cellular enzymes seems to be fullv capable for the synthesis of WEE RNA. The inhibitory action of FPA upon the viral RNA synthesis was considered to reflect the inhibition of the synthesis of a nonenzyme protein essential for the viral RNA synthesis. In that case it would be quite conceivable that the “nonenzyme” protein is actually the viral protein itself. This hypothesis lends itself to further experimental investigations. Apparently, if
116
WECKER.
HUMMELER
the synthesis of viral RNA and viral proteins were dependent upon each other, then FPA should inhibit both in a parallel manner. This could be studied in the polio system. The results were as follows: As already mentioned, at FPA concentrations of 5 pg/ml there was still about 40% of the normal amount of infectious RNA synthesized, and also the brilliance of immune fluorescence was almost that of the controls. At 25 pg/ml of FPA, the RNA represented only about, 5% of the control, and the fluorescence, while still specific, was considerably reduced. At 125 pg/ml of the inhibitor, the infectious RNA was but l%, and specific viral antigens could no longer be demonstrated. Thus, within the limits of such experiments, the synthesis of biologically active viral RNA and antigenically competent viral protein responded indeed in a parallel manner to FPA. While these results are so far in good agreement with the proposed hypothesis of a mutual dependence of viral RNA and viral protein synthesis, numerous other int.erpret,ations are still feasible. For instance, the possibility of an indirect inhibition via enzymes of the polio RNA synthesis has not yet been made as unlikely as in the case of WEE. The apparent similarity of the dose response curves to FPA between the two systems with respect to the formation of infectious virus and infectious RNA only suggests, but does not prove, a similarity of mechanisms. Moreover, direct evidence of WEE viral protein formation in the presence of low FPA concentrations is still lacking. Unless, however, experimental evidence to the contrary should be brought forth, the following hypothesis appears feasible and may be formulated: The synthesis of viral RNA and viral proteins of some animal viruses are interdependent. Even if the proteins formed are somewhat false, e.g., owing to the incorporation of an amino acid analog, the fact that proteins are formed at all also permit.s the synthesis-or at least the accumulation-of viral RNA. This would be the case at low FPA concentrations. If, at higher concentrations, FPA actually interferes with the synthesis of proteins, as indicated also by tracer experi-
AND
GOETZ
ments (Wecker and Schonne, 1961)) the viral RNA synthesis is inhibited as well. ACKNOWLEDGMENTS The authors gratefully appreciate the excellent technical assistance of Miss Jean La Libertk and Mrs. Gisela Lederhilger. REFERENCES W. W., and MAASSAB, H. F. (1955). Growth characteristics of influenza virus. Biochemical differentiation of stages of derelopment. II. J. Ezptl. Med. 102,393402. ACKERIIAES, IV. W., RABSON, A., and KURTZ, H. (1954). Growth characteristics of poliomyelitis virus in HeLa-cell cultures: lack of parallelism in cellular injury and virus increase. J. Exptl. Med. 100,437450. BROWN, F., PLANTEROSE, D. N., and STEWART, D. I,. (1961). Effect of p-fluorophenylalanine on the multiplication of foot and mouth disease virus. Nature 191, 414-415. DARNELL, J. E., JR., LEVINTOW, L., THORBN, M. M., and HOOPER, J. 1,. (1961). The time course of synthesis of poliovirus RNA. Virology 13, 271ACKERMASN,
279. H. O., and SPIEGELMAN, S. (1952). The inhibition of enzyme formation by amino acid analogues. J. Bncteriol. 6p, 207-221. HAUSEN, P., and SCHAFER, W. (1961). Produktion von Virus-Antigen bei der Vermehrung des Miiuse-Encephalomyelitis-(ME&Virus. 2. NnHALVARSOX,
turforsch. 16b,72-73. HINJMA, T., and HWIYELER,
K. (1961). Studies on the complement-fixing antigens of poliomyelitis. III. Intracellular development of antigen. J. Immunol. 87, 367-375. HOLLAND, J. J., HOYER, B. H., MCLAREN, L. C., and SYVERTOS, J. T. (1960). Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. J. Exptl. Med. 112, 821-839. HUM~~ELER, K., and HAYPARIAS, V. V. (1958). Studies on the complement-fixing ant,igens of poliomyelitis. I. Demonstration of type and group specific antigens in native and heated viral preparations. J. Immunol. 81,499505. HUWELER, K., and TUMILOWICZ, J. (1960). Studies on the complement-fixing antigens of poliomyelitis. II. Preparation of type specific anti-N and anti-H indicator sera. J. Immunol. 84, 630-634. HWI.MELER, K., ANDERSON, T. F., and BROWN, R. A. (1962). Identification of poliovirus particles of different antigenicity by specific agglutination as seen in the electron microscope. Virology 16,
84-90. HUPPERT,
fictive
J., and S.~NDERS, F. K. (1958). An inrihonucleip arid component from tumor
VIR$L
RNA AND VIRAL
cells infected with encephalomyocarditis virus. Nature 182,51b517. LEVINTOW, L., and DARNELL, J. E., JR. (1961). Effects of p-fluorophenylalanine on poliovirus replication. Federation Proc. 20, 1. MUNIER, R., and COHEN, G. N. (1959). Incorporation d’analogues structuraux d’amino acides dans les protkines bacthriennes on tours de leur synthese in vitro. Biochim. et Biophys. Acta 31,
378-391. RODRIGUEZ, J., and DEINHARDT, F. (1960). Preparation of a semipermanent mounting medium for 12, 316fluorescent antibody studies. Virology
317. ROIZMAN, B., H~PKEN, W., and MAYER, M. M. (1958). Immunochemical studies of polio virus. II. Kinetics of the formation of infectious and non-infectious Type 1 polio virus in three cell strains of human derivation. J. Zmmunology 80, 38ts395.
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SYNTHESIS
SCHOLTISSEK, C., and ROTT, R. (1961). Influence of p-fluorophenylalanine on the production of viral ribonucleic acid and in the utilizability of viral protein during multiplication of fowl plague virus. Nature 191, 1023-1024. VOGT, M., DULBECCO, R., and WENNER, H. 8. (1957). Mutants of poliomyelitis viruses with reduced efficiency of plating in acid medium and reduced neuropathogenicity. Virology 4, 141-155. WECKER, E. (1960). Eigenschaften einer infektiiisen Nucleinsilure-Fraktion aus Htihnerembryonen, die mit Encephalitis-Virus infiziert wurden. II. Mitt.: Biologische Eigenschaften. 2. Naturforsch.
15b, 71-78. WECKER, E., and SCHONKE, E. (1961). Inhibition of viral RNA synthesis by parafluorophenylalanine. Proc. Natl. Acad. Sci. U. S. 47,27&282. ZIMMERMANN, TH., and SCHKFER, W. (1960). Effect of p-fluorophenylalanine on fowl plague virus multiplicstion. Virology 11, 676698.
17, 110-117 (1962)
Relationship
between
Viral
RNA
EBERHARD The W&tar
Institute
of Anatomy
KLAUS Virus Laboratories
and
Viral
Protein
Synthesis’
WECKER
and Biology,
Philadelphia,
Pennsylvania
HUMMELER
of The Children’s Hospital of Philadelphia, and Medical of The University of Pennsylvania, Philadelphia
OTMAR Virus Laboratories
GOETZ2
of The Chillren’s Accepted
School
January
Hospital
of Philadelphia
24, 1962
Low concentrations of p-fluorophenylalanine (5-10 @g/ml) inhibit the maturation of Western equine encephalomyelitis and poliomyelitis viruses. Much higher concentrations are required (125 pg/ml) to inhibit the synthesis of the respective infectious RNA. By means of fluorescent antibodies against poliovirus, it was found that the formation of the viral antigens and the viral RNA are inhibited in a parallel manner by FPA. The implications of these findings suggesting a mutual dependency of viral protein and viral RNA synthesis are discussed. INTRODUCTION
Significant evidence has been presented to indicate that in HeLa cells infected with the Mahoney strain of poliovirus, the synthesis of viral RNA, viral proteins, and infectious virus particles are almost simultaneous events. Infectious viral RNA can be extracted from the cell only at the time of, or 30 minutes prior to, virus maturation (Darnell et al., 1961). In contrast, with Eastern and Western equine encephalomyelitis virus (EEE and WEE), encephalomyocarditis virus (EMC) , and mouse encephalomyelitis virus (ME), a considerable amount, if not all, of the in‘This work was supported in part by a contract with the United States Army Chemical Corps Biological Laboratories, Fort Detrick, Maryland, a grant from Samuel S. Fels Fund, and grant E-2405 of the National Institutes of Health, United States Public Health Service. ’ On leave of absence from Unirersit&tskinderklinik, Munich, Germany. 110
fectious viral RNA that can be extracted by means of “cold phenol” seems to be derived from an intracellular precursor which appears up to 2 hours prior to the maturation of the first new virus particles (Huppert and Sanders, 1958; Wecker, 1960; Hausen and Schgfer, 1961). Furthermore, with WEE it was shown that the amino acid analog p-fluorophenylalanine can inhibit the synthesis of infectious virus as well as that of infectious viral RNA. It was suggested that this is due to an intimate relationship between the synthesis of a nonenzyme protein and the viral RNA synthesis; e.g., if the former is inhibited by FPA, the latter will also be prevented (Wecker and Schonne, 1961). It was recently reported that when much smaller concentrations of FPA are used, HeLa cells infected with poliovirus still produce a considerable amount of infectious RNA, but no infectious virus (Levintow and Darnell, 1961). Moreover, it was
VIRAL
RNA AND VIRAL
concluded from tracer experiments that under these conditions no viral protein is being synthesized by the inhibited cells, which would signify the dissociation of the synthesis of viral RNA from that of viral protein. This view is in contrast to the notion that viral RNA synthesis may be dependent upon the concurrent synthesis of viral proteins. Such a possibility was suggest,ed by experiments with WEE and more recently with foot-and-mouth disease virus (FMD) (Wecker and Schonne, 1961; Brown et al., 1961). The highly specific and rather sensitive technique of demonstrating intracellular antigens of poliovirus by means of fluorescent antibodies (Hinuma and Hummeler, 1961) offered a possibility of investigating this problem directly. The polio protein occurs in two distinct antigenic manifestations. One, the so-called N antigen (for native) can be converted into H antigen (for heated)1 by exposure of the virus in vitro to 56” C for 30 minutes (Hummeler and Hamparian, 1958). N antigenicity is associated with infectious as well as noninfectious, but structurally complete, virus particles. H antigenicity is displayed by structurally incomplete virus particles that are noninfectious and lack RNA (Hummeler et al., 1962). It is the aim of the present study tal find out whether or not polio RNA can be synthesized in cells inhibited by FPA without the accompanying synthesis of viral proteins, and the further characterization of the antigen found. MATERIAL
AND
METHODS
l’irus. The Mahoney strain of type 1 poliomyelitis virus was used in all experiments. The virus was produced in tissue cultures of HeLa cells. Tissue culture cells. The Sa line of HeLa cells was cultivated on glass in a growth medium consisting of Eagle’s medium in Earle’s bala,nced salt solution (EE) containing 10% calf serum. Inhibition experiments. Monolayers of cells were washed with phosphate-buffered saline (PBS) and exposed for 1 hour at 37” to Mahoney virus at a multiplicity of about 250. After two washings with PBS, EE
PROTEIN
SYNTHESIS
111
lacking the amino acid phenylalanine (PAfree medium) was added containing various concentrations of nn-p-fluorophenylalanine (FPA) (Nutritional Biochemicals Corporation). The controls received PA free medium without the inhibitor. This was considered to be time zero. At various times the medium was removed and the cells were scraped off the glass by means of a rubber policeman. Following one washing in PBS, the cells were resuspended in EE and a sample was taken for virus assay. Then they were immediately frozen in a dry ice-acetone mixture and stored at -50” until extraction for RNA or virus assay. Extraction of infectious Rl\‘A. Each sample contained about log cells in 4 ml of EE. After the cells were thawed, the following mixture was added to each sample: 4 ml of 0.02% Versene; 2 ml of 0.2 M phosphate buffer pH 7.4, and 10 ml of 80% phenol. In this mixture the cells were shaken at 4” for 8 minutes and then centrifuged at 3000 rpm for 10 minutes. The aqueous phase was extracted a second time with 10 ml of 80% phenol as above. After the second extraction, the aqueous phase was mixed with two volumes of ethanol and kept in the icebath until the precipitation of nucleic acid was completed (ea. 2 hours). The centrifuged precipitate was washed once in a mixture of ethanol and 0.02 M phosphate buffer pH 7.2 in the ratio 2: 1 and redissolved in 3 ml of distilled water. To that, 7 ml of M MgSO, in 0.01 &f Tris buffer pH 7.5 were added. This was considered to be a 1:lO dilution of the RNA sample. Subsequently, fivefold dilutions were made in M MgS04 for infectivity assay according to Holland et al. (1960). Infectivity assay for RXA. Monolayers of primary monkey kidney cells were prepared in 60-mm petri dishes using EE + 10% calf serum as growth medium. The medium was removed and the layers were incubated for 15 minutes at 37” in a CO, incubator with 2 ml of physiological saline, containing 0.01 M phosphate buffer pH 7.2 and 0.02 M MgSO, (Mg saline). Then the layers were washed again with Mg saline. For inoculation, 0.2 ml of the respective
112
RECKER,
HUMMELER
RNA dilution in J!f MgS04 were dropped into the center of the dish. The plates were held at room temperature (ca. 20”) for 20 minutes and t,hen washed with EE. Thereafter, they were overlaid with 2.5 ml of 2% agar in water + 2.5 ml of ‘*alkaline overlay medium” according to Vogt et al. (1957). When the agar had solidified, the plates were incubated in a CO, incubator (ea. 4% COO) for 3 days. The cells were then stained by adding 3 ml of neutral red 1: 10 000 in PBS on top of the agar overlay. Plaques could be read after 2-3 hours of further incubation. Virus assay. The samples were thawed and homogenized for 3 minutes in a cooled sonic oscillation chamber. Tenfold dilutions were made in PBS. The monkey kidney monolayers were washed once with PBS before inoculation of 0.2 ml of virus dilution. After 1 hour of incubation, the cells were directly overlaid as described above. Also, staining and reading of the plaques were done as described above. Antisera. Antisera against the N and H antigens of poliomyelitis were prepared in guinea pigs as reported using purified virus preparation (Hummeler and Tumilowicz, 1960). Production of rabbit anti-guinea pig globulin and its conjugation with isothiocyanate have been described previously (Hinuma and Hummeler, 1961). Preparation and fixation of coverslip cultures. Cultures were washed with maintenance medium and infected at a multiplicity of 250. The maintenance medium consisted of 2% calf serum in EE. After incubation at 37” for 1 hour, the inoculum was removed and the cultures were washed twice with PBS. Following this, 2.0 ml of either FPA medium or PA-free medium were added to the tubes. The same amount of maintenance medium was added to some coverslips for control purposes. At varying time intervals after infection, as stated in Results, sets of infected coverslip cultures were harvested and washed twice in phosphate-buffered saline. The washed coverslips were transferred, while still moist, to acetone at -20” and fixed for 10 minutes according to Goetz and Hummeler (to be published).
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Antibody staining. Fixed coverslip cultures were stained by the indirect method. They were overlaid with either anti-N or anti-H guinea pig sera in a dilution of 1:8 and 1: 12, respectively, and incubated in a moist chamber at 37” for 30 minutes. They were then washed in phosphatebuffered saline, and again overlaid with the conjugated rabbit anti-guinea pig globulin in a dilution of 1:4. After furt,her incubation for 30 minutes, the coverslips were washed, dried, and mounted on glass slides, using a recently described semipermanent mounting medium (Rodriguez and Deinhardt, 1960). RESULTS
The Ejfect of Various Concentrations of FPA upon the Synthesis of Infectious RNA and Infectious Viruses In order to inhibit completely the synthesis of infectious WEE RNA, relatively high concentrations of FPA were found to be necessary (600 rg/ml) although concentrations of 10 pg/ml prevent the formation of infectious WEE virus. With poliovirus, concentrations of about 5 pg/ml were reported to inhibit the production of infectious viruses but not that of infectious RNA (Levintow and Darnell, 1961). It was, therefore, of interest to find out whether or not FPA at higher concentrations would inhibit the viral RNA synthesis of polio as has been shown for WEE. This objective was achieved by investigating the respective dose-response curves. Figure 1 shows the results with WEE. While the synthesis of infectious virus is strongly inhibited by FPA concentrations as low as 5 pg/ml, the inhibition of viral RNA synthesis requires much higher doses and follows an altogether different dose-response curve. As shown in Fig. 2, similar results were obtained with poliomyelitis virus. These findings lead to two conclusions: (1) The inhibition of the formation of infectious RNA viruses, e.g., poliomyelitis and WEE by FPA, cannot be explained simply on the basis that this amino acid analog prevents the synthesis of the respective
VIRAL
RNA AND VIRAL
viral RNA: virus synthesis is much more sensitive to FPA than is the respective viral RNA synthesis. (2) As was already known for WEE, the viral RNA synthesis of polio can also be inhibited by FPA. Both types of RNA display similar dose responses. From earlier experiments with WEE, it had been concluded that FPA inhibits the synthesis of the viral RNA by inhibiting the synthesis of a nonenzyme protein, the concurrent formation of this protein, possibly the viral protein, being essential for the simultaneous viral RNA synthesis (Wecker and Schonne, 1961). Brown et al. have recently arrived at similar conclusions from their pertinent studies with foot-andmouth disease virus (Brown et al., 1961). Owing to the availability of highly specific fluorescent antibodies against poliovirus, it now became possible to investigate this problem further: Can the synthesis of viral RNA and viral protein be dissociated by FPA? The results of these studies are described in the following section.
PROTEIN
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SYNTHESIS
FPA pg /ml
FIG. 1. Dose response of WEE virus and RNA synthesis to FPA.
The Effect of Various Concentrations of FPA upon the Synthesis of J’irus-Specific Proteins Two types of experiments were performed. In the first, slides were harvested at different time intervals after infection while 5 pg of FPA was present in the medium. In the second experiment, infected cultures were incubated for 4 hours with varying amounts of FPA. The respective concentrations were 5, 25, and 125 rg/ml. The results of these experiments were as follows: In the first type of experiment, weak perinuclear staining could be seen at 2$$ hours after infection in the PA-free control cells, whereas the cells exposed to FPA did not show specific staining. At the 3-hour interval, the antigens were also evident in the FPA-treated cultures, although the staining was more pronounced in the PA-free controls. This difference was still obvious at 4 hours, but the intensity had increased in both types of preparations. At 5 hours, little difference in staining intensity between PA-free and FPA-treated cells could be seen (Fig. 4,A-D). In spite of the
IO 20 30 40 50 60 70 60 SO 100110 120 FP, g/m, FIG. 2. Dose response of poliovirus RNA synthesis to FPA.
and viral
difference in staining intensity in the earlier hours, it should be pointed out that in the presence of FPA, practically all cells showed synthesis of antigen. It has previously been shown that even with maximal multiplicit,y
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of infection obtainable, only about as% of the HeLa cells show synthesis of antigen (Hinuma and Hummeler, 1961 ‘I. A morphological difference in the localization of antigens was observed. The control cultures showed a normal development of antigen, e.g., a beginning of synthesis around the nucleus at 2’$“&-3 hours pi. and a subsequent distribution of the viral antigen throughout the cytoplasm. This distribution in the cytoplasm was delayed in the FPA-treated cells. Although the intensity of staining increased markedly with time, the antigens were confined perinuclearly for a longer time. Only at the end of the observation time, that is at the fifth hour, could antigen be detected throughout the cytoplasm. This was particularly true for the N antigen, whereas H antigen was still primarily confined to a perinuclear location. The observed delay of antigen production in the presence of 5 pg FPA/ml was reflected by a similar delay of viral RNA synthesis as demonstrated in Fig. 3. The second type of experiment was done with varying amounts of FPA and harvests 4 hours after infection. At the 5-pg concentration of FPA, results were obtained simi-
5.01
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FIG. 4. Fluorescent antibody studies of polioinfected HcLa cells using anti-N guinea pig scrum. $. Control in PA-free medium, 4 hours p.i. B. With 5 rg/ml of FPA, 4 hours pi. C. Control in PA-free medium, 5 hours p.i. D. With 5 rg/mI of FPA, 5 hours p.i. E. With 25 pg/ml of FPA, 5 hours p.i. F. With 125 fig/ml of FPA, 5 hours pi.
lar to those of the first experiment. The controls showed the expected amount of staining for N and H antigens. A considerable degree of cytopathic effect was apparent at 4 hours p.i. in the controls whereas none was observed in the presence of FPA. With 25 pg/ml of FPA the staining for both antigens showed a marked decrease in intensity, but very little if any was to be seen with 125 pg/ml (Fig. 4,DF). Within the limits of quantitation, it is thus apparent that the synthesis of viral proteins, e.g., the viral antigens and that of viral RNA, responds in a similar fashion to increasing amounts of the inhibitor. DISCUSSION
FIG. 3. Synthesis of poliovirus ence and in the absence of FPA.
RNA
in the pres-
The amino acid analog p-fluorophenylalanine (FPA) is a known inhibitor of t,he
VIRAL
RNA
AND
VIRAL
synthesis of infectious animal viruses (Ackermann and Maassab, 1955; Ackermann et al., 1954; Zimmermann and Schiifer, 1960; Wecker and Schonne, 1961; Levintow and Darnell, 1961; Brown et al., 1961; Scholtissek and Rott, 1961). Three main mechanisms of its inhibitory action suggest’ed by results with these and other systems have been discussed: 1. FPA is incorporated into protein, rendering it nonfunctional (Munier and Cohen, 1959; Zimmermann and Schafer, 1960; Scholtissek and Rott, 1961). 2. FPA inhibits the synthesis of proteins as studied in bacterial systems (Halvarson and Spiegelman, 1952) and with poliovirus (Levintow and Darnell, 1961). In the latter case low FPA concentrations (5 pg/ml) inhibit the formation of infectious virus but still permit the synthesis of infectious viral RNA. Levintow and Darnell also studied the protein constitution of mature poliovirus particles produced following reversion of the inhibition by FPA. They found that none of the proteins incorporated into such viruses had been synthesized during the inhibition, but only subsequent to the reversion. This led to the conclusion that presumably no viral proteins at all were formed in the presence of FPA. 3. FPA also inhibits the synthesis of viral RNA as demonstrated in the case of WEE, fowl plague, and foot-and-mouth disease viruses (Wecker apd Schonne, 1961; Scholtissek and Rott, 1961; Brown et al., 1961). In these experiments much higher concentrations of the inhibitor have been used (125 &ml or more). The present work was aimed at gaining additions1 evidence about the inhibitory mechanism of FPA on virus synthesis which could possibly lead to a more generalized understanding. The second possibility cited above offered the easiest access to further experistudies. Fluorescent antibodies mental against poliovirus demonstrated that at low FPA concentrations, which inhibit the formation of infectious virus but still permit the synthesis of a substantial amount of infectious RNA, there is also a consid-
PROTEIN
SYNTHESIS
115
erable amount of viral antigens produced. The cells displayed a bright fluorescence, using antisera to both the N and the H antigens. The demonstration of N antigenicity is of particular significance. N antigens are regularly associated with polioviruses which are infectious or at least structurally complete (Hummeler et al., 1962). Furthermore, N antigens undoubtedly represent a native viral protein. The presence of H antigens might be explained on the basis of conversion from N antigens during the fixation of the cells. This was observed before an improved technique became available whereby the still-moist cells are fixed in acetone at -20” (Goetz and Hummeler, 1961). On the other hand, crude poliovirus preparations also contain both types of antigens without undergoing any manipulation (Roizman et al., 1958; Hummeler and Hamparian, 1958). At any rate, it seems that FPA at low concentrations (about 5 pg/ml) does not inhibit the viral protein synthesis completely. Rather, the proteins formed, although antigenically competent, do not appear to be acceptable for subsequent incorporation into virus, perhaps because they contain some FPA. This interpretation of the experiments brings them into agreement with those mentioned in point (1’1. Greater difficulties are presented by attempting to integrate the results that FPA at higher concentrations (125 pg/ml or more) ran inhibit the synthesis of viral RNA as well. In the case of WEE, this inhibition could not be attributed to an effect of FPA on the synthesis of novel enzymes which might be necessary for the synthesis of the novel type RNA, i.e., the viral RNA. Rather, it was suggested that the set of normal cellular enzymes seems to be fullv capable for the synthesis of WEE RNA. The inhibitory action of FPA upon the viral RNA synthesis was considered to reflect the inhibition of the synthesis of a nonenzyme protein essential for the viral RNA synthesis. In that case it would be quite conceivable that the “nonenzyme” protein is actually the viral protein itself. This hypothesis lends itself to further experimental investigations. Apparently, if
116
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the synthesis of viral RNA and viral proteins were dependent upon each other, then FPA should inhibit both in a parallel manner. This could be studied in the polio system. The results were as follows: As already mentioned, at FPA concentrations of 5 pg/ml there was still about 40% of the normal amount of infectious RNA synthesized, and also the brilliance of immune fluorescence was almost that of the controls. At 25 pg/ml of FPA, the RNA represented only about, 5% of the control, and the fluorescence, while still specific, was considerably reduced. At 125 pg/ml of the inhibitor, the infectious RNA was but l%, and specific viral antigens could no longer be demonstrated. Thus, within the limits of such experiments, the synthesis of biologically active viral RNA and antigenically competent viral protein responded indeed in a parallel manner to FPA. While these results are so far in good agreement with the proposed hypothesis of a mutual dependence of viral RNA and viral protein synthesis, numerous other int.erpret,ations are still feasible. For instance, the possibility of an indirect inhibition via enzymes of the polio RNA synthesis has not yet been made as unlikely as in the case of WEE. The apparent similarity of the dose response curves to FPA between the two systems with respect to the formation of infectious virus and infectious RNA only suggests, but does not prove, a similarity of mechanisms. Moreover, direct evidence of WEE viral protein formation in the presence of low FPA concentrations is still lacking. Unless, however, experimental evidence to the contrary should be brought forth, the following hypothesis appears feasible and may be formulated: The synthesis of viral RNA and viral proteins of some animal viruses are interdependent. Even if the proteins formed are somewhat false, e.g., owing to the incorporation of an amino acid analog, the fact that proteins are formed at all also permit.s the synthesis-or at least the accumulation-of viral RNA. This would be the case at low FPA concentrations. If, at higher concentrations, FPA actually interferes with the synthesis of proteins, as indicated also by tracer experi-
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ments (Wecker and Schonne, 1961)) the viral RNA synthesis is inhibited as well. ACKNOWLEDGMENTS The authors gratefully appreciate the excellent technical assistance of Miss Jean La Libertk and Mrs. Gisela Lederhilger. REFERENCES W. W., and MAASSAB, H. F. (1955). Growth characteristics of influenza virus. Biochemical differentiation of stages of derelopment. II. J. Ezptl. Med. 102,393402. ACKERIIAES, IV. W., RABSON, A., and KURTZ, H. (1954). Growth characteristics of poliomyelitis virus in HeLa-cell cultures: lack of parallelism in cellular injury and virus increase. J. Exptl. Med. 100,437450. BROWN, F., PLANTEROSE, D. N., and STEWART, D. I,. (1961). Effect of p-fluorophenylalanine on the multiplication of foot and mouth disease virus. Nature 191, 414-415. DARNELL, J. E., JR., LEVINTOW, L., THORBN, M. M., and HOOPER, J. 1,. (1961). The time course of synthesis of poliovirus RNA. Virology 13, 271ACKERMASN,
279. H. O., and SPIEGELMAN, S. (1952). The inhibition of enzyme formation by amino acid analogues. J. Bncteriol. 6p, 207-221. HAUSEN, P., and SCHAFER, W. (1961). Produktion von Virus-Antigen bei der Vermehrung des Miiuse-Encephalomyelitis-(ME&Virus. 2. NnHALVARSOX,
turforsch. 16b,72-73. HINJMA, T., and HWIYELER,
K. (1961). Studies on the complement-fixing antigens of poliomyelitis. III. Intracellular development of antigen. J. Immunol. 87, 367-375. HOLLAND, J. J., HOYER, B. H., MCLAREN, L. C., and SYVERTOS, J. T. (1960). Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. J. Exptl. Med. 112, 821-839. HUM~~ELER, K., and HAYPARIAS, V. V. (1958). Studies on the complement-fixing ant,igens of poliomyelitis. I. Demonstration of type and group specific antigens in native and heated viral preparations. J. Immunol. 81,499505. HUWELER, K., and TUMILOWICZ, J. (1960). Studies on the complement-fixing antigens of poliomyelitis. II. Preparation of type specific anti-N and anti-H indicator sera. J. Immunol. 84, 630-634. HWI.MELER, K., ANDERSON, T. F., and BROWN, R. A. (1962). Identification of poliovirus particles of different antigenicity by specific agglutination as seen in the electron microscope. Virology 16,
84-90. HUPPERT,
fictive
J., and S.~NDERS, F. K. (1958). An inrihonucleip arid component from tumor
VIR$L
RNA AND VIRAL
cells infected with encephalomyocarditis virus. Nature 182,51b517. LEVINTOW, L., and DARNELL, J. E., JR. (1961). Effects of p-fluorophenylalanine on poliovirus replication. Federation Proc. 20, 1. MUNIER, R., and COHEN, G. N. (1959). Incorporation d’analogues structuraux d’amino acides dans les protkines bacthriennes on tours de leur synthese in vitro. Biochim. et Biophys. Acta 31,
378-391. RODRIGUEZ, J., and DEINHARDT, F. (1960). Preparation of a semipermanent mounting medium for 12, 316fluorescent antibody studies. Virology
317. ROIZMAN, B., H~PKEN, W., and MAYER, M. M. (1958). Immunochemical studies of polio virus. II. Kinetics of the formation of infectious and non-infectious Type 1 polio virus in three cell strains of human derivation. J. Zmmunology 80, 38ts395.
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SCHOLTISSEK, C., and ROTT, R. (1961). Influence of p-fluorophenylalanine on the production of viral ribonucleic acid and in the utilizability of viral protein during multiplication of fowl plague virus. Nature 191, 1023-1024. VOGT, M., DULBECCO, R., and WENNER, H. 8. (1957). Mutants of poliomyelitis viruses with reduced efficiency of plating in acid medium and reduced neuropathogenicity. Virology 4, 141-155. WECKER, E. (1960). Eigenschaften einer infektiiisen Nucleinsilure-Fraktion aus Htihnerembryonen, die mit Encephalitis-Virus infiziert wurden. II. Mitt.: Biologische Eigenschaften. 2. Naturforsch.
15b, 71-78. WECKER, E., and SCHONKE, E. (1961). Inhibition of viral RNA synthesis by parafluorophenylalanine. Proc. Natl. Acad. Sci. U. S. 47,27&282. ZIMMERMANN, TH., and SCHKFER, W. (1960). Effect of p-fluorophenylalanine on fowl plague virus multiplicstion. Virology 11, 676698.