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A study on the infectivity of encephalomyocarditis virus double-stranded RNA with selectively inactivated strands
VIROLOGY
73, 528-531 (1976)
SHORT
COMMUNICATIONS
A Study on the Infectivity of Encephalomyocarditis Stranded RNA with Selectively Inactivated K. M. CHUMAKOV Department of Virology and Laboratory of Bioorganic Poliomyelitis and Viral Encephalitides, U.S.S.R.
AND
V.
I.
Virus DoubleStrands’
AGOL’
Chemistry, Moscow State University, and Institute Academy of Medical Sciences, Moscow, U.S.S.R.
Accepted April
of
29,1976
“Heteroduplex” molecules of encephalomyocarditis virus double-stranded RNA with uv-inactivated either “plus” or “minus” strands have been prepared. Duplex molecules with inactivated plus strands are not infectious, whereas the infectivity of duplexes with intact plus strands is essentially the same irrespective of minus strands being intact or inactivated.
unless it is more detailed. For example, if transcription is accomplished by a conservative mechanism, then the “minus” strand of the heteroduplex molecule should serve as the carrier of genetic information. A semiconservative transcription mechanism, on the other hand, may result in the expression of both plus and minus strands inasmuch as either the displaced parental plus strand or the plus strand newly synthesized on a minus template may induce the viral RNA replication process. Best et al. (5) have constructed heteroduplex RNA molecules whose plus and minus strands were derived from genetically different strains of poliovirus. It was the plus RNA strand that had been expressed in the virus progeny harvested after infection with such heteroduplexes. On the other hand, Bechet, while working with EMC virus, reported an increase in the infectivity of a preparation of minus RNA strands as a result of their annealing with inactivated (fragmented) plus strands (6, 7). This result may be interpreted as indicating the possibility of expression of the gentic information contained in the minus strand of a duplex RNA molecule, in an apparent contradiction with the conclusions of Best et al. (5). This discrepancy might have been due to differences in
The infectivity of encephalomyocarditis (EMC) virus double-stranded replicative form (RF) RNA molecules was originally described by Montagnier and Sanders as early as 1963 (2). Since then, this observation has been amply confirmed in many picornavirus systems but the mechanisms underlying the infectivity of duplex RNA molecules is not yet understood. Two groups of hypothesis are usually discussed in this regard (3,4): (i) translation of the parental “plus” strand somehow released from the duplex is the primary virus-specific synthetic process in the RF RNA-infected cells; (ii) transcription of the input RF RNA by preexisting cellular polymerases is the primary process. The former hypothesis predicts that the genetic content of the plus, translatable, RNA strand of the duplex should be transferred to the virus progeny obtained upon infection with heteroduplex molecules; no specific prediction can be made for such experiments on the basis of the latter hypothesis ’ Reported in part at the Soviet-American Symposium “Structure and Functions of Nucleic Acids,” Kiev, September 30-October 4, 1975, and at a Conference of the Institute of Poliomyelitis and Viral Encephalitides, Moscow, October 21-23, 1975 (1). 2 Author to whom requests for reprints should be addesssed. 528 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
529
COMMUNICATIONS
either the two viruses studies or the techniques employed. We undertook a study of the expression of genetic information of plus and minus strands of EMC virus RF RNA. For this purpose, we have constructed heteroduplex RNA molecules with either plus or minus strand selectively inactivated with uv, and have studied their infectivity. EMC virus RF RNA was isolated from virus-infected Krebs-II cells by hot phenolsodium dodecyl sulfate (SDS) method (8). A fraction of RNA which was soluble in 2 M LiCl was subjected to gel filtration on a Sepharose 2B column equilibrated with 2.5 x lop2 M EDTA, pH 7.0. RF RNA that was eluted in the void volume was shown to be homogenous as judged by sucrose density gradient centrifugation or agarose gel electrophoresis. Denaturation3 of the bulk of RF RNA was achieved by incubation in 90% dimethyl sulfoxide (DMSO), 1O-3 M HEPES, 2 x lop4 M EDTA, pH 7.0, at 30 for 1 min. Then the mixture was diluted fivefold with the solution containing 0.1 M NaCI, lop2 M Tris-HCl, 10e3M EDTA, pH 7.5, 0.01% SDS, and single-stranded RNA molecules were separated from the remaining double-stranded molecules by chromatography on cellulose CF 11 (10). Some 80% of the RNA material was eluted as single-stranded molecules; they were pooled and precipitated with ethanol in the presence of carrier ribosomal RNA from Krebs II cells. The procedure of separation of plus and minus RNA strands was based on an observation made by L. I. Romanova in this laboratory that EMC virus RNA could be specifically adsorbed on poly(U)-cellulose, most probably due to the presence of poly(A) tracts in these RNA molecules 3 An earlier work from this laboratory had described cross-linking between the complementary strands of EMC virus RF RNA which prevented effective separation of strands in a portion of RF RNA molecules under denaturing conditions (8). After discovery of a long poly(C) sequence(s) in EMC virion RNA (9), we have found that the complementary strands of the majority of EMC virus RF RNA molecules, although perhaps of not all, could be separated under conditions when poly(G) poly(C) duplexes were melted. A more detailed account of this study will be published elsewhere.
(II 1. Poly(U)-cellulose was prepared as described (12,13). The results of chromatography on poly(U)-cellulose of a denatured preparation of EMC virus RF RNA are shown on Fig. 1. It is seen that approximately half of the labeled material was not retained on the column under high salt conditions favoring the formation of poly(U) *poly(A) complexes. The other half of the material bound to the column under high salt conditions and eluted under low salt conditions. The first peak should correspond to minus RNA molecules and fragments of plus RNA molecules lacking poly(A) tracts. The second peak should contain intact plus strands and poly(A)-containing fragments thereof. The infectivity of these RNA fractions were estimated by a plaque assay in agar-suspended Krebs II cells pretreated /c- “HIGH SALT’+ 4oc
“LOW SALT” 4 2OT -3 P 0
( H I I
I I
5
m FRACTIONS
15
FIG. 1. Separation of complementary strands of EMC virus RF RNA by poly(U)-cellulose chromatography. A melted preparation of RF RNA (see text) was dissolved in a high salt buffer solution containing 0.3 M NaCl, 10e2 M Tris-HCl, lo-” M EDTA, pH 7.5, 0.01% SDS, and was loaded on a poly(U)-cellulose column (0.3 x 5 cm) at 4”. The column was washed off with the same buffer and fractions of 1.0 ml were collected. Separate experiments had shown that under these conditions about 98% of EMC virion RNA and less than 1% of Krebs II cells ribosomal RNA were absorbed on poly(U)-cellulose. The elution of adsorbed RNA was carried out with the same buffer but containing no NaCl (low salt buffer) at 20”. Aliquots of 25 ~1 were used for the determinations of infectivity (O- -0) and acid-insoluble radioactivity (0-O).
530
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COMMUNICATIONS
with DEAE-dextran at a concentration of 2.5 mg/ml. The details of this procedure will be described elsewhere. This method is highly sensitive and yields up to 3 x lo8 and 2 x 10” PFU/pg of EMC virus virion RNA and RF RNA, respectively. As expected, almost all infectivity (98%) was associated with the second peak (Fig. 1). To separate intact plus and minus strands from their fragments, the crude preparations of these strands were fractionated in a sucrose density gradient (Fig. 2). The peak fractions were pooled and subjected to another cycle of poly(U)cellulose chromatography. The purity of the preparation of minus strands obtained was checked in the following way. The ability of labeled material from this preparation to hybridize with excess unlabeled virion RNA was investigated. The virion RNA was isolated from purified (14) EMC virus by phenol-SDS deproteinizations and was fractionated by sucrose density gra-
dient centrifugation. The conditions of annealing were as described in the legend to Table 1. The extent of annealing was evaluated by the degree of resistance of labeled RNA to digestion by pancreatic RNAse (5 pg/ml, 30 min, 30”) after adjusting the hybridization mixture to 0.3 M NaCl and 10% DMSO. The results of this experiment (not shown) indicated that not less than 95% of the label in the preparation of minus strands hybridized with virion RNA. The specific infectivities of the final preparations of minus and plus strands were about 5.0 x lo4 and 5.5 x 10’ PFU/pg, respectively. Thus, the preparation of minus strands appeared to contain not more than 0.1% of intact infectious plus RNA molecules. TABLE INFECTIVITY
Expt. no.
2
FRACTIONS
FIG. 2. Isolation
of intact molecules from poly(A)-lacking and poly(A)-containing RNA species in melted preparations of EMC virus RF RNA. RNA material from peaks I (a) and II (b) of Fig. 1 were separately dissolved in 0.1 ml of a solution containing 0.1 ml NaCl, lo-’ M Tris-HCl, 1OV M EDTA, pH 7.5, 0.01% SDS, and were layered onto a 520% sucrose concentration gradient. The centrifugation was carried out for 80 min at 60,000 rpm at 20” in a Beckman SW 65 rotor. Two-drop fractions were collected after the puncture of the bottom of the tubes and acid-insoluble radioactivity in 5-~1 aliquots of each fraction was determined. The peak (shadowed) fractions were pooled and used for the next purification step.
RF RNA
RNA species in the annealing mixture Plus strands
1
1
OF EMC VIRUS HETERODUPLEXES"
Intact Intact Intact uv-Inactivated uv-Inactivated None* Intact Intact Intact uv-Inactivated uv-Inactivated Noneb
Minus strands Intact uv-Inactivated None Intact uv-Inactivated Intact Intact uv-Inactivated None Intact uv-Inactivated Intact
Infec(“;1”FtZ, ml) 310 295 13 <3 <3 <3 420 514 <3 <3 <3 <3
u The intact or uv-inactivated minus strands were transferred to a hybridization buffer (5 x lo-* M NaCl, 2 x lo-* M Tris-HCl, IO-” M EDTA, pH 7.5) by means of gel filtration on Sephadex G-25. The solution containing about 0.01 wg of minus RNA per ml was supplemented with a loo-fold excess of either intact or uv-inactivated EMC virion RNA. Then DMSO was added to a final concentration of 65% (v/ v), the mixture was heated for 1 min at loo”, and then incubated at 37.5” for 3 hr. After precipitation with 4 volumes of ethanol, RNA samples were dissolved in 0.1 M NaCl, lo-* M Tris-HCl, lo-” M EDTA, pH 7.3, and treated with pancreatic RNAse (10e3 pg/ml, 20”, 30 min). b These control samples contained a loo-fold excess of 18 S ribosomal RNA from Krebs II cells, instead of plus RNA, for estimation of self-annealing in the preparation of minus strands.
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531
COMMUNICATIONS
The aliquots of RNA preparations to be inactivated were irradiated by two uv lamps (Tungsram Germicid “F”, Hungary, 15 W) at a distance of 10 cm for 10 min. Under these conditions the infectivity dropped approximately 104-fold in 2.5 and 10 min for virion RNA and RF RNA, respectively. Then the preparations of intact or uv-treated minus RNA strands were separately annealed with a loo-fold excess of intact or uv-treated virion RNA. As stated above, under annealing conditions used, not less than 95% of the minus RNA strands was transformed into a duplex form. It was possible that some of these duplexes were not perfect. To inactivate infectivity due to these incomplete duplexes as well as to the single-stranded virion RNA present in large amounts, the annealed preparations were treated with pancreatic RNAse under conditions where 80% of the material of labeled virion RNA was converted to an acid-soluble form with the complete loss of infectivity, while the infectivity of RF RNA remained unchanged. The conditions of annealing and RNAse treatment are described in the legend to Table 1. The results of the assay of infectivity of different artificially prepared duplex RNA complexes are presented in Table 1. It is clearly seen that the duplex molecules with inactivated plus strands are not infectious, whereas the infectivity of duplexes with an intact plus strand is essentially the same irrespective of the minus strand being intact or inactivated. In agreement with Best et al. (5), it can be concluded that it is the plus strand that is largely, if not exclusively, expressed in the progeny upon infection with picornavirus RF RNA. The infectivity of samples obtained after annealing of a preparation of minus
strands with fragmented plus strands and not treated with RNAse, was also determined. In disagreement with Bechet (6, 71, we did not find any increase in infectivity (due to the admixture of about 0.1% of plus strands in the preparation of minus strands) after such annealing. The infectivity was essentially the same when fragments of either intact or uv-inactivated plus strands were used. Thus, if complexes formed by intact minus strands and fragmented plus strands are infectious, their specific infectivity should be very low.
REFERENCES 1. CHUMAKOV, K. M., In “Problems of Medical Virology,” pp. 36-37. Institute of Poliomyelitis and Viral Encephalitides, Moscow, 1975. 2. MONTAGNIER, L., and SANDERS, F. K., Nature (London) 199, 664-667 (1963). 3. AGOL, V. I., Usp. Sovrem. Biol. 72, 315-338 (1971). 4. BISHOP, J. M., and LEVINTOW, L., Progress in Med. Viral. 13, 1-82 (1972). 5. BEST, M., EVANS, B., and BISHOP, J. M., Virology 47, 592-603 (1972). 6. BECHET, J.-M., C. R. Acad. Sci. (Paris) 274, 1761-1764 (1972). 7. BECHET, J.-M., Virology 48, 855-857 (1972). 8. AGOL, V. I., ROMANOVA, L. I., CUMAKOV, I. M., DUNAEVSKAYA, L. D., and BOGDANOV, A. A., J. Mol. Biol. 72, 77-89 (1972). 9. PORTER, A., CAREY, N., and FELLNER, P., Nature (London) 248, 675-678 (1974). 10. FRANKLIN, R. M., Proc. Nat. Acad. Sci. USA 55, 1504-1511 (1966). II. GILLESPIE, D., TAKEMOTO, K., ROBERT, M., and GALLO, R. C., Science 179,1328-1330 (1973). 12. SHELDON,R., JURALE, C., and KATES, S., Proc. Nat. Acad. Sci. USA 69, 417-421 (1972). 13. FEDOROFF, N. V., and ZINDER, N. D., Proc. Nat. Acad. Sci. USA 68, 1838-1843 (1971). 14. KERR, I. M., and MARTIN, E. M., J. Viral. 9, 559-561 (1972).
73, 528-531 (1976)
SHORT
COMMUNICATIONS
A Study on the Infectivity of Encephalomyocarditis Stranded RNA with Selectively Inactivated K. M. CHUMAKOV Department of Virology and Laboratory of Bioorganic Poliomyelitis and Viral Encephalitides, U.S.S.R.
AND
V.
I.
Virus DoubleStrands’
AGOL’
Chemistry, Moscow State University, and Institute Academy of Medical Sciences, Moscow, U.S.S.R.
Accepted April
of
29,1976
“Heteroduplex” molecules of encephalomyocarditis virus double-stranded RNA with uv-inactivated either “plus” or “minus” strands have been prepared. Duplex molecules with inactivated plus strands are not infectious, whereas the infectivity of duplexes with intact plus strands is essentially the same irrespective of minus strands being intact or inactivated.
unless it is more detailed. For example, if transcription is accomplished by a conservative mechanism, then the “minus” strand of the heteroduplex molecule should serve as the carrier of genetic information. A semiconservative transcription mechanism, on the other hand, may result in the expression of both plus and minus strands inasmuch as either the displaced parental plus strand or the plus strand newly synthesized on a minus template may induce the viral RNA replication process. Best et al. (5) have constructed heteroduplex RNA molecules whose plus and minus strands were derived from genetically different strains of poliovirus. It was the plus RNA strand that had been expressed in the virus progeny harvested after infection with such heteroduplexes. On the other hand, Bechet, while working with EMC virus, reported an increase in the infectivity of a preparation of minus RNA strands as a result of their annealing with inactivated (fragmented) plus strands (6, 7). This result may be interpreted as indicating the possibility of expression of the gentic information contained in the minus strand of a duplex RNA molecule, in an apparent contradiction with the conclusions of Best et al. (5). This discrepancy might have been due to differences in
The infectivity of encephalomyocarditis (EMC) virus double-stranded replicative form (RF) RNA molecules was originally described by Montagnier and Sanders as early as 1963 (2). Since then, this observation has been amply confirmed in many picornavirus systems but the mechanisms underlying the infectivity of duplex RNA molecules is not yet understood. Two groups of hypothesis are usually discussed in this regard (3,4): (i) translation of the parental “plus” strand somehow released from the duplex is the primary virus-specific synthetic process in the RF RNA-infected cells; (ii) transcription of the input RF RNA by preexisting cellular polymerases is the primary process. The former hypothesis predicts that the genetic content of the plus, translatable, RNA strand of the duplex should be transferred to the virus progeny obtained upon infection with heteroduplex molecules; no specific prediction can be made for such experiments on the basis of the latter hypothesis ’ Reported in part at the Soviet-American Symposium “Structure and Functions of Nucleic Acids,” Kiev, September 30-October 4, 1975, and at a Conference of the Institute of Poliomyelitis and Viral Encephalitides, Moscow, October 21-23, 1975 (1). 2 Author to whom requests for reprints should be addesssed. 528 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
529
COMMUNICATIONS
either the two viruses studies or the techniques employed. We undertook a study of the expression of genetic information of plus and minus strands of EMC virus RF RNA. For this purpose, we have constructed heteroduplex RNA molecules with either plus or minus strand selectively inactivated with uv, and have studied their infectivity. EMC virus RF RNA was isolated from virus-infected Krebs-II cells by hot phenolsodium dodecyl sulfate (SDS) method (8). A fraction of RNA which was soluble in 2 M LiCl was subjected to gel filtration on a Sepharose 2B column equilibrated with 2.5 x lop2 M EDTA, pH 7.0. RF RNA that was eluted in the void volume was shown to be homogenous as judged by sucrose density gradient centrifugation or agarose gel electrophoresis. Denaturation3 of the bulk of RF RNA was achieved by incubation in 90% dimethyl sulfoxide (DMSO), 1O-3 M HEPES, 2 x lop4 M EDTA, pH 7.0, at 30 for 1 min. Then the mixture was diluted fivefold with the solution containing 0.1 M NaCI, lop2 M Tris-HCl, 10e3M EDTA, pH 7.5, 0.01% SDS, and single-stranded RNA molecules were separated from the remaining double-stranded molecules by chromatography on cellulose CF 11 (10). Some 80% of the RNA material was eluted as single-stranded molecules; they were pooled and precipitated with ethanol in the presence of carrier ribosomal RNA from Krebs II cells. The procedure of separation of plus and minus RNA strands was based on an observation made by L. I. Romanova in this laboratory that EMC virus RNA could be specifically adsorbed on poly(U)-cellulose, most probably due to the presence of poly(A) tracts in these RNA molecules 3 An earlier work from this laboratory had described cross-linking between the complementary strands of EMC virus RF RNA which prevented effective separation of strands in a portion of RF RNA molecules under denaturing conditions (8). After discovery of a long poly(C) sequence(s) in EMC virion RNA (9), we have found that the complementary strands of the majority of EMC virus RF RNA molecules, although perhaps of not all, could be separated under conditions when poly(G) poly(C) duplexes were melted. A more detailed account of this study will be published elsewhere.
(II 1. Poly(U)-cellulose was prepared as described (12,13). The results of chromatography on poly(U)-cellulose of a denatured preparation of EMC virus RF RNA are shown on Fig. 1. It is seen that approximately half of the labeled material was not retained on the column under high salt conditions favoring the formation of poly(U) *poly(A) complexes. The other half of the material bound to the column under high salt conditions and eluted under low salt conditions. The first peak should correspond to minus RNA molecules and fragments of plus RNA molecules lacking poly(A) tracts. The second peak should contain intact plus strands and poly(A)-containing fragments thereof. The infectivity of these RNA fractions were estimated by a plaque assay in agar-suspended Krebs II cells pretreated /c- “HIGH SALT’+ 4oc
“LOW SALT” 4 2OT -3 P 0
( H I I
I I
5
m FRACTIONS
15
FIG. 1. Separation of complementary strands of EMC virus RF RNA by poly(U)-cellulose chromatography. A melted preparation of RF RNA (see text) was dissolved in a high salt buffer solution containing 0.3 M NaCl, 10e2 M Tris-HCl, lo-” M EDTA, pH 7.5, 0.01% SDS, and was loaded on a poly(U)-cellulose column (0.3 x 5 cm) at 4”. The column was washed off with the same buffer and fractions of 1.0 ml were collected. Separate experiments had shown that under these conditions about 98% of EMC virion RNA and less than 1% of Krebs II cells ribosomal RNA were absorbed on poly(U)-cellulose. The elution of adsorbed RNA was carried out with the same buffer but containing no NaCl (low salt buffer) at 20”. Aliquots of 25 ~1 were used for the determinations of infectivity (O- -0) and acid-insoluble radioactivity (0-O).
530
SHORT
COMMUNICATIONS
with DEAE-dextran at a concentration of 2.5 mg/ml. The details of this procedure will be described elsewhere. This method is highly sensitive and yields up to 3 x lo8 and 2 x 10” PFU/pg of EMC virus virion RNA and RF RNA, respectively. As expected, almost all infectivity (98%) was associated with the second peak (Fig. 1). To separate intact plus and minus strands from their fragments, the crude preparations of these strands were fractionated in a sucrose density gradient (Fig. 2). The peak fractions were pooled and subjected to another cycle of poly(U)cellulose chromatography. The purity of the preparation of minus strands obtained was checked in the following way. The ability of labeled material from this preparation to hybridize with excess unlabeled virion RNA was investigated. The virion RNA was isolated from purified (14) EMC virus by phenol-SDS deproteinizations and was fractionated by sucrose density gra-
dient centrifugation. The conditions of annealing were as described in the legend to Table 1. The extent of annealing was evaluated by the degree of resistance of labeled RNA to digestion by pancreatic RNAse (5 pg/ml, 30 min, 30”) after adjusting the hybridization mixture to 0.3 M NaCl and 10% DMSO. The results of this experiment (not shown) indicated that not less than 95% of the label in the preparation of minus strands hybridized with virion RNA. The specific infectivities of the final preparations of minus and plus strands were about 5.0 x lo4 and 5.5 x 10’ PFU/pg, respectively. Thus, the preparation of minus strands appeared to contain not more than 0.1% of intact infectious plus RNA molecules. TABLE INFECTIVITY
Expt. no.
2
FRACTIONS
FIG. 2. Isolation
of intact molecules from poly(A)-lacking and poly(A)-containing RNA species in melted preparations of EMC virus RF RNA. RNA material from peaks I (a) and II (b) of Fig. 1 were separately dissolved in 0.1 ml of a solution containing 0.1 ml NaCl, lo-’ M Tris-HCl, 1OV M EDTA, pH 7.5, 0.01% SDS, and were layered onto a 520% sucrose concentration gradient. The centrifugation was carried out for 80 min at 60,000 rpm at 20” in a Beckman SW 65 rotor. Two-drop fractions were collected after the puncture of the bottom of the tubes and acid-insoluble radioactivity in 5-~1 aliquots of each fraction was determined. The peak (shadowed) fractions were pooled and used for the next purification step.
RF RNA
RNA species in the annealing mixture Plus strands
1
1
OF EMC VIRUS HETERODUPLEXES"
Intact Intact Intact uv-Inactivated uv-Inactivated None* Intact Intact Intact uv-Inactivated uv-Inactivated Noneb
Minus strands Intact uv-Inactivated None Intact uv-Inactivated Intact Intact uv-Inactivated None Intact uv-Inactivated Intact
Infec(“;1”FtZ, ml) 310 295 13 <3 <3 <3 420 514 <3 <3 <3 <3
u The intact or uv-inactivated minus strands were transferred to a hybridization buffer (5 x lo-* M NaCl, 2 x lo-* M Tris-HCl, IO-” M EDTA, pH 7.5) by means of gel filtration on Sephadex G-25. The solution containing about 0.01 wg of minus RNA per ml was supplemented with a loo-fold excess of either intact or uv-inactivated EMC virion RNA. Then DMSO was added to a final concentration of 65% (v/ v), the mixture was heated for 1 min at loo”, and then incubated at 37.5” for 3 hr. After precipitation with 4 volumes of ethanol, RNA samples were dissolved in 0.1 M NaCl, lo-* M Tris-HCl, lo-” M EDTA, pH 7.3, and treated with pancreatic RNAse (10e3 pg/ml, 20”, 30 min). b These control samples contained a loo-fold excess of 18 S ribosomal RNA from Krebs II cells, instead of plus RNA, for estimation of self-annealing in the preparation of minus strands.
SHORT
531
COMMUNICATIONS
The aliquots of RNA preparations to be inactivated were irradiated by two uv lamps (Tungsram Germicid “F”, Hungary, 15 W) at a distance of 10 cm for 10 min. Under these conditions the infectivity dropped approximately 104-fold in 2.5 and 10 min for virion RNA and RF RNA, respectively. Then the preparations of intact or uv-treated minus RNA strands were separately annealed with a loo-fold excess of intact or uv-treated virion RNA. As stated above, under annealing conditions used, not less than 95% of the minus RNA strands was transformed into a duplex form. It was possible that some of these duplexes were not perfect. To inactivate infectivity due to these incomplete duplexes as well as to the single-stranded virion RNA present in large amounts, the annealed preparations were treated with pancreatic RNAse under conditions where 80% of the material of labeled virion RNA was converted to an acid-soluble form with the complete loss of infectivity, while the infectivity of RF RNA remained unchanged. The conditions of annealing and RNAse treatment are described in the legend to Table 1. The results of the assay of infectivity of different artificially prepared duplex RNA complexes are presented in Table 1. It is clearly seen that the duplex molecules with inactivated plus strands are not infectious, whereas the infectivity of duplexes with an intact plus strand is essentially the same irrespective of the minus strand being intact or inactivated. In agreement with Best et al. (5), it can be concluded that it is the plus strand that is largely, if not exclusively, expressed in the progeny upon infection with picornavirus RF RNA. The infectivity of samples obtained after annealing of a preparation of minus
strands with fragmented plus strands and not treated with RNAse, was also determined. In disagreement with Bechet (6, 71, we did not find any increase in infectivity (due to the admixture of about 0.1% of plus strands in the preparation of minus strands) after such annealing. The infectivity was essentially the same when fragments of either intact or uv-inactivated plus strands were used. Thus, if complexes formed by intact minus strands and fragmented plus strands are infectious, their specific infectivity should be very low.
REFERENCES 1. CHUMAKOV, K. M., In “Problems of Medical Virology,” pp. 36-37. Institute of Poliomyelitis and Viral Encephalitides, Moscow, 1975. 2. MONTAGNIER, L., and SANDERS, F. K., Nature (London) 199, 664-667 (1963). 3. AGOL, V. I., Usp. Sovrem. Biol. 72, 315-338 (1971). 4. BISHOP, J. M., and LEVINTOW, L., Progress in Med. Viral. 13, 1-82 (1972). 5. BEST, M., EVANS, B., and BISHOP, J. M., Virology 47, 592-603 (1972). 6. BECHET, J.-M., C. R. Acad. Sci. (Paris) 274, 1761-1764 (1972). 7. BECHET, J.-M., Virology 48, 855-857 (1972). 8. AGOL, V. I., ROMANOVA, L. I., CUMAKOV, I. M., DUNAEVSKAYA, L. D., and BOGDANOV, A. A., J. Mol. Biol. 72, 77-89 (1972). 9. PORTER, A., CAREY, N., and FELLNER, P., Nature (London) 248, 675-678 (1974). 10. FRANKLIN, R. M., Proc. Nat. Acad. Sci. USA 55, 1504-1511 (1966). II. GILLESPIE, D., TAKEMOTO, K., ROBERT, M., and GALLO, R. C., Science 179,1328-1330 (1973). 12. SHELDON,R., JURALE, C., and KATES, S., Proc. Nat. Acad. Sci. USA 69, 417-421 (1972). 13. FEDOROFF, N. V., and ZINDER, N. D., Proc. Nat. Acad. Sci. USA 68, 1838-1843 (1971). 14. KERR, I. M., and MARTIN, E. M., J. Viral. 9, 559-561 (1972).