VIROLOGY
85,434-444
(1978)
Structural Studies of the RNA Component of the Poliovirus Replication Complex I. Purification
and Biochemical
R. E. LUNDQUIST
AND
Characterization
J. V. MAIZEL,
JR.’
Section on Molecular Structure, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Accepted November I, 1977 Substantial purification of the RNA component of the poliovirus replication complex is achieved by agarose chromatography of the high salt precipitated replication complex. The viral RNA isolated in the agarose-purified fraction is predominantly derived from in vivo replicating structures. Purified poliovirus RNA polymerase activity is associated with an endogenous RNA template that resembles poliovirus replicative intermediate RNA. INTRODUCTION
Cytoplasmic extracts from poliovirus-infected HeLa cells contain an enzymatic activity that synthesizes viral RNA, utilizing an endogenous viral RNA template (Baltimore et al., 1963). Viral RNA synthesis in vitro is limited to elongation of RNA chains initiated in uiuo (Girard, 1969). Deproteinization of this ribonucleoprotein complex, designated the poliovirus replication complex (RC), yields replicative intermediate (RI) RNA (Girard et al., 1967). Two species of double-stranded viral RNA can be isolated from poliovirus-infected cells (reviewed by Levintow, 1974). RI consists of a double-stranded RNA core with associated single-stranded RNA and sediments in sucrose gradients as a heterogeneous population with a peak at 25 S. Replicative form (RF) RNA is completely double-stranded and sediments at 18 S in sucrose gradients. The biological roles of these molecules have been suggested by their labeling kinetics in uiuo (Baltimore and Girard, 1966; Oberg and Philipson, 1969). Short pulses of &dine are preferentially incorporated into RI, demonstrating that RI is derived from the viral replicating structure. In contrast, RF incorporates urI Author addressed.
to whom
requests
for
reprints
should
be 434
oo42-a22/78/ae52-o43$02.00/0 Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
idine slowly and accumulates late in infection, implying that it is an end product of viral replication. The structure of poliovirus RI suggests a semiconservative in uiuo replicating structure that is predominantly doublestranded, containing one complete template strand and one complete complementary strand, in addition to several nascent chains (Baltimore, 1969; Bishop and Levintow, 1971). An alternative single-stranded model is based on the suggestion that the double-stranded nature of RI is an artifact generated during phenol or detergent extraction of a predominantly single-stranded replicating structure (Weissmann et al., 1968; Oberg and Philipson, 1971; Thach et al., 1974). RI and poliovirus RC are derived from the same viral replicating structure (Girard et al., 1967). However, the latter has been isolated using milder purification procedures which preserve enzymatic activity. Therefore, the structure of the RNA component of the purified RC should be a better model than RI for the in uiuo replicating structure. Characterization of the poliovirus RC in the electron microscope is described in the following paper (Meyer et al., 1978), and biochemical analysis is described in this paper. Both approaches demonstrate that the RNA component of the purified polio-
ISOLATION
OF POLIO
virus RC is analogous to poliovirus RI. Furthermore, the observed properties of this RNA do not support predictions of the single-stranded model.
REPLICATION
COMPLEX
435
sayed using the NCS (Amersham) method of Schrier and Wilson (1973). Phenol extraction. (a) Virion RNA. CsCl-purified poliovirus (0.8 mg/ml) in 0.02 M NaHzP04 (pH 7.2), 1% sodium dodecyl sulfate, 5 m&f EDTA, and 0.3 M NaCl MATERIALS AND METHODS was extracted three times with an equal volume of phenol/chloroform/isoamyl alLabeling of poliovirus-infected HeLa cells. HeLa S3 cells at 5 x lo5 cells/ml were cohol (50/50/l) at room temperature. The spun down, washed twice with Earle’s iso- aqueous layer was dialyzed overnight tonic salt solution (ESS), resuspended at 5 against 10 mM Tris-HCl, 10 m&f NaCl, pH 7.4, and then precipitated at -20” by adding x lo6 cells/ml in Eagle’s spinner medium, and infected with 100-150 PFU/cell of 2.8 vol of 95% ethanol. (b) Agarose-excluded peak. The high poliovirus Type 1. Actinomycin D (4 pg/ml) and guanidine (2 mM) were added 10 and salt pellet from 1.7 x lo”-infected cells was 15 min after infection. Thirty minutes after dissolved in 4.0 ml and chromatographed infection, 5% fetal calf serum and 1% of 0.2 on Sepharose 2B. The combined excluded M glutamine were added. After 90 min, the peak was adjusted to a volume of 24 ml of cells were spun down, washed once with 0.15 M NaCl, 0.5% sodium dodecyl sulfate, ESS, and resuspended in Eagle’s medium 5 mit4 EDTA, and 10 mM Tris-HCl, pH containing 0.3 mg/liter of methionine, 5% 7.4. After three extractions with buffer-satfetal calf serum, 1% of 0.2 M glutamine, and urated phenol at room temperature, the 5 mM each of HEPES, PIPES, and BES, aqueous layer was dialyzed overnight pH 7.4. Cells were harvested at 135 min against 10 mM NaCl, 10 n-& Tris-HCl, pH after guanidine reversal (equivalent to 175 7.4. The dialyzed sample was concentrated min postinfection). For uniform labeling of by lyophilization and the pellet was disviral RNA, [3H]uridine (20 pCi/ml) was solved in 1 ml of HzO. RNA was precipiadded 1 hr before cells were harvested. tated with 2.8 ml of 95% ethanol at -20”. Short label pulses utilized 50 pCi/ml of [3H]Hybridization reaction. Hybridization uridine. reactions were carried out as follows. AliPreparation of the viral replication com- quots (20 ~1) of the phenol-extracted agaplex. Purification of the poliovirus RC by rose-excluded peak fraction (, J. M., and LEVINTOW, L. (1971). Replicative forms of viral RNA. Structure and function. Progr. Med. Virol. 13, l-82. CALIGUIRI, L. A. (1974). Analysis of RNA associated with the poliovirus replication complexes. Virology 58, 526-535. CROUCH, R. J. (1974). Ribonuclease III does not degrade deoxyribonucleic acid-ribonucleic acid hybrids. J. Biol. Chem. 249, 1314-1316. DUNN, J. J. (1976). RNAse III cleavage of single stranded RNA. J. Biol. Chem. 251, 3807-3814. EDY, V. G., SZEKELY, M., LOVINY, T., and DREYER, C. (1976). Action of nucleases on double-stranded RNA. Eur. J. Biochem. 61,563-572. GIRARD, M. (1969). In uiuo synthesis of poliovirus ribonucleic acid: Role of the replicative intermediate. J. Virol. 3, 376-384. GIRARD, M., BAI,TIMORE, D., and DARNELL, J. E. (1967). The poliovirus replication complex: Site for
AND
MAIZEL
synthesis of poliovirus RNA. J. Mol. Biol. 24,59-74. GRANBOULAN, N., and GIRARD, M. (1969) Molecular weight, of poliovirus ribonucleic acid. ?J, Virol. 4, 475-479. LEVINTOW, L. (1974). The reproduction of picornaviruses. In “Comprehensive Virology” (H. FraenkelConrat and R. R. Wagner, eds.), Vol. 2. Plenum, New York. LUNDQUIST, R. E., EHRENFELD, E., and MAIZEL, J. V. (1974). Isolation of a viral polypeptide associated with the poliovirus replication complex. Proc. Nat. Acad. Sci. USA 71,4774-4777. MF,YEH, J., LLJNDQ~JIST, R. E., and MAIZEI,, J. V. (1978). Structural studies of the RNA component of the poliovirus replication complex: II. Characterization by electron microscopy and autoradiography. Virology 85, 445-455. MITCHELI,, W. R., and TEHSHAK, D. R. (1973). Synthesis of complementary RNA by poliovirus polymerase in vitro. Virology 56.386-389. OBERG, B., and PHILIPSON, L. (1969). Replication of poliovirus RNA studied by gel filtration and electrophoresis. Eur. J. Biochem. 11, 305-315. OBERG, B., and PHIIXPSON, L. (1971). Replicative structures of poliovirus RNA in vivo. J. Mol. Biol. 58, 725-737. SAVAGE, T., GHANBOULAN, N., and GIHARD, M. (1971). Architecture of the poliovirus replicative intermediate RNA. Biochemie 53, 533-543. SCHHIER, B. K., and WILSON, S. H. (1973). Investigation of methods for measurement of radioactivity in tritiated DNA and applications to assays for DNA polymerase activity. Anal. Biochem. 56, 196-207. SUMMERS, D. F., MAIZIX, J. V., and DAKNEI.L, J. E. (1967). The decrease in size and synthetic activity of poliovirus polysomes late in the infectious cycle. Virology 31,427-435. THACH, S., DOBBEI
85,434-444
(1978)
Structural Studies of the RNA Component of the Poliovirus Replication Complex I. Purification
and Biochemical
R. E. LUNDQUIST
AND
Characterization
J. V. MAIZEL,
JR.’
Section on Molecular Structure, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Accepted November I, 1977 Substantial purification of the RNA component of the poliovirus replication complex is achieved by agarose chromatography of the high salt precipitated replication complex. The viral RNA isolated in the agarose-purified fraction is predominantly derived from in vivo replicating structures. Purified poliovirus RNA polymerase activity is associated with an endogenous RNA template that resembles poliovirus replicative intermediate RNA. INTRODUCTION
Cytoplasmic extracts from poliovirus-infected HeLa cells contain an enzymatic activity that synthesizes viral RNA, utilizing an endogenous viral RNA template (Baltimore et al., 1963). Viral RNA synthesis in vitro is limited to elongation of RNA chains initiated in uiuo (Girard, 1969). Deproteinization of this ribonucleoprotein complex, designated the poliovirus replication complex (RC), yields replicative intermediate (RI) RNA (Girard et al., 1967). Two species of double-stranded viral RNA can be isolated from poliovirus-infected cells (reviewed by Levintow, 1974). RI consists of a double-stranded RNA core with associated single-stranded RNA and sediments in sucrose gradients as a heterogeneous population with a peak at 25 S. Replicative form (RF) RNA is completely double-stranded and sediments at 18 S in sucrose gradients. The biological roles of these molecules have been suggested by their labeling kinetics in uiuo (Baltimore and Girard, 1966; Oberg and Philipson, 1969). Short pulses of &dine are preferentially incorporated into RI, demonstrating that RI is derived from the viral replicating structure. In contrast, RF incorporates urI Author addressed.
to whom
requests
for
reprints
should
be 434
oo42-a22/78/ae52-o43$02.00/0 Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
idine slowly and accumulates late in infection, implying that it is an end product of viral replication. The structure of poliovirus RI suggests a semiconservative in uiuo replicating structure that is predominantly doublestranded, containing one complete template strand and one complete complementary strand, in addition to several nascent chains (Baltimore, 1969; Bishop and Levintow, 1971). An alternative single-stranded model is based on the suggestion that the double-stranded nature of RI is an artifact generated during phenol or detergent extraction of a predominantly single-stranded replicating structure (Weissmann et al., 1968; Oberg and Philipson, 1971; Thach et al., 1974). RI and poliovirus RC are derived from the same viral replicating structure (Girard et al., 1967). However, the latter has been isolated using milder purification procedures which preserve enzymatic activity. Therefore, the structure of the RNA component of the purified RC should be a better model than RI for the in uiuo replicating structure. Characterization of the poliovirus RC in the electron microscope is described in the following paper (Meyer et al., 1978), and biochemical analysis is described in this paper. Both approaches demonstrate that the RNA component of the purified polio-
ISOLATION
OF POLIO
virus RC is analogous to poliovirus RI. Furthermore, the observed properties of this RNA do not support predictions of the single-stranded model.
REPLICATION
COMPLEX
435
sayed using the NCS (Amersham) method of Schrier and Wilson (1973). Phenol extraction. (a) Virion RNA. CsCl-purified poliovirus (0.8 mg/ml) in 0.02 M NaHzP04 (pH 7.2), 1% sodium dodecyl sulfate, 5 m&f EDTA, and 0.3 M NaCl MATERIALS AND METHODS was extracted three times with an equal volume of phenol/chloroform/isoamyl alLabeling of poliovirus-infected HeLa cells. HeLa S3 cells at 5 x lo5 cells/ml were cohol (50/50/l) at room temperature. The spun down, washed twice with Earle’s iso- aqueous layer was dialyzed overnight tonic salt solution (ESS), resuspended at 5 against 10 mM Tris-HCl, 10 m&f NaCl, pH 7.4, and then precipitated at -20” by adding x lo6 cells/ml in Eagle’s spinner medium, and infected with 100-150 PFU/cell of 2.8 vol of 95% ethanol. (b) Agarose-excluded peak. The high poliovirus Type 1. Actinomycin D (4 pg/ml) and guanidine (2 mM) were added 10 and salt pellet from 1.7 x lo”-infected cells was 15 min after infection. Thirty minutes after dissolved in 4.0 ml and chromatographed infection, 5% fetal calf serum and 1% of 0.2 on Sepharose 2B. The combined excluded M glutamine were added. After 90 min, the peak was adjusted to a volume of 24 ml of cells were spun down, washed once with 0.15 M NaCl, 0.5% sodium dodecyl sulfate, ESS, and resuspended in Eagle’s medium 5 mit4 EDTA, and 10 mM Tris-HCl, pH containing 0.3 mg/liter of methionine, 5% 7.4. After three extractions with buffer-satfetal calf serum, 1% of 0.2 M glutamine, and urated phenol at room temperature, the 5 mM each of HEPES, PIPES, and BES, aqueous layer was dialyzed overnight pH 7.4. Cells were harvested at 135 min against 10 mM NaCl, 10 n-& Tris-HCl, pH after guanidine reversal (equivalent to 175 7.4. The dialyzed sample was concentrated min postinfection). For uniform labeling of by lyophilization and the pellet was disviral RNA, [3H]uridine (20 pCi/ml) was solved in 1 ml of HzO. RNA was precipiadded 1 hr before cells were harvested. tated with 2.8 ml of 95% ethanol at -20”. Short label pulses utilized 50 pCi/ml of [3H]Hybridization reaction. Hybridization uridine. reactions were carried out as follows. AliPreparation of the viral replication com- quots (20 ~1) of the phenol-extracted agaplex. Purification of the poliovirus RC by rose-excluded peak fraction (, J. M., and LEVINTOW, L. (1971). Replicative forms of viral RNA. Structure and function. Progr. Med. Virol. 13, l-82. CALIGUIRI, L. A. (1974). Analysis of RNA associated with the poliovirus replication complexes. Virology 58, 526-535. CROUCH, R. J. (1974). Ribonuclease III does not degrade deoxyribonucleic acid-ribonucleic acid hybrids. J. Biol. Chem. 249, 1314-1316. DUNN, J. J. (1976). RNAse III cleavage of single stranded RNA. J. Biol. Chem. 251, 3807-3814. EDY, V. G., SZEKELY, M., LOVINY, T., and DREYER, C. (1976). Action of nucleases on double-stranded RNA. Eur. J. Biochem. 61,563-572. GIRARD, M. (1969). In uiuo synthesis of poliovirus ribonucleic acid: Role of the replicative intermediate. J. Virol. 3, 376-384. GIRARD, M., BAI,TIMORE, D., and DARNELL, J. E. (1967). The poliovirus replication complex: Site for
AND
MAIZEL
synthesis of poliovirus RNA. J. Mol. Biol. 24,59-74. GRANBOULAN, N., and GIRARD, M. (1969) Molecular weight, of poliovirus ribonucleic acid. ?J, Virol. 4, 475-479. LEVINTOW, L. (1974). The reproduction of picornaviruses. In “Comprehensive Virology” (H. FraenkelConrat and R. R. Wagner, eds.), Vol. 2. Plenum, New York. LUNDQUIST, R. E., EHRENFELD, E., and MAIZEL, J. V. (1974). Isolation of a viral polypeptide associated with the poliovirus replication complex. Proc. Nat. Acad. Sci. USA 71,4774-4777. MF,YEH, J., LLJNDQ~JIST, R. E., and MAIZEI,, J. V. (1978). Structural studies of the RNA component of the poliovirus replication complex: II. Characterization by electron microscopy and autoradiography. Virology 85, 445-455. MITCHELI,, W. R., and TEHSHAK, D. R. (1973). Synthesis of complementary RNA by poliovirus polymerase in vitro. Virology 56.386-389. OBERG, B., and PHILIPSON, L. (1969). Replication of poliovirus RNA studied by gel filtration and electrophoresis. Eur. J. Biochem. 11, 305-315. OBERG, B., and PHIIXPSON, L. (1971). Replicative structures of poliovirus RNA in vivo. J. Mol. Biol. 58, 725-737. SAVAGE, T., GHANBOULAN, N., and GIHARD, M. (1971). Architecture of the poliovirus replicative intermediate RNA. Biochemie 53, 533-543. SCHHIER, B. K., and WILSON, S. H. (1973). Investigation of methods for measurement of radioactivity in tritiated DNA and applications to assays for DNA polymerase activity. Anal. Biochem. 56, 196-207. SUMMERS, D. F., MAIZIX, J. V., and DAKNEI.L, J. E. (1967). The decrease in size and synthetic activity of poliovirus polysomes late in the infectious cycle. Virology 31,427-435. THACH, S., DOBBEI
