Identification and partial characterization of a new (50 S) subviral particle in Mengo virus-infected L cells

VIROLOGY

85, 286-295

(1978)

Identification

and Partial Characterization of a New (50 S) Subviral Particle in Mengo Virus-Infected L Cells

PATRICK Department

W. K. LEE, of Biochemistry,

EVA

University Accepted

PAUCHA,’ of Alberta, October

AND

JOHN

S. COLTER”

Edmonton,

Alberta

T6G 2H7

Canada

20,1977

A previously undetected subviral particle has been found in Mengo virus-infected L cells by sucrose density gradient centrifugal analysis of cytoplasmic supernatants (Sz,) prepared from cells after labeling with 13Hlamino acids during the early to mid-log phase of virus production. The particle (designated the “50 S particle” from its position between the ribosomal subunits in the gradient), together with mature virions (150 S) and previously described 14 S particles (McGregor et al., 19751, can be recovered from the S,, fraction by high-speed centrifugation. It contains no RNA and is composed of equimolar amounts of the polypeptides E, a, and y. The results of conventional pulsechase experiments suggest that it may be a precursor in the assembly of Mengo virions, but more convincing evidence that this is the case was obtained from experiments in which the chase was carried out in the presence of cordycepin (3’-deoxyadenosine). In the presence of this inhibitor of viral RNA synthesis, there is a significant accumulation of 50 S particles, and when the inhibition is reversed, a quantitative transfer of radiolabel from 50 S particles to mature virions ensues. The recovery of 50 S particles (and of mature virions) from cell homogenates is strongly dependent upon the concentration of KC1 in the suspending buffer; only trace amounts are recovered at concentrations of less than 60 miV, while maximum recovery is achieved at a concentration of 100

INTRODUCTION

Information regarding the assembly of picornaviruses has come largely from the characterization of subviral particles isolated from infected cells. In the case of poliovirus, the most extensively studied of this group of viruses, a number of subviral particles believed to be precursors in the assembly process have been described. These include 5 and 14 S structures (Phillips et al., 1968), empty capsids having a sedimentation coefficient of 73 S (Maize1 et al., 1967; Jacobson and Baltimore, 1968), and a 125 S particle which, unlike the 5, 14, and 73 S particles, contains viral RNA (Fernandez-Tomas and Baltimore, 1973). All of these subviral particles contain 1 Present address: Imperial Cancer Research Fund Laboratories, P. 0. Box 123, Lincoln’s Inn Fields, London WCBA 3PX England 2 To whom requests for reprints should be addressed. 0042-6822/78/0851-0286$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

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equimolar amounts of the viral polypeptides VP-l, VP-3, and VP-O, the latter being the immediate precursor of capsid polypeptides VP-2 and VP-4. It has been shown that the 14 S particles can aggregate in vitro to form 73 S particles (Phillips, 1969 and 1971) and that the assembly is enhanced by the addition of rough membranes from infected cells (Perlin and Phillips, 1973). Although empty capsids are found regularly in cells infected with poliovirus, as well as in cells infected with other picornaviruses, e.g., FMDV (Rowlands et al., 1975) and bovine enterovirus 1 (Su and Taylor, 1976), there is no convincing evidence that comparable structures are produced in cells infected with members of the cardiovirus subgroup of picornaviruses. Prather and Taylor (1975) reported the detection of 80 and 125 S particles in lysates of Mengo virus-infected MadinDarby bovine kidney cells, which are con-

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sidered to be nonpermissive for Mengo the same medium. Cells were pulse-lavirus, since the viral output is only about beled by incubation for either 20 min (petri dish cultures) or 30 min (roller bottle cul5 PFU/cell. Since neither the polypeptide tures) in amino acid-deficient Eagle’s mecomposition nor the kinetics of formation of these particles was established, their dium containing 1% HS and r3H]amino possible role in the assembly process re- acids (New England Nuclear, NET-250; 30 mains unclear. However, McGregor et al. &i/ml). Labeling was done at either 4.5 or 5 hr postinfection in the case of petri (1975) found two capsid precursors in EMC virus-infected HeLa cells. These have sed- dish cultures and at 5.5 hr postinfection imentation coefficients of 13 and 14 S and with cells grown and infected in roller polypeptide compositions of (A), and (E, a, bottles. In both cases the cultures were Y)~, respectively; polypeptide A is the pre- incubated in amino acid-deficient medium for the hour immediately preceding the cursor of all capsid polypeptides in cardiovirus systems, and E is the immediate labeling period. When labeling was folprecursor of capsid polypeptides p ang 6. lowed by a chase period, the monolayers (after removal of the radioactive medium) In this paper we describe the detection of a 50 S particle in Mengo virus-infected were washed once with PBS and incubated L cells and present evidence from pulsefor the desired period of time in BME-5% chase experiments and from compositional HS. analysis suggesting that the particle may Fractionation of cells and isolation of be an intermediate in the assembly of subviral particles. The cells were harMengo virions. vested from roller bottles by trypsinization, collected into ice-cold BME + 5% HS, pelleted by low-speed centrifugation, and MATERIALS AND METHODS washed sequentially with ice-cold PBS and Cells and virus. Cells of Earle’s L-929 RSB (reticulocyte standard buffer, 10 mM pH 7.4, 10 mM KCl, 1.5 n-&Y strain of mouse fibroblasts were grown in Tris-HCl, suspension culture in calcium-free Eagle’s MgCl,) before being resuspended in hobuffer (1 n-&f Tris-HCl, pH minimum essential medium (MEM) and mogenizing as monolayers in Eagle’s basal minimum 7.4, 1 mit! KCl, and 1.5 m&f MgCI,). The essential medium (BME); both media were monolayers grown in petri dishes were washed with PBS and RSB, after which supplemented with 5% horse serum (HS). the cells were scraped off the dishes into Monolayer cultures were grown in either 100 x l&mm plastic petri dishes or 110 x homogenizing buffer. 425-mm glass roller bottles. Cells were homogenized in an all-glass The virus used was the plaque variant Dounce homogenizer with a tightly fitting pestle, and the tonicity of the homogenate of Mengo virus designated as M-Mengo (Ellem and Colter, 1961). It was propa- was adjusted to give final concentrations pH 7.4, 100 n-&f KCl, gated in roller bottle cultures of L cells of 20 mM Tris-HCl, and purified by the method described by 5 m&f MgCl?, and 6 mM P-mercaptoethano1 (TBS) by adding the appropriate volZiola and Scraba (1974). Infection of monolayers and labeling of ume of 10x concentrated TBS. After the viral polypeptides. Cells grown in either addition of Nonidet P40 and sodium deoxpetri dishes or roller bottles were infected ycholate to give final concentrations of 1 and 0.5%, respectively, the nuclei were at an estimated multiplicity of 100 PFU/ removed by low-speed centrifugation, and cell with Mengo virus suspended in virus diluent. The virus diluent used was the the supernatant was separated into cytoplasmic supernatant (S,,) and cytoplasmic buffered (pH 7.6) balanced salt solution described by Dulbecco and Vogt (1954) pellet (P,,) as described by Roumiantzeff et al. (1971) by centrifugation for 30 min at containing 0.2% bovine plasma albumin, fraction V. After incubation for 1 hr at 37”, 20,000 g (13,000 rpm, JA-20 rotor, Beckthe monolayers were washed with warm man 521 centrifuge). Material having a coefficient of 14 S or larger (37“) BME-5% HS and then incubated in sedimentation

288

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was collected from the S,, fraction by centrifugation for 5 hr at 45,000 rpm (type 50 rotor; Beckman L5-65 centrifuge). The pellet so obtained is referred to herein as the P,, fraction. Sucrose density gradient analyses. Centrifugal analyses of S,, and P,, fractions isolated from cells grown in petri dishes were carried out by layering 0.5- to O.&ml aliquots on 15-ml linear 15-45% sucrose gradients (in TBS) and centrifuging them for 13.5 hr at 20,000 rpm (SW 27.1 rotor; Beckman L5-65 centrifuge). The fractions (0.5 ml) were collected using a Beckman gradient fractionator linked to an LKB peristaltic pump and fraction collector. The single analysis of an So fraction from a roller bottle culture as described here was carried out on a 34-ml 15-45% sucrose gradient. Centrifugation was for 12 hr at 20,000 rpm (Beckman SW 27 rotor), and the gradient was fractionated (l-ml fraction) using an ISCO Model D gradient fractionator equipped with a Model UA-2 uv monitor (Instrumentation Specialties Co. Inc., Lincoln, Nebraska). Following gradient centrifugation of the S2,, preparations, 200-~1 aliquots of the gradient fractions were applied to filter paper disks (Whatman No. 3, 2.3 cm diameter) which were air-dried and washed sequentially with 10% TCA, 5% TCA, ethanol, and acetone before being placed in scintillation vials. After centrifugation of the P,, preparations, 200-~1 aliquots of the gradient fractions were added directly to scintillation vials. In both cases, radioactivity was measured in the presence of 10 ml of Aquasol scintillation fluid (New England Nuclear Corp.) in a Beckman liquid scintillation spectrometer (Model LS-230). Polypeptide analysis. The polypeptide composition of Mengo virions and of the subviral structures recovered from the sucrose density gradients was determined by SDS-polyacrylamide gel electrophoresis carried out according to the procedure of Weber and Osborn (19691, using 7.5% gels (21 cm long and polymerized in 6-mm-i.d. glass tubes) that had been acid-washed and coated with dichlorodimethylsilane. The gels were preelectrophoresed for 1 hr

AND

COLTER

at 8 mA/gel before the samples were applied. Gradient fractions containing labeled virions or subviral particles were made 1.5% in SDS, 2% in P-mercaptoethanol, lop3 M in phenylmethylsulfonyl fluoride (PMSF), and 0.002% in bromophenol blue and were heated at 100” for 5 min before being applied to the gels. Electrophoresis was carried out at 4 mA/gel for the first hour, after which the current was increased to and held at 8 mA/gel for an additional 16 hr. The gels were fractionated using the automatic Aliquogel fractionator (Gilson Medical Electronics, Inc.), and the fractions were incubated overnight at 65” in 0.5 ml of 30% H202, after which the radioactivity in each was measured as described earlier. Cordycepin was purchased from Sigma Chemical Company, St. Louis, Missouri. RESULTS

Identification of subviral particles. The data obtained from sucrose density gradient centrifugal analysis of the SZOfraction prepared from a roller bottle culture of L cells, which were pulse-labeled for 30 min at 5.5 hr after infection with Mengo virus (m.o.i. -100) and harvested after a chase period of 60 min, are illustrated in Fig. 1. The fractions were monitored for OD,,,, and for radioactivity. In addition to the intact virions (150 8, a well-defined peak of labeled material was found at a position between those occupied by the 40 and 60 S ribosomal subunits, which are resolved clearly from each other and from 80 S ribosomes in a 15-45% sucrose density gradient. From the position of this peak in this gradient, we have called the material contained therein “50 S particles.” When particulate material present in the SZOfraction was sedimented by highspeed centrifugation and the pellet (P,, fraction) was analyzed by sucrose density gradient centrifugation, the results shown in Fig. 2 were obtained. In addition to the virus (150 S) and 50 S peaks, a well-defined peak of more slowly sedimenting material was observed. On the basis of compositional analysis (see following section) and position in the gradient, it seems clear

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150 s

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FIG. 1. Sedimentation analysis of a cytoplasmic supernatant (S,,) prepared from a roller bottle culture of Mengo virus-infected L cells. Cells were labeled for 30 min with [3Hlamino acids (30 &i/ml) at 5.5 hr postinfection, and the S,, fraction (in TBS) was isolated after a 60-min chase. Centrifugation through a 34-ml 15-45% linear sucrose gradient prepared over a 3-ml 60% sucrose cushion was at 20,000 rpm for 12 hr at 4” (Beckman SW 27 rotor). Sedimentation was from right to left. The gradient was fractionated and analyzed for acid-insoluble radioactivity (O-0) and absorbance at 260 nm C-h

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clear, NEC-445; 5 ~Cilml). After incubation for an additional 30 min in the absence of labeled amino acids but in the presence of L3Hluridine, the cells were harvested and a P, fraction was isolated therefrom and analyzed by sucrose density gradient centrifugation. The results illustrated in Fig. 4 show clearly that the 50 S particle contains no viral RNA. Confirmatory evidence was obtained by centrifuging 50 S particles (recovered by high-speed centrifugation from pooled sucrose density gradient fractions) to equilibrium in a CsCl density gradient. The 50 S particle was found to have a buoyant density of 1.296 g/cm3, which is substantially lower than that of the intact virion (1.330 g/cm31 but very close to that expected of a particle containing protein only. Precursor role of 50 S particles: Pulsechase experiments. Since the polypeptide

composition of the 50 S particle suggested that it might be an intermediate in the assembly of Mengo virions, conventional

that the material in this peak is identical to the 14 S particles shown by McGregor et al. (1975) to be present in extracts of EMC virus-infected cells. Composition of the subviral

particles.

Peak fractions from each of the 14,50, and 150 S peaks obtained by sucrose density gradient centrifugation of a P,, preparation were pooled separately and analyzed by SDS-PAGE. The results illustrated in Fig. 3 show that the 14 and 50 S particles contain equimolar amounts of polypeptides E, cr, and y , The mature virions as shown earlier by Ziola and Scraba (1974) contain approximately equimolar amounts of polypeptides (Y, /3, y , and 6 plus a small quantity of polypeptide E, the immediate precursor of p and 6 (Butterworth et al., 1971; Paucha et al., 1974). To determine whether the 50 S particle contained any viral RNA, L3Hluridine (10 @i/ml) was added to a culture of infected cells 1 hr before they were pulse-labeled for 20 min (at 5 hr postinfection) with 14Clabeled amino acids (New England Nu-

0

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20 NUMBER

30

FIG. 2. Sedimentation analysis of virus-specific particles isolated from Mengo virus-infected cells. An S,,, fraction, isolated from a roller bottle culture as indicated in the legend to Fig. 1, was centrifuged for 5 hr at 45,000 rpm at 4’ (Beckman type 50 rotor). The pellet (P,, fraction) ,was resuspended in TBS and layered on a 15-ml l&45% linear sucrose gradient, which was then centrifuged at 20,000 ‘pm for 13.5 hr at 4” (Beckman SW 27.1 rotor). Sedimentation was from right to left.

290

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2kk DISTANCE

150 MIGRATED

200

, mm,

FIG. 3. SDS-polyacrylamide gel electrophoretic analysis of 14, 50, and 150 S particles. Samples of the peak fractions from the sucrose density gradient illustrated in Fig. 2 were solubilized by heating them in SDS and then analyzed as outlined under Materials and Methods. Migration was from left to right.

pulse-chase experiments were carried out in an attempt to demonstrate a precursorproduct relationship between the two subviral particles and intact virions. Replicate monolayer cultures of infected cells were pulse labeled with [3H]amino acids for 20 min at 4.5 hr postinfection, and cytoplasmic supernatants prepared immediately after the labeling period and after subsequent chase periods of 30, 60, and 110 min were analyzed by sucrose density gradient centrifugation. SzOrather than P,, fractions were used in this study, because the recovery of 14 S particles in the P,, fraction was found to be less than quantitative. Illustrative data are presented in Fig. 5. No labeled virions were produced during a 20-min labeling period (Fig. 5A), but radioactivity was found in the 50 S peak and in the top fractions, which contained a mixture of viral polypeptides in addition to the 14 S particles. Analysis of the S,, fraction prepared after a 30-min chase period (Fig. 5B) revealed the appearance of a virus peak (150 9, an increase in the amount of label in the 50 S peak, and a

AND COLTER

loss of radioactivity from the top fractions. With longer chase periods (Figs. 5C and 5D1, a progressive and essentially quantitative transfer of radioactivity from both the top fractions and the 50 S peak into the virions was observed. Very little of the 50 S material remained after a llO-min chase (panel D), and the residual radioactivity in the top fractions reflected the presence of labeled viral, nonstructural proteins (no 14 S particles could be detected in the P,, fractions prepared after a chase period of 110 min). These data show clearly that during a chase following a 20-min labeling period, there is a progressive flow of radioactivity from 14 S and 50 S particles into mature virions. The data suggest but do not prove that the 50 S particle is an intermediate between the 14 S particles and virions on the assembly pathway. More convincing evidence that this is the case was obtained from similar studies in which the effects of cordycepin were examined. Effect of cordycepin on viral RNA synthesis and on the formation of 50 S parti-

cles. Reports that the adenosine analog cordycepin (3’-deoxyadenosine) inhibits I

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FIG. 4. Sedimentation analysis of a P,, fraction isolated from Mengo virus-infected cells labeled with L3Hluridine and 14C-amino acids. Cells were labeled with L3Hluridine from 4 to 5 hr postinfection, then pulse labeled for 20 min with ‘X-labeled amino acids, and harvested after an additional incubation period of 30 min in medium containing 13H]uridine. Centrifugation through a 15-ml 15-45% linear sucrose density gradient was for 13.5 hr at 20,000 rpm (Beckman SW 27.1 rotor). Gradient fractions were analyzed for acid-insoluble ‘C-counts per minute (0-O) and 3H-counts per minute (0-O).

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FIG. 5. Sedimentation analysis of min with [aH]amino acids at 4.5 hr density gradients was for 13.5 hr at immediately after the labeling period C), and 110 min (panel D).

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So fractions prepared from Mengo virus-infected cells labeled for 20 postinfection. Centrifugation through X-ml 1545% linear sucrose 20,000 r-pm (Beckman SW 27.1 rotor). SzO fractions were prepared (panel A) and after chase periods of 30 min (panel B), 60 min (panel

the replication of a number of viruses including human rhinovirus (Nair and Owens, 19741, Newcastle disease and Sendai viruses (Mahy et al., 19731, vaccinia virus (Nevins and Joklik, 19751, and poliovirus (Nair and Panicali, 1976) prompted us to examine the effects of this compound in our system. It was found that cordycepin inhibits the synthesis of viral ribonucleates in Mengo-infected cells and that at a concentration of 200 pg/ml, inhibition is rapid and efficient (85-90%). This observation is illustrated by the data shown in Fig. 6, as is the fact that the inhibition can be relieved simply by removing the medium containing the cordycepin and washing the monolayers twice with normal medium. After removal of the inhibitor,

viral RNA synthesis resumes immediately and at a rate comparable to that in control cultures. The effect of cordycepin on the formation of 50 S particles was then examined. Replicate cultures of infected cells were pulse labeled with [3H]amino acids at 4.5 hr postinfection, and cytoplasmic supernatants (S,,) were prepared (i) immediately after the labeling period, (ii) after chase periods of 45 and 135 min in BME-5% HS, (iii) after chase periods of 45 and 135 min in BMEd% HS containing 200 pg of cordycepin/ml, and (iv) after a chase period of 45 min in BME-5% HS containing 200 pg of cordycepin/ml followed (after washing) by an additional chase period of 90 min in BME-5% HS. The results of sucrose den-

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HOURS

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FIG. 6. Effect of cordycepin on the synthesis of viral ribonucleates. 13H]Uridine (10 &i/ml) was added to replicate cultures of Mengo virus-infected cells at 2 hr postinfection. At 4.5 hr postinfection, cordycepin (200 pg/ml) was added to some of the cultures, and at 5.5 hr postinfection, half of the cultures to which cordycepin had been added were washed twice with culture medium and reincubated in medium containing [3H]uridine. At various times from 3 to 7 hr postinfection, the cultures were harvested, the cells were lysed, and the lysates were analyzed for acid-insoluble radioactivity. (O-O), no cordycepin added; (O-O), cordycepin added at 4.5 hr postinfection; (A-A), cordycepin added at 4.5 postinfection and removed by washing at 5.5 hr postinfection.

sity gradient analyses of the S2,, fractions are illustrated in Fig. 7. The presence of cordycepin during a 45min chase period was found to reduce sharply the number of mature virions produced and to result in a significant accumulation of 50 S particles (Fig. 7B). When cordycepin was removed after a chase period of 45 min, and when the cultures were incubated for an additional 90 min in the absence of the inhibitor, a quantitative transfer of radioactivity from the 50 S to the virion peak was observed (Fig. 70. The profile (not shown) obtained with the SZo fraction from cells that were incubated for 135 min in the presence of cordycepin did not differ significantly from that shown in Fig. 7B. The size of the virus peak remained unchanged, while the size of the 50 S peak increased only slightly. Effect of KC1 concentration on recovery

AND COLTER

particles. During the course of this study it was found that the concentration of KC1 employed has a marked effect on the release of virus-specific particles into the S,,, fraction and thus on their recovery in the P,, pellet. Infected cultures were pulse labeled with [3H]amino acids for 20 min at 5 hr postinfection, after which they were incubated for an additional 75 min in BME-5% HS containing cordycepin (200 kg/ml) before being harvested and homogenized as outlined under Materials and Methods. The homogenate was then divided into six aliquots, the KC1 concentration therein was adjusted to 0, 20, 40, 60, 80, and 100 mM, respectively, and the P,, fractions were isolated from each as described earlier. Sucrose density gradient analyses gave the results shown in Fig. 8. The concentration of KC1 in the suspending buffer appears to have little or no effect on the amount of 14 S material present in the P,, fraction but does have a rather profound effect on the recovery of both virions and 50 S particles. Only trace amounts of both were obtained when the KC1 concentration in the cell homogenate was 60 mM or less. Significant quantities of both were recovered in the presence of 80 n&f KCI, and the maximum recovery was obtained at 100 m&f KC1 (higher concentrations of the salt did not increase the yield further). of virus-specific

DISCUSSION

The results presented herein show clearly that two subviral particles can be isolated from Mengo virus-infected cells: a 14 S particle which corresponds to the 14 S particle found in cells infected with poliovirus (Phillips et al., 1968), EMC virus (McGregor et al ., 1975), and a human rhinovirus (McGregor and Rueckert, 1977) and a 50 S particle which has not been described previously. Both particles contain equimolar amounts of the polypeptides e, o, and y , which correspond precisely to the composition of the previously described 14 S particles and to that of the 73 S poliovirus empty capsids (Maize1 et al., 1967; Jacobson and Baltimore, 1968; Phillips and Fennell, 1973) and of the 125 S provirion described by Fernandez-Tomas and Baltimore (1973).

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FIG. 7. Sedimentation analysis of Se,, fractions prepared from Mengo virus-infected cells pulse-labeled for 20 min with 13Hlamino acids at 4.5 hr postinfection. Centrifugation through 15-458 sucrose gradients was for 13.5 hr at 20,000 rpm (Beckman SW 27.1 rotor). (A) SzOfraction prepared immediately after the labeling period. (B) Sz, fractions prepared after a chase period of 45 min in (0) BME-5% HS and (0) BME-5% HS containing 200 pg of cordycepin/ml. (C) &, fractions prepared after (0) a chase period of 135 min in BME-5% HS and (0) a chase period of 45 min in BME-5% HS containing 200 pg of cordycepin/ml followed by 90 min in BME-5% HS after removal of the cordycepin. 14 s 1o.

0 mM

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.

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FIG. 8. Effect of KC1 concentration on the recovery of virus-specific particles from Mengo virus-infected cells. Infected cells were pulse labeled for 20 min with [3HJamino acids at 5 hr postinfection, chased for 75 min in the presence of cordycepin, and then homogenized in homogenizing buffer. The homogenate was then divided into six aliquots in which the KC1 concentration was adjusted to 0 (cl), 20, 40, 60, 80, and 100 mM, respectively before P,, fractions were isolated. The P4:, fractions were suspended in TBS and analyzed in 15-458 sucrose gradients (in TBS). Centrifugation was at 20,000 rpm for 13.5 hr (Beckman SW 27.1 rotor).

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In considering the morphogenesis of picornaviruses as a group, the one common denominator is the 14 S particle, which, it seems clear, occupies an early position in the assembly pathway of the entero-, cardio-, and rhinoviruses (McGregor and Rueckert, 1977). The precise role of the 73 S empty capsids in the poliovirus system is somewhat less secure. Jacobson and Baltimore (1968) found that 73 S capsids accumulate in infected cells in the presence of 3 mM guanidine hydrochloride, and since the labeled protein was found to be chased into mature virions upon removal of the guanidine, they concluded that the 73 S particle is a precursor in the assembly process. However, Ghendon et al. (1972) reported that 14 S rather than 73 S particles accumulate in poliovirus-infected MiO cells (from rhesus monkey tonsils) in the presence of guanidine, and that when the guanidine is removed, the accumulated 14 S material chases into virions without the appearance of 73 S capsids. These experiments, coupled with the fact that there is no completely convincing evidence that empty capsids are produced in cardiovirus systems, has led to the suggestion that the 73 S particles may not be intermediates in the assembly process (for a review, see Casjens and King, 1975). The results of the pulse-chase experiments described here suggest that the 50 S particle may be a true intermediate in the assembly pathway of Mengo virus. Following a brief labeling period, label can be detected in 50 S particles before it appears in mature virions, and during a subsequent chase, label flows from both 14 and 50 S particles into the virions. The results of those experiments in which viral RNA synthesis was blocked by cordycepin during the chase period are even more suggestive. The observations that 50 S particles accumulate under these conditions and that the label chases into virions when the inhibition of RNA synthesis is reversed are very similar to those made by Jacobson and Baltimore (1968) with guanidine-inhibited poliovirus-infected cells and lead us to suggest that the 50 S particle is indeed a precursor of the mature Mengo virion. In view of the low value of the sedimen-

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tation coefficient for the 50 S particle, it may seem unlikely that the 50 S particle described here is analogous to the 73 S particle found in the poliovirus system. However, we have found that when 50 S particles are purified by equilibrium centrifugation in a CsCl density gradient and then reexamined in a sucrose density gradient, they exhibit a marked increase (to between 70 and 80 S) in sedimentation coefficient. A similar shift in sedimentation coefficient has also been observed when, during the isolation of the S,,, fraction, the KC1 concentration was increased to 200 mM. Although we cannot at present rule out the possibility that this sharp increase in sedimentation coefficient at high salt concentrations is due to some sort of aggregation phenomenon, we think it more likely that it reflects a marked conformational change from what would be a highly asymmetric 50 S particle,to a more compact 70-80 S particle. In this regard it is interesting to note that Su and Taylor (1976) showed that 45 S particles, isolated in buffer containing 10 mM NaCl from bovine enterovirus l-infected cells,. can be converted to 80 S particles by dlalysis against buffer containing 150 mM NaCl. The finding that the release of 50 S particles and of newly assembled virions (but not of 14 S particles) into the S,,, fraction is strongly dependent on the KC1 concentration of the buffer in which the manipulations are carried out is compatible with the hypothesis that the 50 S particles are intermediates in the assembly process. Available evidence suggests that at least in the poliovirus system, viral RNA replication and particle formation are coupled processes that occur in association with the smooth cytoplasmic membranes (Caliguiri and Compans, 1973). Our interpretation of the data presented here is that the 14 S precursor particles aggregate on smooth cytoplasmic membranes to form 50 S particles, which in turn interact with newly synthesized viral RNA to form mature virions. The 50 S particles, like newly synthesized virions, are released from membranes only at KC1 concentrations of 80 mM or higher and at salt concentrations of 150 mM or higher,

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undergo a conformational change to produce 70-80 S particles. Further experiments designed to provide supporting evidence for these hypotheses are in progress. ACKNOWLEDGMENTS We wish to thank Pat Carpenter and Irene Korolak for assistance in culturing the cells and preparing the virus pools used in these studies, which were supported by a grant (MT 1191) from the Medical Research Council of Canada. REFERENCES BUTTERWORTH, B. and RUECKERT,

E., HALL, L., STOLTZFUS, C. M., R. R. (1971). Virus specific proteins synthesized in encephalomyocarditis virusinfected HeLa Cells. Proc. Nat. Acad. Sci. USA

68, 3083-3087. CALIGUIRI, L.

A., and COMPANS, R. W. (1973). The formation of poliovirus particles in association with the RNA replication complexes. J. Gen. Viral. 21, 99-108. CASJENS, S., and KING, J. (1975). Virus assembly. Annu. Rev. B&hem. 44, 555-611. DULBECCO, R., and VOCT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Elep. Med. 98, 167-182. ELLEM, K. A. O., and COLTER, J. S. (1961). The isolation of three variants of Mengovirus differing in plaque morphology and hemagglutinating characteristics. Virology 15, 340-347. FERNANDEZ-TOMAS, C. B., and BALTIMORE, D. (1973). Morphogenesis of poliovirus: II, Demonstration of a new intermediate, the provirion. J. Viral. 12, 1122-1130. GHENDON, Y., YAKOBSON, E., and MIKHEJEVA, A. (1972). Study of some stages of poliovirus morphogenesis in MiO cells. J. Viral. 10, 261-266. JACOBSON, M. F., and BALTIMORE, D. (1968). Morphogenesis of poliovirus: I, Association of viral RNA with coat protein. J. Mol. Biol. 33, 369-378. MAHY, B. W. J., Cox, N. J., ARMSTRONG, S. J., and BARRY, R. D. (1973). Multiplication of influenza virus in the presence of cordycepin, an inhibitor of cellular RNA synthesis. Nature New Biol. 243, 172-174. MAIZEL, J. V., PHILLIPS, B. A., and SUMMERS, D. F. (1967). Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1. Virology 32, 692-699. MCGREGOR, S., HALL, L., and RUECKERT, R. R. (1975). Evidence for the existence of protomers in the assembly of encephalomyocarditis virus. J. Virol. 15, 1107-1120. MCGREGOR, S., and RUECKERT, R. R. (1977). Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits. J. Viral. 21, 548-553.

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