JOURNAL OF VIROLOGY, May 1992, P. 2740-27470022-538X/92/052740-08$02.00/0Copyright ©3 1992, American Society for Microbiology

Structural and Functional Characterization of the PoliovirusReplication Complex

KURT BIENZ,* DENISE EGGER, THOMAS PFISTER, AND MONICA TROXLER

Institute for Medical Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland

Received 18 November 1991/Accepted 10 February 1992

Two populations of membrane-bound replication complexes were isolated from poliovirus-infected HEp-2cells by sucrose gradient centrifugation. The two fractions show similar ultrastructural features: the replicationcomplex is enclosed in a rosettelike shell of virus-induced vesicles and contains a very tightly packed secondmembrane system (compact membranes). The vesicular fraction, which bands in 30% sucrose, containsreplicative intermediate (RI) and 36S RNA. The fraction banding in 45% sucrose contains only minute amountsof RI and contains mainly 36S RNA, two-thirds of which is encapsidated. In vitro, the two fractions showsimilar RNA synthesizing capacities and produce 36S plus-strand RNA. Dissolving the membranes within andaround synthetically active replication complexes with sodium deoxycholate abolishes the completion of 36SRNA but still allows elongation in the RI. Our findings suggest an architecture of the replication complex thathas the nascent plus strands on the RI enclosed in the compact membranes and the replication forks wrappedadditionally in protein. Plus-strand RNA can be localized by in situ hybridization with a biotinylated riboprobebetween the replication complex and the rosette of the virus-induced vesicles. It was found that the progeny

RNA strands are set free soon after completion from the replication complex at the sites where the compactmembranes within the replication complex are in close contact with the surrounding virus-induced vesicles.

The replication of poliovirus RNA is carried out by theprimer-dependent viral polymerase 3DPo' and proceedsasymmetrically in two distinct steps. First, the input plus-strand (genomic) RNA is copied into a minus strand, whichleads to the formation of the double-stranded replicativeform (RF) (1, 24). This reaction can be studied in vitro in a

reconstituted system (28) and does not depend on anycellular component or structure. The next step, however,which produces progeny RNA strands of positive polarity, isfully dependent on specialized cellular membranous struc-tures (25). It starts from the RF and proceeds in the partiallydouble-stranded replicative intermediate (RI) (11), which isconfined in the viral replication complex. The replicationcomplex was identified by electron microscopy (EM) (3) andfound to be surrounded by and stretched out inside a rosetteof virus-induced cytoplasmic vesicles (6). The structuralconfiguration of the replication complex is essential forongoing plus-strand RNA synthesis and is maintained by themembranes of the virus-induced vesicles in concert withviral protein 2C (6).The strict dependence of the functional replication com-

plex on cellular membranous structures has hitherto pre-cluded the use of truly reconstituted in vitro transcriptionsystems to study viral plus-strand RNA synthesis. By usingappropriate subcellular fractions of infected cells, however,36S genomic RNA can be produced in vitro (6, 10, 11, 24,25), and thus at least some of the requirements for thissynthetic activity can be investigated.

In the present study, we used such subcellular fractions toelucidate the roles of different components of the replicationcomplex and the surrounding vesicles in viral plus-strandRNA synthesis. By EM immunocytochemistry and in situhybridization, we visualized the spatial relation of viralmacromolecules involved in RNA synthesis with respect toeach other and to the cellular (yet virus-induced) structures.

* Corresponding author.

This allowed us to draw conclusions about the functionalinteraction of the replication complex with its associatedmembranes. In addition to the known virus-induced vesi-cles, we could identify a second membrane system withinthe replication complex. This membrane system is verytightly packed and appears to interact with the surroundingvirus-induced vesicles in the last steps of completion of the36S progeny RNA. We also found that elongation of plus-strand RNA at the RI proceeds in the absence of mem-

branes, whereas completion and liberation of 36S RNA are

dependent on membranes.

MATERIALS AND METHODS

Cells and virus and in vivo labeling of viral RNA. HEp-2cells and poliovirus type 1 (Mahoney) were grown in sus-

pension cultures. The multiplicity of infection for the exper-iments was 30 PFU per cell. To label viral RNA, 5 p.g ofactinomycin D (Merck Sharp & Dohme, Rahway, N.J.) perml was added to infected cells 30 min before the addition of50 p.Ci of [5-3H]uridine (Amersham, Amersham, UnitedKingdom) per ml. The label was added at 2.75 h postinfec-tion and left until the cells were harvested at 4 h postinfec-tion.

Isolation and in vitro RNA synthesis of replication com-

plexes attached to virus-induced vesicles. Preparation of cy-toplasmic extracts by Dounce homogenization and low-speed centrifugation and isolation of the vesicle-attachedreplication complexes were described previously (6). Inshort, cytoplasmic extracts were centrifuged onto a doublesucrose cushion (30% sucrose layered over 45% sucrose inreticulocyte standard buffer) for 1 h at 55,000 rpm in an

SW55 rotor. The vesicular fractions synthesized viral RNAwhen introduced into a cell-free transcription system (6, 24)containing all four nucleotides and an ATP-generating sys-tem. To monitor in vitro RNA synthesis, 50 p.Ci of [5,6-3H]UTP (Amersham) per 40 p.l was added to the transcrip-tion system.

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STRUCTURE OF THE POLIOVIRUS REPLICATION COMPLEX 2741

In some experiments, membranes were dissolved by treat-ing the vesicular fraction with 0.5% sodium deoxycholate(DOC; Difco) for 30 min on ice (5). To test the accessibilityof viral RNA, the native vesicular fraction was treated with100 ,ug of bovine pancreas RNase A (Boehringer Mannheim)per ml for 15 min at 25°C.

Isolation and characterization of viral RNA. In vivo- or invitro-synthesized viral RNA was isolated by the phenol-chloroform-isoamyl alcohol method (23), precipitated withethanol, redissolved in water, adjusted to 10 mM Tris-HCl(pH 7.4)-i mM EDTA-2 mM dithiothreitol-1 U of RNaseinhibitor from human placenta (Boehringer Mannheim) perp.l, and loaded on 5 to 20% sucrose gradients in 10 mMTris-HCI (pH 7.4)-i mM EDTA. The gradients were centri-fuged for 2.5 h at 50,000 rpm in an SW55 rotor and fraction-ated from the bottom, and the radioactivity was measured ina liquid scintillation counter. Sedimentation coefficient stan-dards included 3H-labeled poliovirus 36S RNA isolated frompurified virions (7) and Escherichia coli 16S and 23S rRNAobtained from Boehringer Mannheim.

Biotinylated RNA probes for in situ hybridization. For insitu hybridization on the negatively stained preparations ofvesicles and replication complexes, the RNA probe utilizedfor sectioned material was used (26). In short, from aplasmid (pVR106 [22]) containing full-length polioviruscDNA, nucleotides 670 to 2099 were excised with BamHI,cloned into a pGEM-3 vector (Promega Biotec) between theSP6 and T7 promoters, and linearized with SspI at position1639 of the insert. The sequence between nucleotides 1639and 2099 was transcribed in vitro into RNA of negativepolarity with 50 U of T7 RNA polymerase (BRL) and 1 mMbiotin-11-UTP (Enzo).MAb. Monoclonal antibodies (MAb) against proteins of

the P2 genomic region of poliovirus were characterizedpreviously (3, 19). They recognize an epitope in protein 2Cand its precursors 2BC and P2. They will be referred tohereafter as 2C-MAb. The hybridoma culture supernatantswere used at a 1:2 dilution for immunocytochemistry.EM. Specimens for EM histochemistry, immunocyto-

chemistry (IEM), and in situ hybridization were prepared asdescribed previously (6) by adsorbing the vesicle fractionsonto nitrocellulose (Parlodion)-coated EM grids. The prep-arations were negatively stained with 1% phosphotungsticacid, pH 7.0, before being viewed in a Siemens 102 EMoperated at 100 or 125 kV.

(i) Detection of viral P2 proteins by IEM. For immunocyto-chemical labeling, nonspecific binding sites on the prepara-tions were blocked with normal goat serum and bovineserum albumin. Next, the grids were incubated with 2C-MAb and then with goat anti-mouse antibodies coupled to5-nm colloidal gold (Janssen Pharmaceutica) as describedpreviously (6).

(ii) Detection of RNA with RNase-gold. For RNA detectionwith RNase-gold (2), the same types of specimen and block-ing reaction utilized for IEM were used. To visualize RNA,grids were incubated on RNase coupled to 15-nm gold (E-YLaboratories, San Mateo, Calif.). Care was taken to keep allsolutions as RNase free as possible.

(iii) Double labeling for RNA and P2 protein detection. TheRNase-gold and the 2C-MAb were mixed and applied to thepreparation. After being washed, the grid was treated with5-nm gold-labeled anti-mouse antibody and negativelystained as described above.

(iv) EM in situ hybridization. The vesicle fractions de-scribed above were also used for in situ hybridization, whichwas performed essentially as described previously for ul-

dpm45% ves.

40000

20000

dpm30% ves.

*20000

*10000

30fractions

FIG. 1. RNA content of vesicular (ves.) fractions isolated frompoliovirus-infected HEp-2 cells. The cells were labeled with [3H]uridine from 2.75 to 4 h postinfection, and RNA of the vesicularfractions was analyzed, after phenol extraction, by centrifugationthrough 5 to 20% sucrose gradients. The 30% vesicular fraction (A)contains 36S RNA and RI sedimenting at 27 to 30S. The 45%vesicular fraction (A) contains 36S RNA and a small shoulder(fractions 20 to 22) of presumably RI RNA.

trathin sections (26). Some modifications proved to be es-sential to retain ultrastructural preservation of the native,and thus rather fragile, vesicles. After in vitro transcription,the vesicular fractions were washed once by centrifugationand resuspended in phosphate-buffered saline (PBS). UVcross-linking minimized the loss of RNA in the subsequentsteps. The material was then adsorbed to EM grids, andhybridization was performed for 2 h at 58°C by floating thegrids on 10-,ul drops of a hybridization solution containing4x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate), 13.5% formamide, and the biotinylated riboprobe.After the grids were washed with PBS, biotin detection wasperformed immunocytochemically with anti-biotin antibod-ies coupled to 6-nm gold (AURION, Wageningen, TheNetherlands). As a negative control, an unrelated biotiny-lated RNA probe (vaccinia virus thymidine kinase) wasused.

RESULTS

Content of viral RNA species in two vesicle populations. Thepoliovirus replication complex, attached to vesicular mem-branes, is found in two distinct populations of vesicles (6); itbands in 30 or 45% sucrose and is referred to as the 30 or45% fraction, respectively. We investigated possible differ-ences in content of viral RNA by phenol extraction followedby sucrose gradient centrifugation of in vivo [3H]uridine-labeled RNA. Figure 1 shows that the 30% fraction contains36S genomic RNA in addition to RI sedimenting at 27 to 30S.The 45% fractions contained largely 36S genomic RNA anda small amount of more slowly sedimenting RNA of presum-ably RI configuration.RNase treatment after phenol extraction indicated that in

both vesicle preparations, no double-stranded RNA was

detectable (not shown). If the vesicle preparations were

treated with RNase A before phenol extraction (Fig. 2), the36S RNA in the 30% fraction was found to be fully accessibleto the enzyme, whereas the RI was protected. In the 45%fraction, about two-thirds of the 36S RNA was found to beprotected against RNase (compare the corresponding 36S

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2742 BIENZ ET AL.

dpm36 - 23 - 16S

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FIG. 2. RNase treatment of a 30% vesicular fraction (A) digestspractically all of the 36S RNA but leaves the RI intact. RNasetreatment of a 45% vesicular fraction (A) digests about one-third ofthe 36S RNA, leaving the remainder intact because it is encapsi-dated (data not shown). RNA was analyzed as described in thelegend to Fig. 1.

peaks in Fig. 1 and 2). The protected RNA of the native,non-phenol-extracted 45% fraction sedimented at 150S (datanot shown) and thus was encapsidated in virions.

Transcriptional activity of the 30 and 45% fractions in vitro.The amount and type of RNA synthesized in vitro weremonitored for both vesicular preparations. The 30% frac-tions incorporated [3H]UTP at first into RI and graduallyalso into 36S RNA (Fig. 3). The 45% fractions, whichcontained predominantly 36S RNA, showed kinetics of RIand 36S RNA synthesis in vitro (Fig. 4) similar to those ofthe 30% fraction. After approximately 60 min, both systemsbecame exhausted. The in vitro-synthesized 36S RNA of the30% fraction as well as that of the 45% fraction was found tobe fully sensitive to RNase, whereas the RI of both fractionswas protected (not shown). Thus, during in vitro transcrip-tion, both fractions react to RNase treatment in a mannersimilar to that of freshly isolated 30% fraction.The combined data from Fig. 1 to 4 are interpreted to

0 1 0 20 30fractions

FIG. 3. In vitro RNA synthesis of a 30% vesicular fraction for 15min (*), 30 min (-), 45 min (A), and 60 min (-). [3H]UTP isincorporated first into RI and gradually also into 36S RNA. RNAwas analyzed as described in the legend to Fig. 1.

dpm40000 36S

30000-

20000-

10000

00 10 20 30

fractions

FIG. 4. In vitro RNA synthesis of a 45% vesicular fraction for 15min (O), 30 min (O), 45 min (A), and 60 min (0). The kinetics andpattern of plus-strand RNA synthesis are very similar to thoseobtained with 30% fractions. RNA was analyzed as described in thelegend to Fig. 1.

mean that the 30% fraction, since it contains RI, synthesizesRNA in vivo and continues the synthesis in vitro, whereasthe 45% fraction seems to restart its plus-strand RNAsynthesis in vitro. Whether this happens as elongation, e.g.,on a small amount of RI (trailing shoulder in Fig. 1) or asinitiation on RF or RI cannot be determined at present (seeDiscussion).

Ultrastructural aspects of the 30 and 45% vesicles. Asstated earlier (6), the poliovirus replication complex is func-tional only if, by means of viral protein 2C, it is associatedwith the membranes of the virus-induced vesicles. In thecurrent investigation, we found that the virus-induced vesi-cies are not the only membrane system associated with thereplication complex. A second type of membrane was ob-served in the replication complex of both vesicular fractions(Fig. 5). These membranes are tightly packed and appear assmall (approximately 50-nm), clustered, vesiclelike bodies(compact membranes). Like the membranes of the virus-induced vesicles, these compact membranes were labeledwith 2C-MAb (Fig. 5). The label was found predominantly atthe border of the replication complex at the sites where thereis intimate contact between the compact membranes of thereplication complex and the membranes of the virus-inducedvesicles.The ultrastructural aspects of the compact membranes

within the replication complex are the same in the 30 and45% fractions and also do not change during in vitro tran-scription. In ultrathin sections of poliovirus-infected cells,the compact membranes appear tightly coiled and do nothave a clearly defined shape (data not shown).DOC treatment of vesicular fractions. To test whether the

replication complex-associated membranes described aboveare involved in the observed protection of the RI, a freshlyisolated, RI-containing 30% fraction was treated with DOCfor 30 min at 0°C. This rendered the RI partially RNasesusceptible, as the RI could now be converted by RNase intoan 18S core (10) (Fig. 6). After phenol extraction, the 18Score was RNase sensitive (data not shown), which indicatesthat this 18S structure is derived from the RI (and not fromthe RF) and that it is enclosed in presumably proteinaceousmaterial.Together with the findings illustrated in Fig. 2, these

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STRUCTURE OF THE POLIOVIRUS REPLICATION COMPLEX 2743

~~~U1

FIG. 5. Electron micrograph of a poliovirus replication complex surrounded by virus-induced vesicles (V) and containing a second, compact

membrane system (arrowheads). Immunocytochemical labeling with 2C-MLAb and 5-nm colloidal gold shows that both membrane types are

labeled. The micrograph shows a 45% vesicular fraction. No morphological differences from the 30% fractions could be found. Bar, 100 nm.

findings indicate that the mature 36S RNA is located outsideof the replication complex, whereas the nascent plus strandsof the RI are sequestered by membranes. DOC treatmentconverts the replication complex into a membrane-deficient

dpm 36 - 23- 16S

200 -_ _ _ - -

20000 t U

0 1 0 20 30fractions

FIG. 6. RNase treatment of a 30%vesicular fraction digests only36S RNA and leaves the RI intact (A) (Fig. 2). The RI can bedigested to an 18S core only after DOC treatment (-), indicatingthat membranes protect the growing plus strands. RNA was ana-lyzed as described in the legend to Fig. 1.

matrix containing the RI with protruding, and thus RNase-sensitive, nascent RNA strands.

Since the 30 and 45% fractions are morphologically and invitro also functionally identical, the function(s) of the repli-cation complex-associated membranes during in vitro viralRNA synthesis was investigated with 45% vesicular frac-tions.DOC was added to an in vitro transcription system after 30

min of ongoing in vitro RNA synthesis. During further RNAsynthesis, the amount of radioactivity increased in the RI,whereas the amount of 36S RNA remained stable at the levelreached when DOC was added (Fig. 7). Thus, it can beconcluded that the membranes of the transcriptionally activereplication complexes not only sequester the nascent plusstrands but also are involved in their detachment in becom-ing 36S RNA.

Ultrastructural analysis of replication complexes aftertreatment with DOC shows that the virus-induced vesicles aswell as the compact vesiclelike structures within the repli-cation complex have completely disappeared (Fig. 8). Thisindicates that these compact structures indeed consist ofmembranes. The membrane-deficient matrix of the replica-tion complex still contains P2 protein at its periphery.

Role of membranes and location of progeny virus RNA inthe replication complex as revealed by RNase-gold histochem-istry and in situ hybridization. The observations presented sofar indicate that the replication complex is a membrane-containing structure that encloses the RI tightly and is

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2744 BIENZ ET AL.

dpm36 23 - 16S

30000 - l l

20000-

10000 L

0'0 10 20 30

fractionsFIG. 7. In vitro RNA synthesis of a 45% vesicular fraction was

allowed to proceed for 30 min, and RI and 36S RNA were made (A).To a parallel tube (O), DOC was added after 30 min of in vitrosynthesis, and the reaction was allowed to proceed for another 15min; only RI continued to incorporate [3H]UTP, whereas radioac-tivity did not increase in the 36S moiety. RNA was analyzed asdescribed in the legend to Fig. 1.

surrounded by a rosette of virus-induced vesicles. On thebasis of the ultrastructural data (Fig. 5), it seemed to us thatthe resistance of the nascent plus-strand RNA against RNasereported above might be mediated not by the rosettes oflarge, virus-induced vesicles (which do not seem to be in anextremely tight arrangement) but by the densely packedcompact membranes within the replication complex.To identify the membrane system protecting the RI and at

the same time to obtain more information about the topog-raphy of the replication complex, we explored the location of

the 36S RNA after in vitro RNA synthesis, adopting thefollowing rationale. Since only the 36S RNA and not the RIwas found to be accessible to RNase, only the 36S RNA canbe located by histochemistry and in situ hybridization. Theobserved location of the 36S RNA would then be indicativeof the tightness of the two membrane systems.

In Fig. 9, RNA in general was identified by a histochem-ical reaction using RNase coupled to colloidal gold. In Fig.10, viral 36S plus-strand RNA was localized specifically byin situ hybridization with a biotinylated riboprobe comple-mentary to a part of the P1 genomic region. Both reactionsclearly show their respective targets located within therosette of the virus-induced vesicles, thus demonstrating therather loose and in any case permeable configuration of thevesicular rosette and suggesting that the central compactmembranes are the protecting agents of the RI. It is note-worthy that the 36S RNA is set free at several distinct sitesat the periphery of the replication complex. This can beexplained by assuming a topography of the viral RNAreplication in which the template moves through the repli-cation complex and has the progeny RNA strands fixed atdefined sites.

DISCUSSION

From poliovirus-infected cells, two populations of mem-brane-bound replication complexes can be isolated. Onepopulation bands in 30% sucrose and actively synthesizes36S plus-strand RNA in vivo and in vitro. The fractionbanding in 45% sucrose contains capsid precursors andvirions (20) and besides encapsidated and free 36S RNAcontains little, if any, RI. In vivo, the 45% fraction istherefore considered to be transcriptionally silent ("burntout") but contains progeny RNA in the process of becomingencapsidated. In vitro, it is capable of resuming RNA

I ..*, ..4~f V

-kN .

.0-

FIG. 8. Electron micrograph of a DOC-treated replication complex (45% fraction). The membranes of the virus-induced vesicles as wellas the compact membranes within the replication complex are dissolved. The immunocytochemical label with 2C-MAb is at the surface ofthe core structure of the replication complex. Bar, 100 nm.

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STRUCTURE OF THE POLIOVIRUS REPLICATION COMPLEX 2745

FIG. 9. A vesicle (V)-bound replication complex (RC) as in Fig.5 but doubly labeled with 2C-MAb (small gold grains) and withRNase-gold (large gold grains). The 36S RNA is located between thevirus-induced vesicles and the replication complex. Bar, 100 nm.

synthesis and producing 36S RNA (Fig. 4). It is not clearwhether in vitro the plus-strand RNA is elongated only on asmall amount of RI or whether there is true initiation, whichcould take place either on preexisting RI or on RF, whichcould already be present in small quantities in vivo or couldhave been freshly synthesized in vitro.The two types of viral replication complexes show similar

ultrastructural features. They are enclosed in a rosettelikeshell of virus-induced vesicles, which are attached to thereplication complex by means of viral protein 2C (6). In thepresent investigation, we identified a second, very compactmembrane system within the replication complex itself (Fig.5). It encloses tightly the RI, which in concert with viralproteins represents the actual plus-strand RNA-synthesizingmachinery. This was shown by our DOC experiments:removal of all membranes with DOC leaves the core of thereplication complex, which still incorporates [3H]UTP. Thefinding that under these conditions [3H]UTP was incorpo-rated only into RI and not into 36S RNA indicates thatelongation takes place but that no completed 36S RNA isreleased. The elongation proceeds for quite some time, albeitat a reduced rate compared with that of non-DOC-treatedreplication complexes or intact cells.

If DOC-treated cores of replication complexes devoid ofmembranes are treated with RNase A, the nascent plusstrands at the RI are exposed to the action of the enzyme,whereas the partially double-stranded template with thereplication forks remains protected from the RNase by asubstance removable by phenol. RNase treatment of non-DOC-treated, transcriptionally active replication complexes

FIG. 10. A vesicle (V)-bound replication complex (RC) (45% fraction) on which in situ hybridization with a biotinylated RNA probe wasperformed. The probe is visualized with 6-nm gold-coupled anti-biotin antibodies. The hybridization signal, localizing the 36S RNA, is foundon the surface of the replication complex but within the rosette of the virus-induced vesicles. (Inset) Higher magnification. The hybridizationsignal is found where the compact membranes (CM) of the replication complex are in close contact with the virus-induced vesicles. Bars, 100nm.

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2746 BIENZ ET AL.

digests only 36S RNA while leaving the entire RI intact. Thismeans that freshly completed 36S RNA is rapidly set free.Since the completed 36S RNA is found by RNase-gold aswell as by in situ hybridization within the rosette of virus-induced vesicles but outside the replication complex per se,it is possible that the compact membranes within the repli-cation complex have to interact with the virus-inducedvesicles for the detachment of the 36S RNA from thetemplate.At the same site of this putative interaction, the presence

of protein 2C (and the other 2C-containing viral P2 proteins)could consistently be demonstrated by immunocytochemis-try (Fig. 5 and 9). Its presence in the replication complexeven after DOC treatment indicates that it is not onlymembrane associated but also bound to other components.This is in agreement with the findings that it is RNAassociated and that it attaches viral RNA to the surface ofthe virus-induced vesicles (6). The 2C-RNA association isconsistent with the finding that 2C contains NTP-binding (8)as well as helicase (12) motifs. It might well be that 2C is alsoinvolved in the liberation of the RNA from the replicationcomplex and possibly in preparatory steps for encapsidation(16).Whatever the exact functions of P2 proteins 2BC, 2B, and

2C might ultimately be, the action of one or more of themwas found to be essential for viral plus-strand RNA synthe-sis (6, 13, 15); for hepatitis A virus (9, 14), rhinovirus (17),and poliovirus (21) replication in cell culture; and for expres-sion of cytopathic effects (5). Since these P2 protein-medi-ated processes are interconnected (4), one might speculatethat these proteins do not function on their own but functiononly in concert with each other and/or other viral or evencellular proteins. This would also explain why, despitenumerous attempts, e.g., by mutational analysis, it is still notpossible to attribute a biochemically defined role to each ofthese proteins.

In our in vitro system, encapsidation of progeny RNAcould not be observed. This is in contrast with a recentlypublished report (18) and might be due to several experimen-tal differences. Our in vitro system is a transcription system,not a coupled transcription-translation system, and it waskept going for a much shorter time. An important aspect ofthis study could be the finding that the 36S RNA is set freefrom the replication complex immediately after its comple-tion. This is in agreement with the finding, demonstrated byin situ hybridization on infected cells, that a pool of freegenomic RNA was present in the vicinity of the replicationcomplex (27). In the in vitro system, this RNA then might belost for encapsidation, because the RNA-synthesizing enti-ties are rather diluted in vitro compared with the in vivosituation.The data presented here allow us to propose the following

model of the poliovirus replication complex. The replicationcomplex contains a compact membrane system which en-closes the RI with its nascent plus-strand RNA. The repli-cation forks are in addition tightly enclosed in protein. Thecompact membranes are involved in the liberation of thecompleted 36S RNA from the RI and from the replicationcomplex. The progeny strands are set free at different pointson the replication complex's surface, i.e., at the sites wherethe replication complex is attached to the virus-inducedvesicles. During RNA synthesis, the RI would thus have tomove (turn) through, or together with, the replication com-plex with respect to the virus-induced vesicles.

ACKNOWLEDGMENTSWe thank Bea Herzog for excellent technical assistance.This work was supported by grant 31-29910.90 from the Swiss

National Science Foundation. M.T. was supported by a grant fromthe Freiwillige Akademische Gesellschaft Basel, Basel, Switzer-land.

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