Vol. 66, No. 8

JOURNAL OF VIROLOGY, Aug. 1992, p. 5040-5046

0022-538X/92/085040-07$02.00/0 Copyright © 1992, American Society for Microbiology

A Poliovirus Replicon Containing the Chloramphenicol Acetyltransferase Gene Can Be Used To Study the Replication and Encapsidation of Poliovirus RNA NEIL PERCY, WENDY S. BARCLAY, MICHAEL SULLIVAN, AND JEFFREY W. ALMOND* Department of Microbiology, University of Reading, Whiteknights, P. O. Box 228, Reading RG6 2AJ, United Kingdom Received 28 February 1992/Accepted 10 May 1992

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation ofvirus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.

The genome of poliovirus is a single-stranded positivesense RNA molecule of approximately 7,500 nucleotides. It has a virus-coded protein, VPg, covalently attached to its 5' terminus and a poly(A) tract at its 3' terminus. The body of the RNA comprises a 5' noncoding region (NCR) of 743 nucleotides, a large open reading frame encoding a virus polyprotein with an Mr of 220,000 and a 3' NCR of 72 nucleotides (10, 21, 24). The open reading frame is divided into 3 regions on the basis of the primary posttranslational cleavages of the virus-coded polyprotein. Thus, the P1 region encodes the structural proteins of the virus capsid and the P2 and P3 regions encode the nonstructural proteins, which include two proteases and an RNA polymerase (10). The 5' NCR of the poliovirus genome is highly conserved in primary and secondary structures, suggesting that it plays a central role in virus replication (26). Indeed, it has been demonstrated that the central approximately 300 nucleotides promote the internal entry of ribosomes required for capindependent translation (18). In addition, the 5'-terminal 100 nucleotides have been shown to form a cruciform structure essential in the plus strand for RNA synthesis (1). Although the replication of poliovirus RNA has been successfully studied in classical genetics, these studies have been limited by the need to generate infectious virus particles. Nowadays, highly infectious RNAs can be generated from cloned cDNAs in vitro (27), so that it is possible to study a single cycle of replication of mutated genomes created by reverse genetics. Using these methods, Kaplan and Racaniello (9) constructed a series of subgenomic poliovirus replicons from which large regions of P1 were deleted. These experiments demonstrated that the P1 region is not essential for genome replication. This conclusion is in agreement with studies on naturally occurring (8) and artificially engineered (7) defective interfering (DI) particles which also suggest that both the P2 and the P3 regions must be con*

Corresponding author.

served and translated for deleted RNAs to retain replication competence. In principle, similar transfection methods could be used to characterize further the role of the 5' NCR in virus replication following site-specific mutagenesis. In practice, however, mutations which severely affect translation or replication are difficult to assay over a single cycle, especially when complementing helper virus is present. Furthermore, it is often impossible to recover RNAs which are poor replicons as DI particles because of their inability to compete with helper virus. Such problems could be partly overcome by the insertion of a reporter gene into the mutated poliovirus replicon, which would allow it to be distinguished unambiguously from helper virus and would enable its replication to be monitored by a simple enzyme assay. Our principal objective in the present study was to determine whether a foreign gene, chloramphenicol acetyltransferase (CAT), could be inserted into a nonessential region of the poliovirus genome and replicated and expressed following transfection. We then exploited the CAT-containing genome to investigate the role of certain 5' NCR stem-loop structures (20, 22, 23) in virus replication. Finally, we determined whether the CATcontaining genome could be packaged efficiently into the capsids of superinfecting helper virus. We propose that the poliovirus replicon described here will be of value in studying the replication and packaging of poliovirus RNA.

MATERIALS AND METHODS Construction of plasmids. The plasmid pT7FLC contains a full-length infectious cDNA of the poliovirus type 3 genome in a modified pBR322 vector. The virus sequences are derived from a clone of poliovirus type 3 P3/Leon/37 (pOLIO Leon [25]), except that the sequences between N278 and N2766 were taken from a full-length clone of Sabin poliovirus type 3 (28), in which the SstI site at N1900 had been removed by mutagenesis (unpublished data). In addition, the AatII site of pBR322 (N4286) had been replaced by a NotI 5040

POLIOVIRUS CAT REPLICON

VOL. 66, 1992

T7 a

CAT

ampp4K1 pT7FLC/REP

I

ori Sal

b CAT

P2

GG5'NCR IF77 i

P3 3'NCR A(30)

inframe fusion c

CAT M

G

T

AO

G

Y

ATG GGA GCT CAA ATC ACT GGA TAT 756

IUS OF POLIO-CAT FUSION

NTEMI

N-TERMIN

743

VP3

CAT

S D N H G G A T CM GGA GGT GCG ACG TCA GAC MC CAC 0

C-TERMIN

IUS OF CAT-POLIO FUSION

1805

POLIOVIRUS-CAT REP'LICON FIG. 1. (a) The structure of plasmid pT7FLC/REP. The 17 promoter (black triangle) precedes a full-ler igth copy of the poliovirus type 3 genome (filled) into which has been inserted the gene encoding CAT (hatched region) between po isitions 743 and 1805. (b) The RNA produced in vitro following transc -ription of pT7FLCIREP linearized with SalI. (c) The sequences at thi e N terminus of the CAT protein and the CAT/VP3 boundary after in isertion of the CAT gene into the P1 region of the poliovirus genonne in plasmid pT7FLC/ REP. At the N terminus of the CAT gene,, a unique SstI site was created. This allowed the CAT gene to be iinserted on an SstI-AatII

Ppoly-

fragment so that the first four amino acids of the poliovirus protein replaced those of CAT. At the C terminus of CAT, the termination codon was replaced by the sequ ience encoding Gin-Gly.

site. These two modifications result! ed in a vector with unique NotI and SstI sites. CAT sequeinces for insertion into pT7FLC were derived from the vecto irpRSVCAT (6), mutated such that an SstI site was introduiced at the 5' terminus of the CAT gene. These manipulatic )ns resulted in three amino acid changes at the N terminu,s of the CAT protein (Fig. lc). Mutations were also introc luced to remove the termination codon, to incorporate a 3ln-Gly cleavage site toward the carboxyl end of the CAT protein, and to introduce an AatII site at the 3' end of the CAT gene (Fig. lc). The CAT sequences were then introdiaced into pT7FLC by replacing the SstI-AatII fragment by thte modified CAT gene to generate plasmid pT7FLC/REP (Fiig. la). Plasmid pT7pol-FLC/REP, which c:arries a 200-bp deletion in 3D pol, was constructed by dilgesting pT7FLC/REP DNA with the unique enzymes XbaI aind XhoI, blunt ending the vector fragment, and religating All derivatives of pT7FLC and pT7FLC/REP harboring mutations in the 5' NCR were generated by site-directead mutagenesis of a

5041

NotI-SstI subclone of the 5' NCR in M13. Mutations were ligated into the parent plasmids via the unique NotI and SstI sites. Cells and viruses. Ohio HeLa cells were grown in Eagle's minimal essential medium (EMEM) containing 5% fetal calf serum. For plaque assays, 6-well plates were inoculated with 200 ,l of diluted virus and overlaid 30 min later with EMEM plus 2.5% fetal calf serum and 2% agarose. After incubation at 34°C for 3 to 4 days, the cell monolayers were stained with crystal violet. For superinfection studies, confluent 6-cm dishes of Ohio HeLa cells were infected with 200 ,ul of diluted virus to achieve a multiplicity of infection of approximately S PFU per cell. After 30 min, the inoculum was removed and replaced with EMEM. Analysis of RNA by dot-blot hybridization. Transfected cells were harvested and lysed by being resuspended in a solution containing 0.1 M NaCl, 0.1 M Tris (pH 7.5), and 1% Nonidet P-40 on ice for 15 min. Cell debris and nuclei were removed by centrifugation, and the supernatants were extracted with an equal volume of phenol-chloroform. After precipitation with ethanol, the nucleic acid was collected by centrifugation, resuspended in water, and combined with an equal volume of 10x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 17% formaldehyde. The RNAs were then heated at 65°C for 15 min, dotted onto presoaked nylon filters (Hybond N; Amersham), and cross-linked by UV irradiation. The filters were then prehybridized for 2 h in 5 x SSPE (lx SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]), 50% deionized formamide, 5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS) containing 500 ,ug of denatured salmon sperm DNA. For generation of a CAT-specific probe, 100 ng of a 510-bp SstI-NcoI fragment from pT7FLC/REP was labelled with [a- P]CT? (50 ,uCi; Amersham). The probe was denatured by boiling and hybridized to filters for 8 to 18 h at 50°C. The filters were washed at 65°C with diluted SSPE containing 0.5% SDS until no further radioactivity was detected in the wash buffer and then air dried and subjected to autoradiography. Analysis of [3SJmethionine-labelied polypeptides. Transfected or infected Ohio HeLa cells on 60-mm dishes were labelled at 4 to 6 h postinfection by adding 1 ml of methionine-free EMEM containing 50 ,uCi of [35S]methionine. At 6 h, the cells were harvested by scraping and lysed in 1 ml of radioimmunoprecipitation assay buffer and cell debris was removed by centrifugation. To 400 Rl of this extract, 4 ,u of a 1:10 dilution of anti-CAT rabbit polyclonal antibody (SPrime-3Prime Inc.) was added, and the mixture was incubated at 37°C for 1 h. A 100-,u volume of a 10% solution containing prewashed Staphylococcus aureus cells (Pansorbin) was added, followed by incubation for 1 h further at 37°C and washing three times with radioimmunoprecipitation assay buffer. After being pelletted, the mixtures were boiled in 20 ,ul of Laemmli buffer for 3 min and analyzed by autoradiography following electrophoresis on an SDS-polyacrylamide gel (12% acrylamide). Analysis of CAT enzyme activity. Transfected cell pellets were resuspended in 100 pl of Tris-HCl (pH 7.8) and lysed by rapid freeze-thawing. After centrifugation at 13,000 rpm for 15 min, 2 ,ul of the supernatants was used to measure protein concentration with a Coomassie protein assay kit (Pierce). Samples containing 50 ,ug of protein were then incubated with 0.1 ,uCi of [14C]chloramphenicol (50 mCi/ mM) and 0.5 mM acetyl coenzyme A for 2 h. Labelled materials were extracted with 1 ml of ethyl acetate, concen-

5042

J. VIROL.

PERCY ET AL.

DNA 0e*

C

*: 2 4 6 8 24 10

REP

FIG. 2. Replication of FLC/REP RNA. Total cytoplasmic RNAs isolated at various times (2 to 24 h) after transfection with in vitro transcripts of pT7FLC/REP (REP) or from untransfected cells (C). An aliquot of each RNA was spotted onto nylon filters and detected by hybridization to a CAT-specific probe. pT7FLC/REP DNA (DNA) was also spotted onto the filter as a positive hybridization control in 10-fold dilutions ranging from 10 to 0.001 ng (left to right).

were

trated by vacuum-drying, and subjected to thin-layer chromatography with a solvent of chloroform-methanol at 95:5 (vol/vol). Thin-layer chromatography plates were air dried and autoradiographed. RESULTS

Construction of the replication plasmid pT7FLC/REP. On the basis of the structures of naturally occurring defective interfering particles (DIs) of poliovirus (7, 8), the plasmid pT7FLC/REP was designed to retain sequences essential for virus RNA replication and to incorporate the foreign marker gene, CAT. The construction was based on the plasmid pT7FLC, which contains a full-length cDNA of poliovirus P3/Leon/37 (25) under the control of a T7 promoter. When pT7FLC was linearized by digestion with restriction endonuclease Sall, transcription by T7 polymerase in vitro produced a genomelike RNA molecule with 2 additional guanosine residues at its 5' terminus and a poly(A) tail of 30 residues at its 3' terminus (Fig. lb). This RNA has a specific infectivity of 105 PFU/,ug following transfection of Ohio HeLa cells (data not shown). Plasmid pT7FLC/REP was constructed from pT7FLC, with the 657 bases of the CAT coding sequence inserted in frame in place of the 1,049 bases of the P1 region between 756 and 1805 encoding proteins VP4, VP2, and part of VP3 (Fig. lb). The initiation codon and codons 2 to 4 of the poliovirus polyprotein were retained and replaced by amino acids 1 to 4 of CAT, but otherwise the CAT protein forms the amino terminus of the polyprotein. The RNA transcribed from pT7FLC/REP was 408 bases (5%) shorter than full-length poliovirus RNA. This is well within the size range of naturally occurring DIs, which have deletions of between 4.2 and 13.2% of the total genome (8). FLC/REP RNA is amplified. To determine whether RNA transcribed from pT7FLC/REP could act as a replicon, T7 transcripts were transfected onto Ohio HeLa cells, and at various times posttransfection total cytoplasmic RNA was extracted and hybridized to a radiolabelled CAT-specific DNA probe (see Materials and Methods). Figure 2 shows that the amount of CAT-containing RNA increases with time over a 24-h period following transfection. A similar result was obtained if a poliovirus-specific probe was used (data not shown). No increase above base levels of CAT-containing RNA could be detected in cells transfected with replication-defective T7 transcripts (see below). These results illustrate that the CAT gene is replicated when inserted into

the P1 region of a replication-competent genome, presumably by poliovirus RNA polymerase. A functional CAT protein is expressed from FLC/REP RNA. The CAT gene in FLC/REP is positioned so that the CAT protein will form the amino terminus of the polyprotein produced in transfected cells. Since the FLC/REP RNA replicates, it seemed likely that processing of the P2 and P3 proteins would occur normally, but the extent of P1 protein processing could not be predicted. Processing of P1 protein into the component capsid proteins occurs at certain of several Gln-Gly motifs (10) and is effected by the viral protease, 3CD (30). A Gln-Gly motif was incorporated very close to the C terminus of the CAT protein of pT7FLC/REP (Fig. lc), but because of the replacement of VP4, the myristoylation signal was lost. Analysis of myristoyl-deficient mutants has suggested that myristoylation of the polyprotein is necessary for processing (11), although Moscufo et al. (16) have described a mutant with reduced levels of myristoylation that processed P1 normally in vivo. It was therefore of interest to investigate the extent of P1 processing of the FLC/REP polyprotein. The predicted Mr of the unprocessed P1 from FLC/REP is 84,000, whereas P1 produced during infection with P3/Leon/37 has an Mr of 97,000. Processing of the FLC/REP P1 protein would give rise to proteins with Mrs of 51,000 and 34,000 if processing occurs only at authentic poliovirus cleavage sites and proteins with Mrs of 25,000, 26,000, and 34,000 if the Gln-Gly motif near the CAT/VP3 boundary is also used (Fig. 3A). Cells transfected with FLC/REP RNA were therefore pulse-labelled with [35S]methionine, and cytoplasmic extracts were immunoprecipitated with a rabbit polyclonal anti-CAT serum. Polyacrylamide gel electrophoresis revealed a band with an Mr of approximately 51,000 from cells containing FLC/REP but not from poliovirus-infected or mock-infected cells (Fig. 3B). This suggests that the FLC/REP P1 protein is cleaved to produce a CAT-containing fusion protein, probably CATVP3 and VP1, presumably by protease 3CD, but that the Gln-Gly motif near the junction of the CAT and VP3 proteins is not cleaved (Fig. 3A and B). The CAT-VP3 fusion protein was then monitored for enzymic activity by the standard CAT assay. Figure 4 shows the increasing levels of CAT activity present in equal amounts of cell extracts over time, reflecting the increase in levels of RNA presented in Fig. 2. On the basis of a visual comparison with dilutions of a known amount of CAT enzyme, the 10-h sample was estimated to contain approximately 2 U of CAT activity per mg of total extracted cellular protein (Pierce protein assay kit). Thus, the CAT-VP3 fusion protein is enzymatically functional and is expressed at high levels in transfected cells. To demonstrate that the level of CAT activity was dependent on replication of FLC/REP RNA, a polymerase-deficient mutant, pol-FLC/REP, which has an intact 5' end and is therefore translation competent but carries a 200-nucleotide deletion in polymerase 3D, was transfected into Ohio HeLa cells. No CAT activity was detectable after transfection of pol-FLC/REP RNA, even after 10 h. In addition, CAT activity was not detected after transfection of a T7 polymerase reaction mixture from which the enzyme had been omitted, indicating that template DNA alone could not give rise to functional enzyme under the conditions described. CAT assays can be used to study RNA replication of FLC/REP mutants. Since the increase in CAT activity correlates well with the increase in FLC/REP RNA in the cell following transfection, the enzyme assay can be used to

VOL. 66, 1992

POLIOVIRUS CAT REPLICON

5043

A T

25kDa

T

26kDa

CAT

a

34kDa

t

VP1

VP g!n gly

v

51kDa

b

CAT

VP3

l

34kDa

v

VP1 CAT

v

85kDa C

CAT

f

VP3

VP1

B

2

4

6

10

8

24

C

FIG. 4. CAT enzyme activity produced from FLC/REP. Cytoplasmic proteins were extracted at various times (2 to 24 h) after transfection with in vitro transcripts of pT7FLC/REP or from untransfected cells (C), and 50 Fg was analyzed for CAT enzyme activity as described in Materials and Methods. A 1-U amount of commercial CAT (CAT) was included.

after transfection of the various mutant RNAs should indicate an effect of the mutation on RNA replication. Figure 6 shows the CAT activity produced by each mutant. It can be M

1

2

664536-

CAT-CA/VP3 sA. 11.-l

that deletion of stem-loops designated L (N10-34) or A (N51-78) in mutants L- and A-, respectively (Fig. 5), result in a complete loss of CAT activity, suggesting that these structures are essential for RNA replication. Inversion of the whole of stem-loop L (mutant L-inv) resulted in a replicon producing a much-reduced level of CAT activity, whereas inversion of the stem only, leaving the loop intact (mutant L-sinv), had little effect. These results suggest that the primary nucleotide sequence of the loop but not the stem is important for replication of the RNA. Deletion of the loop N188-222 (mutant 220-) had little effect on CAT activity compared with the wild-type FLC/REP, suggesting that this stem-loop is not essential for RNA replication. To confirm these conclusions on the role of stem-loops, each of the mutations was then built into the infectious full-length poliovirus cDNA, pT7FLC, and the viability of the mutant genomes was tested by direct plaque assay of the transfected RNAs. Repeated transfection of mutant RNAs L- or Afailed to produce infectious virus, confirming that these deletions are lethal. Mutants L-sinv and 220- produced virus with a normal plaque size phenotype. Mutant L-inv produced a virus with a minute plaque size, as would be expected from the level of CAT activity produced from the corresponding replicon (Table 1). FLC/REP RNA is packaged into poliovirus capsids. Except for the inclusion of the CAT gene, the FLC/REP replicon is seen

3

!T

29-.

242014-

FIG. 3. CAT expression from FLC/REP RNA. (A) Predicted cleavage products of P1 translated from pT7FLC/REP RNA. Cleavage at the P1/P2, VP3/VP1 boundaries and at the engineered Gln-Gly site at the C terminus of CAT would liberate three proteins with Mrs of 25,000, 26,000, and 34,000 (a), whereas cleavage only at the P1/P2 and VP3/VP1 boundaries would liberate two proteins with Mrs of 51,000 and 34,000 (b). The uncleaved P1 precursor with an Mr of 85,000 (c) is also shown. (B) A CAT-specific protein with an Mr of 51,000 (CAT-VP3 fusion) indicated by the arrow is present in extracts of cells transfected with FLC/REP RNA (lane 3) but is absent from mock-transfected (lane 1) or virus RNA-transfected (lane 2) cell extracts, as detected by immunoprecipitation with anti-CAT rabbit polyclonal antisera and polyacrylamide gel electrophoresis.

RNA replication. A series of mutations of FLC/ REP containing modifications or deletions in the first 250 bases of the 5' NCR were therefore constructed. These mutations were based on the secondary-structure model proposed by Skinner et al. (23) (Fig. 5) and were designed to assess the role of individual stem-loops in poliovirus RNA replication. We first obtained evidence that the mutations did not affect the translatability of the RNA by placing the modified 5' NCRs downstream of a reporter gene in a bicistronic mRNA (19) and by assaying the amount of CAT produced from monocistronic RNAs in vivo (unpublished data). Therefore, any differences in CAT enzyme activity

0

220

DI

measure

A

DII DIII

FIG. 5. The secondary structure of the 5' NCR of poliovirus type 3 as proposed by Skinner et al. (23). The stem-loops referred to in the text as L (N10-34), A (N51-78), and 220 (N188-222) are indicated.

J. VIROL.

PERCY ET AL.

5044

B

A .5

a

* CAT

Fl CR

*.o_

* *

LJ R

A-R

L R

L R

220-R

FIG. 6. CAT enzyme activity produced from 5' NCR mutants of FLC/REP. A 50->xg amount of cytoplasmic proteins extracted 10 h after transfection with 5' NCR mutants of FLC/REP was analyzed for CAT enzyme activity as described in Materials and Methods. The positions of individual mutations are as described in Table 1.

similar in structure to the genome of a poliovirus DI particle. In order to be propagated, DI genomes require capsids provided in trans. It was therefore of interest to examine whether FLC/REP could be packaged into poliovirus capsids provided by helper virus. Cells were therefore superinfected with 5 PFU of virus per cell at 4 h posttransfection with RNA from pT7FLC/REP and incubated overnight. Supernatants were then treated with RNase A (1 mg/ml) to digest any CAT-containing RNA which was free in solution and used to infect fresh cells. Cytoplasmic extracts from these cells contained abundant CAT activity, indicating that they had been infected by encapsidated FLC/REP (Fig. 7). No CAT activity was detected following infection with supernatants from cells which did not receive helper virus. Similarly, CAT was not detected if the supernatants were incubated with a poliovirus type 3 neutralizing antibody or if the cells were pretreated with an anti-poliovirus receptor antibody, monoclonal antibody 303 (15). Thus, infection of cells by FLC/REP was mediated by poliovirus helper capsids infecting via the poliovirus cellular receptor, indicating that the CAT-containing RNA was efficiently packaged. Moreover, the poliovirus capsids containing FLC/REP genomes could be passaged at least four times by infecting fresh monolayers with RNase A-treated supematants. The CAT activity remained high and approximately constant with each passage (Fig. 8). Titration of the multiplicity of virus used for superinfection indicated that efficient propagation of FLC/REP required coinfection of every transfected cell with helper virus. We therefore routinely used 5 PFU per cell. The efficiency of propagation of FLC/REP declined significantly when the multiplicity of superinfection was 0.1 PFU per cell or less (data not shown).

CAT

-0

+PV +PV + anti-PV

CAT

+PV +303

+PV

FIG. 7. Encapsidation of FLC/REP RNA. Supernatants from cells transfected with FLC/REP RNA and superinfected without (+0) or with (+PV) poliovirus type 3 were treated with RNase A (1 mg/ml) and used to infect fresh monolayers. CAT enzyme produced from 50 ,ug of cytoplasmic extract 8 h after infection was analyzed. (A) CAT activity produced after infection with supernatants from cells which had received FLC/REP and poliovirus pretreated with a polyclonal anti-poliovirus type 3 neutralizing antiserum (+PV +anti-PV); (B) CAT activity produced from cells pretreated with an anti-poliovirus receptor monoclonal antibody (MAb 303) before infection with supernatants from cells which had received FLC/REP and poliovirus (+PV +303).

DISCUSSION A poliovirus genome containing a foreign gene in place of a region encoding capsid proteins has been shown to replicate autonomously in cell culture. The presence of the CAT gene as a marker allows the replication of the RNA to be monitored either by probing for increases in CAT-specific RNA or more simply by a CAT enzyme assay. The CAT activity in transfected cells was not due to accumulating translation products from input RNA, because transfection of mutated versions of FLC/REP which were translation competent but deficient in replication did not result in detectable CAT activity. Hence, this system can be used to investigate the effects of mutations in the 5' NCR, for example, on genome replication by a simple enzyme assay. The enzyme activity will be affected by mutations which alter translation as well as by those affecting replication. However, the sequences in the 5' NCR which affect translation have been mapped to the region between bases 320 and 631 (19). On the other hand, the sequences which control

w

TABLE 1. Replication and viability of FLC/REP mutants Deletion

FLC/REP LL-inv L-sinv A.220-

Description

Wild type N10-34 deleted N10-34 inverted N10-18 and N2634 inverted N51-78 deleted N188-222 deleted

of CAT activity)

(plaque

+++ 0 + +++

Large Not viable Minute Large

0 ++

Not viable Large

size)

CAT

+0

+PV

10

2°

30

40

FIG. 8. Passage of encapsidated FLC/REP RNA. CAT activity from 50 ,ug of cytoplasmic extracts of cells 8 h after infection with supernatants from monolayers which received FLC/REP alone (+0) or with poliovirus type 3 (+PV). A total of one-tenth of the supernatants was used to infect fresh monolayers, and CAT activity produced 8 h after infection was analyzed over four passages (1°, 2, 30, and 40).

VOL. 66, 1992

genome replication are generally believed to lie within the first 100 nucleotides of the 5' NCR (1). Even gross deletions within this 100-base region do not decrease translation (19) but were observed to affect CAT production in transfected cells. Although a Gln-Gly motif was incorporated near the CAT/VP3 boundary in FLC/REP, this signal did not appear to be cleaved efficiently, since only a CAT-VP3 fusion protein could be detected in transfected cells. In light of recent results indicating the importance of alanine at the P4 position (2), this result is not surprising. The result suggests, however, that the CAT enzyme is active as a fusion protein. Since the CAT assay lends itself readily to quantitation, small changes in the replication efficiency of genomes should be detectable by this system. Cells transfected with the wild-type replicon produced about 2 U of CAT activity per mg of extracted cellular protein. This level compares favorably with those reported for some plant RNA viruses used as expression vectors (5) and suggests that even poor replicons can be monitored by CAT assay. Indeed, using the CAT assay we were able to detect replication of a mutant which gave rise to minute plaques (L-inv). This mutant had the sequences of the L domain (N10-34) of the 5' terminal clover-leaf inverted. A different mutant, with only the stem sequences inverted (L-sinv) but with wild-type loop sequences, produced high levels of CAT activity and gave rise to large wild-type plaques when built into a complete genome. These observations suggest that the primary sequence exposed at the loop of this structure is important for RNA replication, whereas the sequence of the stem region is not. When the entire N10-34 loop was deleted (mutant L-), the replication was so severely affected that no CAT activity could be detected and viable virus could not be recovered. This was also the case for a mutant that had the A domain (N51-78) of the clover-leaf deleted (mutant A-), whereas loss of the loop 220 (N188-222) had only a marginal effect on CAT activity and no effect on plaque size. This is in agreement with previous reports, which suggested that similar mutations in this region also resulted in viruses with essentially wild-type phenotypes (4). The roles of individual domains of the 5'-terminal clover-leaf structure in RNA replication are currently under further investigation with a series of CAT-containing replicons. It was also possible to demonstrate specific, receptormediated propagation of recombinant genomes indicating that FLC/REP RNA packaged efficiently into homologous capsids. This is in contrast to a recent report which describes the construction of poliovirus replicons carrying regions of human immunodeficiency virus genes within P1. Although Choi et al. (3) showed that the human immunodeficiency virus sequences were amplified, they were unable to demonstrate packaging either by superinfection or by cotransfection of helper virus RNA. The reasons for these contrasting results are not clear. It is possible that insertion of these particular foreign sequences has either removed or disrupted an encapsidation signal in the P1 region. However, since packaging could not be detected for any of four different replicons, this explanation seems unlikely. Moreover, our own unpublished studies suggest that such signals are not present in the P1 region. The approach we describe here has been used already to map important cis-acting sequences of several other viruses. For example, Levis and coworkers (13) also inserted the CAT gene into Sindbis virus and achieved expression of biologically active CAT following transfection of infectious RNAs produced in vitro. Superinfection with helper virus

POLIOVIRUS CAT REPLICON

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enabled the recovery of CAT-containing genomes which had been packaged into capsids provided in trans. Furthermore, in a separate study CAT expression from a Sindbis virus vector increased over seven passages such that an amplification of 1018-fold was achieved (29). More recently, the CAT gene has been amplified, expressed, and packaged when inserted into the genomes of the negative-strand RNA viruses influenza virus (14) and Sendai virus (17). However, unlike the CAT replicons described here, those based on influenza virus genes were unstable, and their CAT activities decreased markedly over three passages (14). This was attributed to the rapid emergence of DI particles when influenza virus was passaged at a high multiplicity of infection. Although not confirmed in the present study, we believe that the FLC/REP genome is amplified at a rate similar to that of wild-type poliovirus, since the size difference between the two RNAs is small: FLC/REP is 408 bases or 5% smaller than poliovirus RNA. Kaplan and Racaniello (9) found that the replication rates of three subgenomic replicons were inversely proportional to their sizes, although this effect was not pronounced unless the deletions were large. The largest of their replicons had a deletion of 1,295 bases (17% of the genome) and yet replicated at a rate only 1.4 times greater than that of the full-length genome. Thus, FLC/REP is unlikely to interfere with the functions of the helper virus. Indeed, the observation that CAT activity does not significantly change over four passages supports this view, and preliminary hybridization data confirm that the numbers of FLC/REP and wild-type genomes are similar and remain constant through each of these passages (18a). The genomes of naturally occurring DI poliovirus particles always contain the sequences coding for capsid protein VP4 (12). However, this region is not required for genome replication, since some RNAs constructed without VP4 coding sequences were able to act as replicons (9). Taken together, these observations have led to speculation that VP4 might contain the signal for encapsidation of poliovirus RNA. Our results show that a recombinant genome lacking VP4 coding sequences is efficiently packaged. Thus, the observations from the study of DI particles may reflect the mechanism of their generation rather than their specific packaging requirements, and the packaging for poliovirus RNA must lie outside the VP4 coding region. The CAT marker gene within FLC/REP provides an unambiguous way to distinguish it from helper virus RNA, and studies to locate the encapsidation signal are currently under way. ACKNOWLEDGMENT This work was supported by the Medical Research Council of Great Britain.

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