Department of Cellular and Developmental Biology, University of Rome `La Sapienza', Viale di Porta Tiburtina 28, 00185-Rome, Italy1
Istituto Superiore di Sanità, 00161-Rome, Italy2
Author for correspondence: Raul Pérez Bercoff.Fax +39 06 446 2306. e-mail bercoff{at}caspur.it
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Abstract |
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Introduction |
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A series of cis-acting elements, known as the `ribosome-landing pad' (Pelletier & Sonenberg, 1988 ), or `internal ribosome entry site' (IRES; Meerovitch et al., 1989
; Jang & Wimmer, 1990
), secure the interaction of the ternary initiation complex with the viral RNA in a cap- and 5'-end-independent manner. Translation then proceeds along a single open reading frame (which spans typically over 8090% of the genome), generating a polyprotein which is subsequently cleaved into the mature peptides (Perez Bercoff, 1987
; Rueckert, 1990
).
As a consequence of such functional organization, an unusually long sequence precedes the region encoding the viral polyprotein, extending between the uncapped 5'-end and the AUG triplet used for the initiation of translation. The 5'-terminal untranslated region (5'-UTR), therefore, may account for more than 10% of the entire genome (730 nt in hepatitis A and 1200 nt in Theiler's murine encephalitis virus), and in every case a series of AUG triplets has been identified in this region of the genome that appear to be devoid of coding capacity: in fact, point mutations that removed the initiator codons one at a time were engineered in poliovirus cDNA infectious clones and these changes had no effect on either the ability of the mutants to direct internal initiation of translation of a reporter gene, or on infectivity of full-length cDNA clones (Pelletier et al., 1988 ).
A closer inspection of the 5'-UTR of poliovirus type 1 (PV1), however, revealed the presence of ten additional mini-cistrons starting with the alternative translation initiation codons GUG, ACG and AUA in the three possible reading frames. In the course of experiments designed to assess the potential role of these mini-cistrons, we came across a double mutation (replacement of the ACG triplet at position 157 with TAA and a transition U to C at position 169) that completely abrogated the infectivity of full-length PV cDNA clones. The effects of these substitutions on the infectivity and the in vitro translation of the viral RNA have been discussed elsewhere (Pierangeli et al., 1998 ). Since the nature of the function(s) lost by PV were unknown, we tried then to complement in trans the lethal defect by cotransfecting cultures of COS-1 cells with the lethal mutant cDNA clone and an expression vector containing just the 5'-UTR of PV2 (Lansing strain) under the transcriptional control of the SV40 late promoter; we report here that the trans-complementation that rescued infectious virus was mediated by RNA recombination through a novel mechanism that we propose to call `primer alignment-and-extension'.
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Methods |
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Plasmids and reagents.
Plasmid pGEM-2 and pGEM-4 were from Promega; the eukaryotic expression vector pSV-L was obtained from Pharmacia. Unless otherwise indicated, restriction and modifying enzymes were used according to the manufacturer's specifications. All DNA manipulations were performed following standard procedures (Sambrook et al., 1989 ).
PCR mutagenesis and plasmid construction.
The infectious plasmid pGEM-Pol, containing a full-length cDNA representation of the PV1genome in the plasmid pGEM-2, was used to construct mutants with modified 5'-UTRs. To this end, the sequences extending between the 5'-end and position 1178 of PV were removed by digestion of the plasmid DNA with SalI and NruI [which cut in the multiple cloning site of the plasmid and at position 1178 of PV cDNA, respectively], followed by dephosphorylation and gel purification. Full-length PV cDNAs with mutated 5'-UTRs were reconstructed by ligation of PCR-generated DNA fragments digested with the same enzymes. The latter were generated in PCRs driven by pairs of complementary oligonucleotides containing the desired mutations. The mutated Lansing 5'-UTRs were generated by PCR using a pSV-GH-5'-UTR (Lansing)-CAT plasmid (Nicholson et al., 1991 ) as a template. After purification, the mutated fragments were digested with the enzymes BglII and HindIII (which cut in the sequences encoding GH and in the intergenic region, respectively) and introduced into the BglIIHindIII, phosphatase-treated plasmid pSV-GH-CAT. The presence of the mutations and the correct insertion of the fragments were ascertained by direct DNA sequencing.
Transfection.
COS-1 cells (2·5x105) in plastic flasks (75 cm2) were transfected with 10 µg of each plasmid DNA as previously described (Pierangeli et al., 1995 ).
Gene expression driven by the PV IRES.
COS-1 cells were transfected with plasmids carrying a wild-type (wt) or mutated PV IRES inserted between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT). The amount of GH present in the supernatants was determined by radioimmunoassay in triplicate 100 µl aliquots (Silveira Carneiro et al., 1995 ). The cells were then detached from the plates, washed in ice-cold PBS, suspended in 300 µl 250 mM TrisHCl (pH 8·3), frozen and thawed three times, and the CAT activity was assayed and quantified as described (Degener et al., 1995
; Silveira Carneiro et al., 1995
).
In vitro transcription.
A cDNA representation of Lansing 5'-UTR inserted in the SmaIEcoRI sites of BS-KS+ vector was used as a template for in vitro transcription in reactions driven by T3 RNA polymerase. The DNA template was linearized by digestion with PvuII (which cleaves in the BS-KS+ plasmid at positions 529 and 977) and gel purified to obtain the ca. 1000 bp fragment containing the T3 promoter and the Lansing 5'-UTR. Following transcription, samples were phenolchloroform extracted, ethanol precipitated and checked by electrophoresis through denaturing formamideagarose gels.
Blocking the 3'-OH ends of RNA with cordycepin.
Gel-purified RNA transcribed in vitro in a final volume of 100 µl was made 50 mM TrisHCl (pH 7·9); 250 mM NaCl; 10 mM MgCl2; 2·5 mM MnCl2; 1 mM DTT; 0·05% BSA. Poly(A) polymerase (4 units) (Boehringer) was then added. The reaction mixture was divided in two equal aliquots, and cordycepin (3'-deoxyadenosine; Sigma) was added to one of them (final concentration 1 mM); the reaction mixtures were then incubated at 37 °C for 1 h. Following phenolchloroform extraction and ethanol precipitation, the integrity of the 3'-blocked RNA was ascertained by electrophoresis through a formamideagarose gel under full denaturing conditions. Both mock- and cordycepin-blocked RNAs (0·50·8 µg per plate) were used as `helper' in rescue experiments as above. This gave a molar ratio of helper RNA:mutated plasmid close to 1:1.
Sequence analysis of viral RNAs.
Stocks of mutated virus derived from plasmid transfection were grown on Vero cells. The clarified supernatants obtained as described above were subjected to two sequential precipitations with 6% PEG, and viral RNA was phenolchloroform extracted as described (Pierangeli et al., 1995 ). The presence of the mutations engineered was confirmed by direct RNA sequencing. Alternatively, cDNA fragments were generated and amplified by RTPCR using pairs of appropriate oligonucleotides. Following removal of the primers and dNTPs, the purified cDNA fragments were sequenced by the dideoxy chain termination procedure using an Amplicycle kit (Perkin-Elmer).
Computer-assisted sequence analysis.
Sequence analysis was performed using the GCG software package (Devereux et al., 1985 ).
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Results |
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Replacement of the putative initiation triplet ACG157159 with the (ochre) termination codon UAA abrogated the infectivity of full-length PV cDNA clones. However, a closer inspection of this non-infectious cDNA revealed that a second point mutation had been fortuitously introduced at position 169 (a transition U to C). Interestingly, neither these changes separately had any effect on the infectivity of PV cDNA clones, and we could recover infectious virus whose genomic RNAs carried either mutations 157159 or 169. Moreover, neither of these substitutions modified the titre or the temperature stability of the mutants, which from this viewpoint did not differ from wt PV (Pierangeli et al., 1998 ).
The concomitant introduction of the (ochre) termination codon 157159 and the point mutation 169 U to C resulted in a lethal mutant, hereafter referred to as Pol-157+169.Mut. Following transfection of COS-1 cells with this construct, we were unable to recover infectious virus even after four or five blind passages of the supernatants in Vero cells.
Efficient internal initiation of translation directed by mutants 157, 169 and 157+169
We wondered whether the combined mutations 157+169 modified the ability of the UTR to direct internal initiation of translation. Accordingly, these mutations were engineered by PCR in the 5'-UTR of PV cDNA, which was then introduced into the intergenic region of the bi-cistronic plasmid pSV-GH-CAT (Nicholson et al., 1991 ), between the genes encoding human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT). Upon transfection of COS-1 cells with these constructs, the CAT activity present in cell lysates was determined and quantified as described (Silveira Carneiro et al., 1995
). As can be seen in Fig. 1
, PV 5'-UTRs with mutation at either positions 157159, 169 or 157+169 were as efficient as the wt UTR in directing expression of the reporter gene, suggesting that the observed loss of infectivity of the full-length cDNA clones could not be ascribed to a defect that determined the loss of ability of these mutants to direct internal initiation of translation.
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COS-1 cells were therefore cotransfected with the non-infectious cDNA clone 157+169.Mut of PV1 (Mahoney) together with the construct that expressed just the 5'-UTR of PV2 (Lansing strain). While none of the `parental' constructs was infectious (Fig. 2), expression of the 5'-UTR of PV2 (Lansing) was able to rescue infectious PV in every cotransfection experiment (Fig. 2)
: a clear cytopathic effect appeared 72 h after transfection and was complete 18 h later. The emerging viruses were propagated on either Vero or HeLa cells, and the viral stocks were titrated by plaque formation as shown in Fig. 2
.
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In every case, the genome of the infectious virus recovered by cotransfection was a recombinant of PV1 Mahoney (as the parental 157+169.Mut), and PV2 (Lansing strain), the PV type of the 5'-UTR that served as helper.
In Fig. 3 the autoradiograph of a sequencing gel illustrates the transition from PV1 (Mahoney, up to nt 597 in this experiment) to PV2 (Lansing, from nt 591). In addition to the bases distinctive to each strain, one can clearly see the `crossing-over', i.e. the region of sequences common to both types, where the recombination event had occurred (nt 603592 in this experiment; base numbering of the Lansing genome, GenBank accession no. M12197).
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The mechanism of the recombination event
The question, therefore, arose as to whether the recombination event responsible for the trans-complementation involved the parental plasmid DNAs, or RNA structures formed during the virus replication cycle. The obvious implication was that in the former case we may be dealing with a mere laboratory artefact, whereas the latter may mimic the frequent recombination observed among different serotypes and variants of PV in the course of natural infections (Lipskaya et al., 1991 ).
To discriminate between these two possibilities, we cotransfected COS-1 cells with the non-infectious PV1 cDNA clone 157+169.Mut and an RNA fragment transcribed in vitro from the 5'-UTR of PV2 (Lansing).
To that end, a cDNA representation of the entire 5'-UTR of the genome of PV2 (Lansing strain) was removed from the plasmid pSV-GH-Lansing-CAT (Nicholson et al., 1991 ) with the enzymes EcoRI and HindIII, and directionally sub-cloned into the phagemid BlueScript KS (Stratagene) digested with the same enzymes. Following propagation of this construct in bacteria, the plasmid DNA was digested with the enzyme PvuII, and a 950 bp fragment containing the T3 promoter followed by the sequences of the 5'-UTR of PV2 was gel purified and used as template for the transcription in vitro of the 5'-UTR of PV2, in reactions driven by T3 RNA polymerase as described in Methods. Typically, 0·50·8µg RNA was cotransfected with the non-infectious PV cDNA clone 157+169, at a molar ratio about 1:1.
As expected, the gel-purified 950 bp cDNA fragment used as template was not infectious by itself (Fig. 4), nor was it able to trans-complement when cotransfected with the non-infectious PV cDNA clone 157+169.Mut (Fig. 4
). This was in marked contrast with the behaviour of the RNA fragment transcribed in vitro which, while non-infectious itself, could consistently rescue infectious virus when cotransfected with the non-infectious cDNA clone (Fig. 4
).
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The mechanism involved in the recombination event driven by short RNA transcripts of positive polarity representing just the 5'-UTR of the PV genome remained obscure, and we wondered whether the helper RNA was used as a template, according to a classical `template switching' model, or else it served to prime a reaction requiring a free 3'-OH end. To discriminate between these possibilities, we blocked the 3'-end of the RNA transcripts with cordycepin (a 3'-deoxy analogue of ATP that blocks chain elongation by introducing a 3'-deoxy end and consequently inhibits formation of the phosphodiester bond), and checked the ability of the RNA so treated to drive a recombination process. We were unable to recover any infectious virus (even after five blind passages) when the helper RNA lacked a free 3'-OH end (Table 1).
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Discussion |
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Whatever the mechanism involved in the suppression introduced by the lesion 157+169 of PV1, the defect was complemented in trans by co-expressing the wt 5'-UTR of the closely related PV2, Lansing strain. As expected, the same mutations introduced in the helper 5'-UTR abolished its ability to rescue in trans. In these constructs, the orientation of the inserts placed under the transcriptional control of the SV40 late promoter was such that only genomic sense RNA (be it the 5'-UTR of the `helper', or the full-length PV1 genome) could be transcribed.
The results reported here provide direct experimental evidence proving that the molecular mechanism responsible for the trans-complementation phenomenon is RNARNA recombination. The use of the 5'-UTR of PV2 Lansing strain as a helper to rescue the inactivated PV1 cDNA allowed us to trace the fate of the parental molecules, excluding the possibility that the rescue was either the mere effect of a contamination by wt PV cDNA, or due to the reversion of the point mutations engineered: in fact, the infectious virus recovered in different experiments always contained recombinant genomic RNAs like the one depicted in Fig. 3.
Not surprisingly, the `crossing-over' region differed from experiment to experiment: nt 348380 were involved in one case, and nt 592603, 606622 and 415442 in other experiments, i.e. stretches of complete sequence identity between PV1 (Mahoney strain) and PV2 (Lansing strain).
The recombination event occurs between RNA molecules, as demonstrated by the fact that infectious virus with recombinant genomes could be recovered upon cotransfection with RNA transcribed in vitro from a cDNA representation of the 5'-UTR of PV2 (Lansing strain). Here again, this result cannot be attributed to a contaminant intact (or re-circularized) plasmid DNA present in the RNA preparation, since the template used for the in vitro transcription was a gel-purified PvuII fragment, 950 bp in length, devoid by itself of any trans-complementation ability as shown in Fig. 4. Since the in vitro transcription generates RNA fragments of variable lengths, several different crossing-over points were found.
The recombination of PV RNA genomes of different serotypes was described as early as 1962 (Hirst, 1962 ), and is known to occur frequently in nature, as previously documented (Lipskaya et al., 1991
). Moreover, there is increasing evidence that recombination of viral RNA genomes does occur with high frequency in natural conditions, a phenomenon supposed to have considerable biological implications (for an extensive review see Bujarski, 1996
). The mechanism involved in RNA recombination, however, has remained elusive: in fact, in the case of coronaviruses (as in that of other RNA viruses) a `copy-choice' (or `template switching') mechanism was supposed to be involved, although (as explicitly pointed out by Lai) there is no direct evidence for such a claim (Lai, 1996
).
Hence, the fact that an RNA fragment of positive polarity with a free 3'-OH but lacking the signals required to initiate minus-strand RNA synthesis (Kirkegaard & Baltimore, 1986 ; Pierangeli et al., 1995
) (and therefore unable to be used as a template to start the construction of a replicative intermediate structure) can be recombined during viral RNA replication requires an explanation.
We propose that the `helper' RNA transcribed from the wt 5'-UTR cDNA hybridizes to homologous sequences in the anti-genomic template, and serves as primer in an extension reaction that yields necessarily a recombinant genome (Fig. 5). Since substitutions 157+169 do not prevent the translation of the non-infectious viral RNA, the mutated genome can be translated, and the viral RNA polymerase so produced transcribes a negative-strand molecule in a totally conventional manner, using as a template the defective RNA. The wt `helper' RNA fragment of genomic sense can then hybridize to homologous regions on the minus-strand, and can serve as a primer that the viral polymerase would then extend. This view is further substantiated by the fact that a free 3'-OH is stringently required, and blocking the 3' end of the `helper' RNA abrogated its ability to rescue infectious PV. This would not be expected to occur if the RNA was to serve as template in a `copy-choice' mechanism, in which case the nature of the 3'-end would be totally indifferent.
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The model we propose differs substantially from the `template switching' one in its different versions. In fact, the `copy choice' model proposed to explain non-homologous recombination observed in bromoviruses postulates the formation of local heteroduplexes between two template RNA strands of the very same polarity, followed by the sliding of the RNA replicase under the heteroduplexed region and the restarting of RNA synthesis of the nascent strand on the acceptor template (Bujarski & Nagy, 1996 ). This model requires the presence, in the first place, of a stretch of palindromic sequences to allow formation of the heteroduplex structure between two RNA strands of the same polarity, and would irremediably result in the synthesis of a deleted recombinant, lacking the entire portion of the genome involved in the heteroduplex region (see Fig. 2
in Bujarski & Nagy, 1996
). This is clearly not the case for the infectious PV recombinants whose `crossing-over' sequences are documented in Fig. 3
.
In its original version, the `copy choice' model postulated that a nascent RNA chain of viral RNA still bound to the polymerase complex `jumped' from one template to another one, crossing over stretches of high sequence identity: this would require that the nascent strand quits the template and is transferred onto a passing `helper' RNA molecule precisely at the same position just transcribed, even though the `helper' (of genomic sense) cannot hybridize to the genomic-sense template. Obviously, such a model cannot explain the rescue experiments reported here, where a deproteinized, phenol-extracted RNA fragment transcribed in vitro (and therefore lacking the RNA polymerase molecule attached to its 3'-end), was able to rescue by recombination the inactivated PV1 cDNA clone, provided a 3'-OH end was available. In this case, the `helper' could just hybridize to the anti-genomic template, and act as a primer that the RNA polymerase would then extend. Several lines of evidence tend to weaken even further the `template switching' mechanism as an explanation of the results we report here: in fact, according to the `copy choice' model recombination should occur preferentially at highly structural RNA sites since these structures promote transcriptional pausing. However, Lai (1996) has rightly reported that in recombinant coronavirus the crossing-over sites were distributed almost randomly. Moreover, AU-rich regions are expected to facilitate the formation of `bubble' structures in the double-stranded core of the replicative intermediate, thus facilitating the jumping of the polymerase-nascent strand complex from one template to a neighbouring one. This has not been observed in the crossing-over sequences, and Fig. 3
illustrates one such transition, MahoneyLansing, over a 23 nt stretch containing just 11 A+U.
The mechanism of `primer alignment-and-extension' can more easily explain the natural occurrence of recombination between viral RNA genomes: in fact, premature termination of transcription can generate RNA fragments of variable lengths, subsequently aligned to, and extended, on a different template thanks to stretches of sequence identity (the `crossing-over' regions), an event that the physical vicinity of the membrane-bound replication complexes on the one hand, and the overabundance of the viral RNA polymerase (60 copies of RNA polymerase per picornavirus particle produced) on the other, may facilitate.
Such a mechanism is reminiscent of that involved in the generation of the nested sub-genomic mRNAs of coronaviruses, bearing the same 70 nt leader at the 5'-termini (Lai, 1992 ). In this case, a process of discontinuous RNA transcription is known to produce a leader sequence 7090 nt in length identical to the 5'-end of the genome, and following hybridization to the minus-strand template over short stretches of sequence identity, the leader RNA is extended, generating the nested, subgenomic mRNAs (Lai, 1996
).
Thus, far from being a mere laboratory artefact, the alignment and extension of RNA fragments appears to be a widely used mechanism under natural conditions. This view is further substantiated by recent studies on the mechanism of synthesis of equine arteritis virus subgenomic mRNAs: mutational analysis of the intergenic conserved sequences has provided convincing evidence of the critical role of duplex formation in priming discontinuous mRNA synthesis (van Marle et al., 1998 ).
The proposed model of RNA recombination by `primer alignment-and-extension' explains our finding that trans-complementation can rescue only infectious poliovirions with recombinant genomes, where the sequences encoding the defective function had been replaced with those provided by the helper.
Three main features not previously envisaged emerge from these findings. It appears, in the first place, that non-replicating RNA fragments can efficiently drive viral RNARNA recombination provided they have a 3'-OH end. The `helper' RNA does not require a bound RNA polymerase complex (a prediction of the `copy-choice' model). Moreover, no open reading frame on such an RNA is required, thus eliminating any need for cis-acting proteins on the rescuing RNA.
The `primer alignment-and extension' model would also predict the reactivation of attenuated strains of related viruses in the course of a co-infection, provided they share common sequences able to serve as a `crossing-over' stretch. This view is reinforced by the observation that intertypic recombination of PV occurs far more frequently in the course of natural infection (including vaccination with the non-neurovirulent Sabin strains) than in cell culture (Tolskaya et al., 1983 ; Agol et al., 1984
; Lipskaya et al., 1991
, and references therein). While the role of selective pressure (which would favour the `fitter' recombinants) cannot be excluded, the possibility must be entertained that the reactivation of `dormant', replication incompetent viruses by a mechanism of RNARNA recombination might be an important pathway of genetic variation in viruses.
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Acknowledgments |
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References |
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Received 22 December 1998;
accepted 9 April 1999.