Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1
Author for correspondence: Paul Britton. Fax +44 1635 577263. e-mail paul.britton{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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Coronaviruses are enveloped RNA viruses with unsegmented single-stranded, positive-sense RNA genomes of 2732 kb that are 5' capped and 3' polyadenylated (Siddell, 1995 ; de Vries et al., 1997
; Lai & Cavanagh, 1997
). The 5' two-thirds of coronavirus genomes encode the polymerase gene producing two polyproteins, Pol1a and Pol1ab, the latter resulting from a -1 frameshift. During coronavirus replication a 3'-coterminal nested set of subgenomic mRNAs is synthesized that encodes other virus proteins. Each subgenomic mRNA has a short non-translated leader sequence (6090 nt), derived from the 5' end of the genome by a discontinuous process. IBV has a genome of 27608 nt (Boursnell et al., 1987
) which produces six subgenomic mRNAs, each containing a 64 nt leader sequence.
Two models have been proposed for the discontinuous mechanism for the addition of coronavirus leader sequences, leader-primed transcription and discontinuous transcription during negative-strand synthesis (Sawicki & Sawicki, 1990 ; van der Most & Spaan, 1995
; Brian & Spaan, 1997
; Lai & Cavanagh, 1997
). Both models require the presence of a consensus sequence, which we previously termed the transcription-associated sequence (TAS; Hiscox et al., 1995
), on the genomic sequence, required for leader sequence acquisition during synthesis of subgenomic mRNAs.
Following high multiplicity passage of coronaviruses, defective RNAs (D-RNAs) or in some cases defective interfering RNAs (DI-RNAs), have been found to be produced; they lack large internal parts of the genome, have complete 5' and 3' UTRs and require helper virus for replication. The internal deletions result in the fusion of different regions of the polymerase gene and varying amounts of the N gene (Makino et al., 1988b , 1990
; van der Most et al., 1991
; Chang et al., 1994
; Pénzes et al., 1994
; Mendez et al., 1996
).
Makino et al. (1986) demonstrated that leader sequences from different murine hepatitis virus (MHV) strains can be exchanged during a mixed infection, indicating that there is some interaction of the MHV polymerase between the heterologous RNAs during replication. Initial work by Makino et al. (1988a
) to determine whether leader sequences can be switched between helper virus and an MHV DI-RNA failed to demonstrate this event using naturally occurring MHV JHM-derived DI-RNAs rescued with MHV A59. All the naturally occurring MHV JHM-derived DI-RNAs had a 9 nt deletion, UUUAUAAAC, downstream of the leader sequence. Replacement of the 9 nt sequence in an MHV JHM-derived DI-RNA, DIssE, resulted in a modified DI-RNA, DE5-w3, that was able to leader switch following rescue with MHV A59 (Makino & Lai, 1989
). This phenomenon of an MHV DI-RNA acquiring the leader sequence of the helper virus was termed leader switching and was believed to be the result of a high-frequency recombination event (Makino & Lai, 1989
). The second coronavirus system from which D-RNAs were isolated involved the Mebus strain of bovine coronavirus (BCoV; Hofmann et al., 1990
). A synthetic BCoV DI-RNA, derived from a naturally occurring Mebus DI-RNA, was modified by the introduction of specific point mutations within the leader sequence to determine their potential effect(s) on replication of the DI-RNA (Chang et al., 1994
). All the mutations appeared to affect replication only minimally, following rescue with homologous Mebus helper virus. However, analysis of the leader sequences on the rescued D-RNAs showed that they had all reverted to the wild-type, Mebus, leader sequence (Chang et al., 1994
).
In a recent review (Brian & Spaan, 1997 ) the authors proposed that an AU-rich region, immediately downstream of the leader junction sequence at the 5' ends of BCoV and MHV DI-RNAs, is involved in leader switching. The authors also indicated that a similar AU-rich region is also present downstream of the leader junction sequence on DI-RNAs of the porcine coronavirus transmissible gastroenteritis virus (TGEV; Mendez et al., 1996
) but not on D-RNAs of IBV (Pénzes et al., 1994
). These observations led the authors to raise the question as to whether leader switching could occur on IBV D-RNAs. At that time the phenomenon of leader switching had not been demonstrated for DI-RNAs from TGEV or D-RNAs from IBV.
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Methods |
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Oligonucleotides.
The oligonucleotides used in this study were obtained from Cruachem or MWG-Biotech and are listed in Table 1.
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Sequences analysis of the 5' end of IBV genomic RNA.
IBV genomic RNA, from strains M41, HV10, H120 or D207, was extracted from virions grown in embryonated eggs (Kottier et al., 1995 ). A slightly modified version of the cRACE method (Maruyama et al., 1995
) was used to determine the very 5' ends of the IBV genomic RNAs. Essentially, 1 µg of each viral RNA was copied into single-stranded cDNAs (402 nt) using oligonucleotide K5UTR-2 and Superscript II RNase H- reverse transcriptase (200 U; GibcoBRL) at 47 °C for 2 h. The RNADNA hybrids were incubated with RNase H (4 U; GibcoBRL) at 37 °C for 20 min and the cDNAs purified on GLASSMAX (GibcoBRL) spin columns. The cDNAs were eluted using 50 µl H2O at 65 °C and circularized using T4 RNA ligase (1 U; USB) in 50 mM TrisHCl (pH 7·5), 10 mM MgCl2, 10 mM DTT, 1 mM ATP and 60 µg/ml BSA at 17 °C for 18 h. The reaction was terminated by the addition of EDTA to a final concentration of 20 mM. The circularized cDNAs were used as templates for PCRs across the ligated ends using oligonucleotides 93/137 and 18A with Pfu DNA polymerase (Stratagene). The PCR products (280 bp) were sequenced, using oligonucleotide 93/137, either directly or following ligation into EcoRV-digested pBluescript SK from which several IBV-derived cDNAs were sequenced. This method produced sequence data corresponding to nt 1151 of the genomic RNAs. To determine sequence data beyond nt 151 each viral RNA was copied into cDNA by RTPCR using Pfu DNA polymerase (Stratagene). Different oligonucleotide pairs were utilized depending on the IBV strain, i.e. oligonucleotides 42+93/102 and 94/155+K5UTR-3 were used for HV10; 5deg-ve+43 for H120; and 93/101+93/102 and 94/155+K5UTR-3 for D207. The PCR products were sequenced either directly or from cloned DNA from which several IBV-derived cDNAs were sequenced.
In vitro transcription and electroporation of primary CK cells.
RNA corresponding to IBV D-RNA CD-61 was synthesized in vitro from 2 µg NotI-linearized pCD-61 (Pénzes et al., 1996 ) in a total volume of 40 µl. This was done using 40 U T7 RNA polymerase (Promega) in 40 mM TrisHCl (pH 7·5), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 30 nmol each of ATP, UTP, CTP and GTP, 400 nmol DTT, 40 U RNasin (Promega) at 37 °C for 2 h. CK cells were grown to 8090% confluence in 25 cm2 tissue culture flasks (Falcon) and infected with 0·5 ml egg-grown IBV helper virus or 1 ml of CK-grown D207 virus. At 8 h p.i. the cells were washed twice with PBSa (172 mM NaCl, 3 mM KCl, 100 mM Na2HPO4, 2 mM KH2PO4, pH 7·2), once with trypsinversene (0·03% trypsin in 0·5 mM EDTA). The IBV-infected cell sheets were disrupted using 1 ml trypsinversene for 35 min at 37 °C, followed by addition of 0·3 ml 10% newborn calf serum, cooled on ice, centrifuged at 2500 r.p.m. for 3 min and the cells resuspended in 0·3 ml ice-cold PBSa. Transcription reactions (40 µl) were mixed with the resuspended IBV-infected cells in 0·4 cm electroporation cuvettes (Invitrogen) for electroporation at 220 V using a BTX Electro Cell Manipulator (model ECM-395). The electroporated IBV-infected cells were placed on ice for 5 min, diluted 1:10 in CK cell medium (Pénzes et al., 1994
), transferred onto tissue culture plates and incubated at 37 °C for 16 h. Virus (V1) in 1 ml of the supernatants was used to infect CK cells and after 2024 h p.i. virus (V2) from the supernatants was passaged on CK cells. This was repeated up to five more passages, V3V7. RNA was isolated from cells (P1P7) infected with each virus (V1V7) passage as outlined in Fig. 1
.
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Sequence analysis of IBV D-RNAs.
Total cellular RNA extracted from P7-infected CK cells containing the D-RNAs, CD-61, CD61M41, CD61H120, CD61HV10 and CD61D207 rescued with helper IBVs, Beaudette, M41, H120, HV10 or D207, respectively, was used for RTPCRs, using oligonucleotide 93/136 for the RT reactions. A variety of oligonucleotides was used for the PCRs to: (a) distinguish D-RNAs from either genomic RNA or subgenomic mRNAs and to confirm the adenosine deletion at nt 749 (A-749) of CD-61 [a characteristic of the D-RNA (Pénzes et al., 1994 )] and (b) to characterize the leader sequence and adjacent 5' UTR on the rescued D-RNAs. The PCRs for (a) used oligonucleotides 93/136 and 93/106, which result in a fragment of 910 bp spanning domains I and II of D-RNA CD-61 (Pénzes et al., 1994
). Oligonucleotide 93/120 was used to sequence part of the 910 bp PCR fragment to confirm the absence of A-749. Oligonucleotides 95/165 and ST4 were used to sequence the domain I/II junction within the 910 bp PCR fragment. The PCRs for (b) used oligonucleotides 93/136 and 94/155, which resulted in a fragment of 1·6 kb extending from within domain II of CD-61 to nt 23 from the 5' end of D-RNA CD-61. Oligonucleotides 93/137, K5UTR-1, K5UTR-2 and K5UTR-3 were used to sequence nt 24342 of the rescued D-RNAs.
Computer analysis of sequence data.
Sequence data were assembled using Gap4 of the Staden Sequence Software Programs (Bonfield et al., 1995 ). Sequences were aligned using ClustalX version 1.64b (Thompson et al., 1997
) and the alignments analysed using GeneDoc version 2.5 (Nicholas & Nicholas, 1997
). Identification of potential RNA structures was determined using RNAdraw version 1.1 (Matzura & Wennborg, 1996
) and MFOLD (Zuker & Stiegler, 1981
; Jaeger et al., 1989
, 1990
; Zuker, 1989a
, b
). The MFOLD program used was part of the Wisconsin package (GCG version 9.0 UNIX software; Genetics Computer Group Inc., 1997) (Devereux et al., 1984
). Both types of analyses work on the basis of free-energy minimization.
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Results |
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Three of the heterologous strains, M41, HV10 and H120, belong to the same serotype, Massachusetts, as Beaudette from which CD-61 was derived, based on either serology of the spike (S) protein (Darbyshire et al., 1979 ) or S gene sequence identity determined from Beaudette (Binns et al., 1985
; Boursnell et al., 1987
), M41 (Binns et al., 1986
; Niesters et al., 1986
) and H120 (Kusters et al., 1989
). The fourth heterologous strain, D207, is of a different antigenic serotype to the Massachusetts strains based on serology of the S protein (Davelaar et al., 1984
) and S gene sequence (Kusters et al., 1989
). Serotyping of IBV strains is entirely based on the S protein/gene and therefore any similarities between the viruses may not be reflected in nucleotide sequences over other regions of the genome.
To confirm whether leader switching occurs during the rescue of IBV D-RNAs we needed to identify potential nucleotide differences, within the leader and adjacent 5' UTR sequences, between the IBV strains. The potential substitutions had to be sufficiently different to act as markers capable of distinguishing resultant sequences, derived from any leader switching events, from both the helper virus and the D-RNA CD-61. Sequences (nt 1151) of the 5' ends of the genomic RNAs were determined for the four heterologous IBV strains using cRACE (Maruyama et al., 1995 ). Further genomic sequence, following nt 151, was determined from IBV strains HV10, H120 and D207 by RTPCR. The genomic sequences (nt 1376) obtained as described here and from previously derived sequence data, for M41 (Pénzes, 1995
) and Beaudette (Boursnell et al., 1987
), were aligned.
Comparison of the aligned sequences identified a variety of nucleotide substitutions between the IBV sequences (Fig. 2). M41 had 3 (4·7%) and 4 (1·3%), HV10 had 11 (17·2%) and 11 (3·5%), H120 had 3 (4·7%) and 19 (6·1%) and D207 had 11 (17·2%) and 8 (2·6%) substitutions within the leader and adjacent 5' UTR sequences, respectively, when compared to the Beaudette sequence. This indicated, except for strain H120, that the proportion of substitutions was higher within the leader sequence than the adjacent 5' UTR. The percentage differences of the complete heterologous IBV 5' UTRs (nt 1528) compared to the Beaudette sequence were 0·9% (M41), 2·8% (HV10), 4·3% (H120) and 1·7% (D207) (data not shown), confirming that there were fewer substitutions within the complete adjacent 5' UTR when compared to the leader sequences. It should be noted that nucleotide substitutions between Beaudette and the four heterologous viruses over other regions of the genome range between 1020%.
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Rescue of D-RNA CD-61 using heterologous IBV helper strains
To investigate whether the heterologous IBV strains would support replication of the Beaudette-derived CD-61 D-RNA, CK cells were infected with the heterologous strains of IBV and electroporated with in vitro T7-transcribed CD-61 D-RNA. Progeny virus (V1) was serially passaged on CK cells, generating progeny virus V2V7 (Fig. 1). Total cellular RNA was extracted from CK cells (P1P7) previously infected with V1V7 virus and examined by Northern blot analysis. An RNA species, corresponding to the size of D-RNA CD-61, was identified in the RNA isolated from P2P7 CK cells infected with the four heterologous strains. The IBV RNAs isolated from P7-infected cells are shown in Fig. 3
; however, it should be noted that the passage number for either initially observing rescue of the D-RNA or detecting the highest amount of D-RNA varied depending on the helper strain used (data not shown). The system was not optimized for the rescue of the D-RNA and was therefore only semi-quantitative. Hence, as can be seen from Fig. 3
, the amounts of CD-61 rescued in the P7 cells varied depending on the heterologous helper virus used, rescue efficiency being least with strain D207. We cannot rule out the possibility of some incompatibility between the sequences involved in either replication or packaging, of the Beaudette-derived D-RNA, and those present in the helper virus D207, resulting in a lower efficiency of rescue.
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Analysis of D-RNA CD-61 leader sequences following rescue by heterologous helper viruses
RNA was isolated from P6 CK cells and analysed by RTPCR using oligonucleotides 93/136 and 94/155. Oligonucleotide 94/155 corresponded to nt 123 of the Beaudette genome. The 1·59 kb RTPCR products represented sequences derived from the rescued D-RNAs from nt 24 in the leader sequence to within domain II.
Sequences representing nt 2464 of the leader and nt 65342 of the adjacent 5' UTR sequences were determined from the 1·59 kb RTPCR products. The 318 nt sequences, derived from D-RNAs CD61M41, CD61H120, CD61HV10 and CD61D207, rescued with the heterologous IBV strains M41, H120, HV10 and D207, respectively, were compared to the corresponding genomic and Beaudette sequences. The results showed that all the rescued D-RNAs had leader sequences corresponding to the specific helper virus and not Beaudette-derived CD-61 sequence (Fig. 4). In contrast, nt 65342 of the adjacent 5' UTR sequences corresponded to Beaudette sequence and were therefore derived from CD-61 (Fig. 4
). The only exception was that D-RNA CD61D207, rescued with D207, had a thymidine residue at nt 94, which corresponded to D207 genomic sequence, and not a cytosine residue corresponding to Beaudette sequence. None of the D207-specific nucleotide substitutions, downstream of nt 94 in the D207 genomic sequence, were present in the rescued CD61D207 sequence. Nucleotide 94 is the first substitution in the adjacent 5' UTR sequence of the D207 genomic sequence 30 nt from the leader junction sequence.
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Identification of potential RNA secondary structures in the IBV 5' UTRs.
Computer analysis of potential RNA secondary structures within the leader and adjacent 5' UTR genomic sequences (nt 1100) of the five IBV strains was carried out. Three stemloop structures were predicted with thermodynamic stabilities of -6·2 to -8·8 kcal/mol for stemloop I, -3·94 kcal/mol for stemloop II and -3·74 kcal/mol for stemloop III (1 cal=4·184 J) (Fig. 5).
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Discussion |
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Coronavirus leader switching was first demonstrated to occur during the replication of an MHV JHM-derived synthetic DI-RNA modified by the inclusion of a 9 nt sequence, deleted from all naturally occurring MHV JHM DI-RNAs, adjacent to the leader sequence. The second report of a coronavirus D-RNA undergoing leader switching was for BCoV using a synthetic DI-RNA derived from the Mebus strain of BCoV (Chang et al., 1994 ). Leader switching, referred to as leader reversion by the authors, was observed to occur following experiments designed to investigate the potential role of various nucleotides on the replication of the D-RNA. A series of point mutations was introduced into the leader sequence of the D-RNA but no potential effect of the mutations could be determined because the replicated D-RNAs were found to have leader sequences identical to the helper virus. The BCoV system differed from the MHV system in that homologous helper virus was used whereas the MHV system used heterologous helper virus.
Chang et al. (1996) showed that, unlike the acquisition of the leader sequence onto coronavirus subgenomic mRNAs, leader switching does not require a complete leader junction sequence. BCoV DI-RNAs containing either a scrambled or a deleted leader junction sequence were replicated, the modified DI-RNAs re-acquiring leader junction sequences as a result of leader switching. The authors concluded that leader switching involves a recombination event over a region of 24 nt comprising the last base of the leader junction sequence and the adjacent 23 nt. Analysis of IBV D-RNAs rescued with heterologous strains indicated that the boundaries of the crossover region were nt 55 and 95, the latter 31 nt downstream of the leader junction sequence. Whilst we cannot rule out the possibility that the IBV leader junction sequence is involved in leader switching it would appear from the work of Chang et al. (1996)
that the event may take place downstream of the IBV leader junction sequence, potentially involving nt 6495.
The BCoV crossover region contains an 8 nt palindromic AU-rich sequence, UUUAUAAA, comprising 8 nt of the 9 nt deleted from the MHV DI-RNAs that did not undergo leader switching (Makino et al., 1988a ). No such AU-rich sequence is present in any of the five IBV genomic sequences. Absence of the AU-rich sequence in the Beaudette sequence led Brian & Spaan (1997)
to query whether IBV D-RNAs could undergo leader switching. Interestingly, the five IBV strains have the sequence AAAUACCU (nt 7481), reminiscent of the palindromic sequence, AAAUAUUU, found in the TGEV 5' UTR sequence. It should be noted that the IBV AAAUACCU sequence is not adjacent but 9 nt downstream of the leader junction sequence whereas the AU-rich sequence in BCV, MHV and TGEV is adjacent to the leader junction sequence. Deletion of the first 7 nt of the 8 nt AU-rich palindromic sequence in a BCoV D-RNA prevented replication. This is in contrast to the situation in MHV where many of the natural MHV JHM DI-RNAs lacked the 8 nt AU-rich palindromic sequence.
Three stemloop structures have been identified within the 5' end of the BCoV DI-RNA (Chang et al., 1994 , 1996
). Deletion mutagenesis of the DI-RNA (Chang et al., 1994
) showed that nt 18 were not required for replication but larger deletions,
12 nt, resulting in loss of stemloop I, abolished replication. Disruption of BCoV DI-RNA stemloop I by base substitution only minimally impaired DI-RNA replication. Analysis of the rescued DI-RNAs showed that they had acquired helper virus-type leader sequence via leader switching. Chang et al. (1994)
concluded that stemloop I is critical for the cis-acting replication signal associated with the leader sequence. Analysis of the 5' UTR of IBV D-RNA CD-61 and genomic RNAs identified three potential stemloop structures (Fig. 5
). However, unlike the BCoV DI-RNA structures, we have no biochemical evidence for the structures in the IBV 5' UTR. Our phylogenetic evidence for the existence of stemloop I in the IBV 5' UTR supports the BCoV data that this structure plays a major role in replication. IBV leader sequences tend to have more nucleotide substitutions than the adjacent 5' UTR, possibly a consequence of some substitutions requiring compensatory substitutions to maintain stemloop I. It would be interesting to know whether MHV DI-RNAs lacking the 9 nt AU-rich sequence are able to replicate, in the absence of leader switching, following the introduction of stemloop I destabilizing mutations.
The demonstration that rescue of IBV D-RNA CD-61 with heterologous virus results in leader switching is similar to the observation that of Chang et al. (1994) for rescue of BCoV DI-RNAs. However, in our case, rather than reversion to wild-type sequence, as for BCoV, the leader sequence acquired by the rescued IBV D-RNA was from the heterologous helper virus. For the IBV system there was no intrinsic requirement for altering the leader sequence as no potentially destabilizing nucleotide substitutions had been introduced. We conclude that it is possible that leader switching is part of the replication mechanism for IBV D-RNAs occurring with both heterologous and homologous helper viruses. We saw no evidence of Beaudette-type leader sequence on any D-RNA following rescue by heterologous virus. RTPCR products derived from the rescued D-RNAs, CD61M41 and CD61H120, were cloned and several IBV-derived cDNAs sequenced to confirm that there were no minor populations of rescued D-RNAs with Beaudette-type leader sequences.
The results presented in this study together with previous observations support the hypothesis that in general coronavirus D-RNAs do not appear to replicate by reiterative replication. IBV D-RNAs leader switch in the absence of introduced point mutations indicating that leader sequence acquisition is part of the replicative process. In this manner it is feasible that coronavirus D-RNAs replicate in a manner analogous to the synthesis of coronavirus subgenomic mRNAs, i.e. that they acquire a leader sequence by a discontinuous process. However, leader acquisition in mRNA synthesis is TAS dependent whereas leader acquisition during D-RNA replication appears to be TAS independent. The discontinuous addition of a leader sequence during replication of D-RNAs is an RNA recombination event since it involves both donor and acceptor templates. The recombination event must, by definition, be extremely efficient at a very early stage and/or there is some positive selection of the chimaeric D-RNA. Otherwise a mixed population of D-RNAs with the two different leader sequences would be found. Therefore, we would propose that replication of coronavirus D-RNAs involves acquisition of the leader sequence from the helper virus, either from a homologous or heterologous virus, as part of the replication cycle, analogous to the manner that subgenomic mRNAs acquire their leader sequence via a discontinuous mechanism. The acquisition of the leader sequence therefore probably occurs via an enzymatic process involving the polymerase. Such a potential activity will only be clarified following the development of a reverse genetics system for coronaviruses.
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Acknowledgments |
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Footnotes |
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c Present address: Departments of Pathology and Cell Biology (BML 342), Yale University School of Medicine, 310 Cedar St, New Haven, CT 06510, USA.
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References |
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Received 10 September 1999;
accepted 23 November 1999.