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 have the largest genomes of any known RNA virus and defective RNAs (D-RNAs), which contain the necessary sequences for replication and packaging into virions in the presence of helper virus, have been used as RNA vectors for modifying coronavirus genomes by targetted recombination (Masters, 1999 ). As a result, recombinant viruses have been obtained for the coronaviruses murine hepatitis virus (MHV) (Fischer et al., 1997
, 1998
; Hsue & Masters, 1997
, 1999
; Koetzner et al., 1992
; Kuo et al., 2000
; Liao & Lai, 1992
; Masters et al., 1994
; Peng et al., 1995a
, b
; Phillips et al., 1999
; van der Most et al., 1992
) and transmissible gastroenteritis virus (TGEV) (Sanchez et al., 1999
).
We have been developing an IBV-based D-RNA system as an RNA vector for both expression of genes and targetted recombination. The IBV system is based on D-RNA CD-61, which contains the sequences necessary for replication and for packaging into virus particles and can therefore be rescued (replicated and packaged into virions) in a helper IBV-dependent manner (Pénzes et al., 1994 , 1996
). Previous work demonstrated that CD-61 can be rescued by heterologous strains of IBV (Stirrups et al., 2000a
), resulting in the phenomenon of leader-switching (Makino & Lai, 1989
), and can be used for the expression of heterologous genes (Stirrups et al., 2000b
). Expression of reporter genes from D-RNAs has been accomplished for a variety of viruses including poliovirus (Barclay et al., 1998
), arteriviruses (Molenkamp et al., 2000
) and coronaviruses (Izeta et al., 1999
; Liao & Lai, 1994
; Lin et al., 1994
; Stirrups et al., 2000b
). The expression of a reporter gene from a D-RNA provides a convenient, highly sensitive and established way of monitoring replication and rescue of the D-RNAs.
Previous work demonstrated the rescue of IBV D-RNAs following electroporation of in vitro T7-transcribed D-RNAs into IBV-infected cells (Pénzes et al., 1996 ; Stirrups et al., 2000a
). Analysis of the amounts of D-RNAs detected by Northern blot analysis (Pénzes et al., 1994
, 1996
; Stirrups et al., 2000a
) or following expression of a reporter gene (Stirrups et al., 2000b
) showed that the efficiency of initial rescue of D-RNAs was low, probably resulting from the small number of cells that were both infected with IBV and electroporated with T7-transcribed RNA. A recombinant fowlpox virus (rFPV)-based system was devised for delivery and initial transcription of D-RNAs, thereby increasing the number of cells potentially containing the D-RNA in order to increase the initial efficiency of rescue by helper IBV. An FPV system was chosen because avian cells are readily infected by FPV and an rFPV is available that expresses T7 RNA polymerase to initiate the in situ synthesis of the D-RNA (Britton et al., 1996
).
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Methods |
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Recombinant DNA techniques.
Recombinant DNA techniques used standard procedures (Ausubel et al., 1987 ; Sambrook et al., 1989
) or the manufacturers instructions.
Construction of plasmids containing modified CD-61.
The hepatitis delta ribozyme (HR) and T7 RNA polymerase termination signal (T7
) sequences were isolated as a discrete restriction fragment from plasmid p2,0 (Pattnaik et al., 1992
) and inserted into pZSL1190 (Pénzes et al., 1996
), resulting in pZSLH
R6. IBV CD-61 cDNA, under the control of the T7 promoter (T7
), was removed from pCD-61 (Pénzes et al., 1996
) as an EcoRINotI fragment, in which the NotI site had been end-filled, and fused to the SmaI site of the H
R in EcoRI/SmaI-digested pZSLH
R6. The resulting construct, pIBV-Vec, contained the modified CD-61 sequence T7
CD-61H
RT7
. The luciferase (Luc) gene, under the control of the Beaudette gene 5 transcription-associated sequence (TAS; Hiscox et al., 1995
), was inserted into the PmaCI site of pIBV-Vec, as described by Stirrups et al. (2000b
), to produce pIBV-Vec-Luc. The T7
CD-61H
RT7
and T7
CD-61LucH
RT7
cassettes were excised from pIBV-Vec and pIBV-Vec-Luc, respectively, and inserted into the ScaI site of the FPV transfer vector pEFL10 (Qingzhong et al., 1994
). Sequence analysis of the resulting plasmids, pEF-CD-61 and pEF-CD-61-Luc, was used to confirm the orientation of each insert.
Rescue of D-RNAs by IBV Beaudette.
In vitro T7-transcribed D-RNAs, corresponding to CD-61 and CD-61Luc, synthesized from 2 µg of pEF-CD-61 and pEF-CD-61-Luc were electroporated into IBV-infected CK cells (P0) as described by Stirrups et al. (2000a) . Progeny virus (V1) from 1 ml of the P0 supernatants was serially passaged on CK cells for up to six passages (P1P6). DNA (2 µg) from pEF-CD-61 and pEF-CD-61-Luc was electroporated, as for RNA electroporation, into CK cells co-infected with IBV and rFPV-T7 for the rescue of transiently transcribed CD-61 and CD-61Luc. Supernatants were filtered (0·2 µm) to remove the FPV and progeny IBV was passaged on CK cells.
Isolation of rFPVs containing IBV D-RNAs.
The FPV transfer vector pEFL10 (Qingzhong et al., 1994 ) contains sequences from the termini of FPV ORF 1, flanking the ScaI cloning site, for the integration of cloned sequences into the FPV genome. The
-galactosidase (lacZ) gene, under the control of the FPV P4b promoter, is present in pEFL10 for detection of potential rFPVs. DNA (510 µg) from pEF-CD-61 and pEF-CD-61-Luc was transfected into FPV FP9-infected CEF cells by using 20 µl lipofectin (1 µg/µl; Gibco BRL) or 25 µl Effectene (1 µg/µl; Qiagen) as described by Qingzhong et al. (1994)
. Potential recombinant viruses were titrated on CEF cells in the presence of X-Gal and blue-staining plaques were purified three times (Boursnell et al., 1990
).
To confirm that the CD-61 and CD-61Luc sequences were within the rFPV genomes, FPV genomic DNA was extracted from rFPV-infected CEF cells. A modified version of the method described by Black et al. (1986) was used to isolate the rFPV genomic DNA. Briefly, CEF cells in 25 cm2 tissue culture flasks (Falcon), infected with rFPV and grown for 4 days, were washed with 10 mM TrisHCl (pH 8·0), 150 mM NaCl and 5 mM EDTA, followed by 300 µl 10 mM TrisHCl (pH 8·0), 10 mM KCl and 5 mM EDTA. The cells were cooled on ice for 10 min and lysed by the addition of 2·5 µl
-mercaptoethanol and 100 µl 10% (v/v) Triton X-100. The lysed cells were centrifuged at 2000 r.p.m. for 5 min at 4 °C to remove the nuclei and the supernatants were centrifuged at 13000 r.p.m. for 1 h at 4 °C. The viral cores were resuspended in 80 µl cold 10 mM TrisHCl (pH 8·0), 1 mM EDTA (TE buffer) followed by 1·5 µl
-mercaptoethanol, 5 µl proteinase K (10 mg/ml) and 20 µl 20% (w/v) Sarkosyl and incubated at 4 °C for 30 min. Following digestion, 140 µl 54% (w/v) sucrose was added to the samples, which were incubated at 55 °C for 2 h, after which 40 µl 5 M NaCl was added. The rFPV DNA was phenol extracted twice, ethanol precipitated and redissolved in TE buffer.
Rescue of CD-61 D-RNAs expressed from rFPV.
CK or Vero cells (P0) were co-infected simultaneously with either rFPV-CD-61 or rFPV-CD-61-Luc, rFPV-T7 and IBV (Beaudette or M41) at 107 p.f.u. for each virus. After 24 h, the supernatants were filtered (0·2 µm) and progeny virus (V1) was serially passaged on CK or Vero cells.
Isolation and analysis of RNAs from infected cells.
Total cellular RNA was extracted from infected cells by the RNeasy method (Qiagen), electrophoresed in denaturing 1% agarose2·2 M formaldehyde gels (Sambrook et al., 1989 ) and Northern blotted onto Hybond-C extra 0·45 µm nitrocellulose membranes (Amersham). IBV-derived RNAs were detected by hybridization with a 32P-labelled 590 bp 3'-UTR probe (Stirrups et al., 2000a
). Luc-containing RNAs were detected with a 1664 bp Luc-specific probe, produced by PCR with oligonucleotides corresponding to the 5' and 3' ends of the Luc gene and labelled with [32P]dCTP by using the random oligonucleotide-primed synthesis method (Feinberg & Vogelstein, 1983
). Alternatively, IBV-derived RNAs were detected non-isotopically following transfer onto Hybond XL nylon membranes (Amersham) by using a 309 bp IBV 3'-UTR probe corresponding to the last 309 nt at the 3' end of the IBV genome. The probe was labelled covalently with psoralenbiotin (BrightStar, Ambion), hybridized to the IBV-derived RNAs at 42 °C for 16 h and detected with streptavidinalkaline phosphatase and an alkaline phosphatase 1,2-dioxetane chemiluminescent substrate (CDP-star, BrightStar, BioDetect, Ambion) by exposure to film at 20 °C for 2 h. The presence of FPV in infected cell lysates was analysed by RTPCR from total cellular RNA with oligonucleotides MASH-48 and MASH-49. Production of a 1·4 kb DNA fragment, derived from the FPV 39K protein gene, was indicative of FPV (Boulanger et al., 1998
).
Analysis of reporter gene expression.
Cells (approximately 2x106) were centrifuged at 2500 r.p.m. and lysed by using 0·5 ml lysis buffer (Promega). Equal volumes (50 µl) of cell lysate and luciferase assay reagent (Promega) were mixed and analysed for luciferase activity with a luminometer (Labtech, model Jade 1253).
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Results |
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Intracellular (in situ) expression of an IBV D-RNA from an rFPV, under the control of the T7 promoter, requires the presence of a T7 sequence at the 3' end to terminate RNA synthesis. However, addition of the 128 nt T7
sequence would potentially affect replication of the D-RNA transcript by the IBV RNA-dependent RNA polymerase. Therefore, the H
R sequence was included between the end of the IBV D-RNA and the T7
sequence to allow self-cleavage of the H
RT7
sequence. The end-filled NotI site of the CD-61 cDNA sequence from pCD-61 (Pénzes et al., 1996
) was ligated to the SmaI-digested H
RT7
sequence, resulting in pIBV-Vec. Synthesis of T7-transcribed RNAs followed by self-cleavage of the H
RT7
sequence would result in a CD-61-type D-RNA with 11 extra residues, four more than on CD-61 derived from NotI-linearized pCD-61.
In order to determine whether modified CD-61 could be rescued, in vitro T7-transcribed RNAs from pIBV-Vec were electroporated into IBV-infected cells and progeny virus was serially passaged. Northern blot analysis of RNA extracted from the cells confirmed rescue of pIBV-Vec-derived CD-61 (data not shown), demonstrating that the additional HRT7
sequence had not affected rescue of CD-61 adversely, presumably resulting from self-cleavage of the sequence by the H
R.
The Luc reporter gene, under the control of an IBV TAS (Stirrups et al., 2000b ), was inserted into the PmaCI site of pIBV-Vec, resulting in pIBV-Vec-Luc. Expression of luciferase was used to monitor replication in P0 cells and the subsequent rescue of CD-61Luc in a convenient and sensitive manner and to determine whether expression of an IBV D-RNA from an rFPV would result in expression of a heterologous gene. Rescue of in vitro T7-transcribed CD-61Luc from pIBV-Vec-Luc was observed following electroporation of the RNA into IBV-infected CK cells and was confirmed by luciferase expression (data not shown).
Rescue of CD-61 from FPV recombination vector
The T7CD-61H
RT7
and T7
CD-61LucH
RT7
cassettes from pIBV-Vec and pIBV-Vec-Luc, respectively, were removed using SalI and, following end repair, were ligated into the ScaI site of the FPV recombination vector pEFL10, thus generating pEF-CD-61 and pEF-CD-61-Luc. The FPV recombination vector pEFL10 contains FPV sequences for the integration of heterologous sequences into the FPV genome via homologous recombination but does not contain an FPV promoter at the ScaI site. Plasmids pEF-CD-61 and pEF-CD-61-Luc were electroporated into CK cells co-infected with rFPV-T7 and IBV and progeny virus was serially passaged on CK cells. Cell lysates were assessed for luciferase activity and the presence of D-RNA. D-RNA CD-61 was detected by Northern blot analysis from P4 cells (data not shown) demonstrating that transiently transcribed CD-61 was capable of being rescued by helper IBV. Transient transcription of CD-61Luc resulted in rescue of the D-RNA and expression of luciferase.
Generation of rFPVs containing CD-61 and CD-61Luc cDNAs
Plasmids pEF-CD-61 and pEF-CD-61-Luc were transfected into CEF cells infected with FPV FP9 and resulting rFPVs, potentially containing the IBV D-RNA cDNA sequences, were identified by a blue plaque phenotype. DNA from plaque-purified rFPVs was analysed by dot-blot analysis with the 32P-labelled 590 bp IBV-specific 3'-UTR probe (Stirrups et al., 2000a ). The rFPVs rFPV-CD-61 and rFPV-CD-61-Luc, containing CD-61 and CD-61Luc, were identified.
Rescue of IBV D-RNA CD-61 from rFPV-CD-61
CK cells were co-infected simultaneously with rFPV-CD-61, rFPV-T7 and IBV. After incubation at 37 °C for 24 h, supernatants were filtered to remove FPV and progeny IBV and any packaged D-RNA was serially passaged on CK cells. RNA extracted from the infected cells was analysed for the presence of IBV-derived RNAs by using the 32P-labelled 590 bp IBV-specific 3'-UTR probe (Fig. 1A). CD-61 was detected initially at P1 and was observed to increase in quantity following serial passage (Fig. 1A
), confirming rescue of rFPV-CD-61-derived CD-61.
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In order to establish further that helper IBV rescued the rFPV-CD-61-derived CD-61, the initial co-infection (P0) was carried out in Vero cells. Vero cells, being of mammalian origin, do not support a productive FPV infection. Progeny virus in supernatants from P0 was filtered to remove any FPV that may have been released into the tissue culture medium through cell disruption and then serially passaged on CK cells. Northern blot analysis detected CD-61 in RNA isolated from the cells (data not shown). The experiment was repeated except that serial passage of P0-derived progeny virus and any potentially packaged CD-61 was carried out in Vero cells rather than CK cells. Analysis of RNA isolated from the Vero cells showed that CD-61 was rescued from P1P5 (Fig. 1B). This confirmed the previous observation that the helper IBV rescued CD-61 and demonstrated that Vero cells were capable of supporting the rescue of an IBV D-RNA. Overall, our results showed that rescue of rFPV-CD-61-derived CD-61 was more efficient than the rescue of electroporated in vitro T7-transcribed CD-61 RNA following analysis of IBV-derived RNAs from infected cells by Northern blot analysis.
In order to confirm that FPV was not passaged beyond P0, total RNA was extracted from P0P2-infected cells and analysed by RTPCR with oligonucleotides MASH-48 and -49, which result in a product of 1·4 kb, specific for the FPV 39K protein gene (Boulanger et al., 1998 ), in the presence of FPV. RTPCR products of 1·4 kb were only amplified from RNA isolated from P0 cells (Fig. 2
). No 1·4 kb RTPCR product was amplified from RNA isolated from cells after P0, confirming that FPV was not being transferred following serial passage of progeny virus in P0 supernatants.
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A derivative of rFPV-CD-61, rFPV-CD-61-Luc, was produced, in which the Luc gene, under the control of an IBV TAS, within the PmaCI site of domain III in CD-61 was integrated into the FPV genome, in order to evaluate the ability of the rFPV system to generate a D-RNA expressing a reporter gene. Following co-infection of CK cells with rFPV-CD-61-Luc, rFPV-T7 and IBV, P0-derived supernatants were filtered and progeny virus was serially passaged on CK cells. Cell lysates were analysed for luciferase activity and RNA isolated from the infected cells was analysed by Northern blot analysis for CD-61Luc D-RNA. Luciferase activity was detected in the cell lysates (Fig. 5) at levels up to 300-fold higher than those observed following rescue of transiently transcribed CD-61Luc from electroporated pEF-CD-61-Luc, which showed a peak luciferase activity of 2·5 RLU (2500-fold higher than background) per 106 cells at P3. This demonstrated that the rFPV system, in addition to helper virus-dependent rescue of IBV D-RNAs, supported expression of a reporter gene from a D-RNA.
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RTPCR analysis with oligonucleotides 43 (Stirrups et al., 2000a ) and Kluc-3 (complementary to nucleotides 357376 of the Luc gene) was carried out on RNA isolated from cells exhibiting luciferase activity following rescue of rFPV-CD-61-derived CD-61Luc. An RTPCR product of 449 bp was generated, indicative of the 5' end of a D-RNA-derived Luc mRNA, since oligonucleotide 43 corresponded to the 5' end of the IBV leader sequence. Sequence analysis of the 449 bp RTPCR product, generated from RNA isolated from luciferase-positive P3 cells, identified the IBV leader sequence fused to the canonical TAS-2 site of the Beaudette gene 5 TAS proximal to the Luc gene (Fig. 6B
). This confirmed the presence of an IBV-transcribed Luc-specific mRNA in luciferase-positive cell lysates following rescue of rFPV-CD-61-derived CD-61Luc, which supported results of the Northern blot analysis, which initially identified such an mRNA. The result corroborated our previous observation that the canonical TAS-2 site of the Beaudette gene 5 TAS is utilized preferentially for acquisition of the leader sequence during synthesis of a D-RNA-derived mRNA (Stirrups et al., 2000b
).
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Discussion |
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The vaccinia virus (VV) T7 RNA polymerase system (Fuerst et al., 1986 , 1987
) was used to express an MHV-derived D-RNA transiently, from transfected DNA, which was subsequently replicated by co-infected MHV helper virus (van der Most et al., 1992
). D-RNAs from the coronavirus TGEV have been rescued successfully by using a two-step amplification system that involves pol II transcription in the nucleus followed by TGEV helper virus replication in the cytoplasm (Izeta et al., 1999
). However, in this system, there is the possibility that cryptic splice sites may be present in the D-RNA sequences, although no obvious splicing was observed following the pol II amplification step with the TGEV D-RNA sequences. The MHV and TGEV results, using transfected DNA to introduce the D-RNA into cells, demonstrated that in situ transcription of a coronavirus D-RNA either by T7 RNA polymerase or pol II resulted in rescue of the D-RNA by a helper coronavirus. The transfection of cells with DNA rather than RNA has been reported to be more efficient, although transfection efficiencies vary depending on cell type.
We chose to use a poxvirus-based method as an alternative delivery system to introduce IBV D-RNAs into primary cells, followed by in situ synthesis of the D-RNA, with the aim of increasing the efficiency of rescue. Poxviruses have been employed successfully for the expression of various genes following insertion of the gene into the poxvirus genome via homologous recombination (Carroll & Moss, 1997 ; Moss, 1992
, 1996
). The expression of the gene can be either direct from a poxvirus promoter or under the control of a T7 promoter, which requires the co-infection of cells with a second recombinant virus expressing T7 RNA polymerase. The most widely used poxvirus system is based on VV; however, an important alternative to VV is FPV. Infectious FPV is only produced from avian cells, making it a more appropriate system for use with IBV. FPV replication is abortive in mammalian cells, with no production of infectious virus (Somogyi et al., 1993
). Cytopathic effects associated with FPV infection occur later in the replication cycle than those observed for VV, potentially decreasing any interference by FPV with the replication of other co-infecting viruses. We elected to use an FPV-based system in which cDNAs corresponding to the IBV D-RNAs are inserted into the FPV genome under the control of a T7 promoter. The system has three steps: (i) introduction of the D-RNA sequence into cells by infection with rFPV, potentially involving 100% of cells; (ii) in situ transcription of the D-RNA by T7 RNA polymerase expressed as a result of co-infection of cells with an rFPV expressing T7 RNA polymerase (Britton et al., 1996
); and (iii) rescue of the D-RNA by co-infecting the FPV-infected cells with helper IBV.
Co-infection of cells with rFPV-T7, IBV and either rFPV-CD-61 or rFPV-CD-61-Luc resulted in the rescue of the D-RNAs CD-61 and CD-61Luc. The D-RNAs were transcribed initially under the control of T7 RNA polymerase and then rescued in a helper IBV-dependent manner, indicating that FPV did not interfere significantly with replication of IBV in co-infected cells. Rescue of rFPV-derived D-RNAs was more efficient than the rescue of electroporated in vitro T7-transcribed D-RNAs or transiently expressed in situ T7-transcribed D-RNAs following transfection of DNA. The rFPV-derived D-RNAs were detected both in larger amounts and in cells at earlier passages and were rescued successfully in Vero cells. Work by Stirrups et al. (2000b) showed that electroporation of in vitro-transcribed CD-61Luc resulted in rescue of the D-RNA, as shown by the expression of luciferase. However, the levels of luciferase activity detected on serial passage never attained the levels observed in P0 cells. No CD-61Luc D-RNA was detected by Northern blot analysis in cells expressing luciferase, although RNAs smaller than CD-61Luc were detectable. In contrast, serial passage of the rFPV-CD-61-Luc-derived D-RNA resulted in observable amounts of the D-RNA, luciferase activities that routinely increased from P0 to P4 and the detection of a Luc-specific mRNA synthesized from CD-61Luc. The observation that the luciferase activities increased on serial passage was similar to previous observations of the expression of CAT protein following rescue of electroporated in vitro T7-transcribed CD-61CAT (Stirrups et al., 2000b
). Although our previous results demonstrated that D-RNA CD-61CAT was detectable on serial passage, no D-RNA-derived CAT-specific mRNA was detected by Northern blot analysis. However, RTPCR analysis detected the CAT-specific mRNA, indicating that the mRNA was either unstable or produced in small amounts. As observed previously with the expression of CAT from CD-61CAT and for the expression of heterologous genes from other coronavirus D-RNAs, expression of luciferase from rFPV-CD-61-Luc-derived CD-61Luc was eventually lost over continued serial passage. The observation that luciferase activity peaked at P4, with gradual loss on further passage, was concordant with the identification of smaller RNAs, detectable with the Luc probe, in RNA isolated from P3 onwards (Fig. 6A
, panel 2).
Overall, our results demonstrate that expression of IBV D-RNAs from an rFPV provides an efficient and alternative method for the rescue of coronavirus D-RNAs. Increasing the number of cells potentially containing the D-RNA increased the efficiency of rescue of a D-RNA by helper IBV.
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Acknowledgments |
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Received 17 May 2000;
accepted 9 August 2000.