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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During replication, all coronaviruses produce a 3'-co-terminal nested set of polycistronic subgenomic mRNAs by a discontinuous transcription mechanism (Baric et al., 1983 ; Sawicki & Sawicki, 1990
, 1998
). In general, only the 5'-most ORF of each coronavirus subgenomic mRNA is translated to produce the structural proteins, spike glycoprotein (S), small membrane protein (E), integral membrane protein (M) and nucleocapsid protein (N), and a number of other potential non-structural proteins of as yet unknown function. As part of the discontinuous transcription mechanism for synthesis of the mRNAs, a common 5'-terminal leader sequence, derived from the 5' end of the genome, is attached to the body sequence of each subgenomic mRNA. Conserved sequences, which we have previously termed the transcription-associated sequence (TAS; Hiscox et al., 1995
), are present along the genomic RNA, corresponding to the sites where the subgenomic mRNAs are produced, and are involved in the acquisition of the leader sequence. The TASs are found proximal to the initiation codon of the first ORF for each particular subgenomic mRNA. However, the distance between the TAS and the AUG varies for each subgenomic mRNA. The canonical octameric IBV TAS, CT(T/G)AACAA, is found 993 nt from the particular initiation codon of the 5'-most ORF for synthesis of the five IBV subgenomic mRNAs.
The availability of complete full-length cDNA copies of RNA virus genomes that can be used for the production of infectious RNA copies has proved a powerful tool for understanding the molecular biology of the viruses and for studying the role of individual genes in pathogenesis. To date, there is no complete coronavirus cDNA available for generation of an infectious RNA. Therefore, we have been developing an alternative strategy, utilizing an IBV defective RNA (D-RNA), CD-61, that functions like a minigenome as a potential RNA vector both as an expression vector and for targetted recombination. IBV D-RNA CD-61 (Pénzes et al., 1994 , 1996
) lacks internal parts of the genome but contains the sequences required for replication and for packaging into virus particles and can therefore be replicated and packaged (rescued) in a helper virus-dependent manner.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oligonucleotides.
The oligonucleotides used in this study were obtained from MWG-Biotech or Pharmacia and are listed in Table 1.
|
Production of gene cassettes.
A unique PmaCI site within the IBV D-RNA CD-61 was initially chosen for insertion of genes. The PmaCI site is within domain III of CD-61 but not within the D-RNA-specific ORF (Pénzes et al., 1994 , 1996
). However, the pZSL1190 sequence of pCD-61 (Pénzes et al., 1996
), into which the CD-61 cDNA was inserted, contained a PmaCI site within the multiple cloning site. A 72 nt PstISfiI fragment containing the PmaCI site was removed from pCD-61 and the two ends were converted to blunt ends and ligated together. A resultant plasmid, pCD-61-PmaCI, was sequenced and found to contain an 81 nt deletion and was subsequently used for the insertion of genes. A second unique site, SnaBI, which was contained in pCD-61-PmaCI but within domain II of CD-61 (Fig. 1b
) and which interrupts the D-RNA-specific ORF, was also used.
|
RNA electroporation of primary CK cells.
T7-derived RNAs corresponding to the various D-RNAs were synthesized in vitro from 2 µg of the corresponding NotI-linearized D-RNA-containing plasmids (Pénzes et al., 1996 ). CK cells (P0) were grown to 8090% confluence in 25 cm2 tissue culture flasks (Falcon) and infected with 0·5 ml Beaudette helper virus (7x107 p.f.u./ml) in allantoic fluid. At 8 h p.i., the cells were electroporated with the transcription reactions (Stirrups et al., 2000
). Following incubation of the electroporated cells for 16 h, virus (V1) in 1 ml of the supernatants was used to infect CK cells (P1) and, after 2024 h p.i., virus (V2) from the supernatants was passaged on CK cells (P2) for up to P12.
Analysis of IBV-derived RNAs.
Total cellular RNA was extracted from the Beaudette-infected CK cells (Pénzes et al., 1994 ) and electrophoresed in denaturing 1% agarose2·2 M formaldehyde gels (Sambrook et al., 1989
). The RNA was Northern blotted onto Hybond-C extra 0·45 µm nitrocellulose membranes (Amersham) and IBV-specific RNAs were detected by hybridization with 32P-labelled DNA. Several different probes were used: (i) a 590 bp IBV 3' probe, to detect all IBV-derived RNAs, corresponding to nt 2701727607 at the 3' end of IBV genome, was generated by PCR with oligonucleotides N1145 and 93/100; (ii) an IBV 5' probe, minus the leader sequence, to detect IBV genomic RNA and D-RNAs consisted of a 1120 bp AgeISphI fragment (nt 3381458); (iii) a 1664 bp Luc-specific probe, to detect D-RNAs containing the Luc gene, was produced by PCR with oligonucleotides corresponding to the 5' and 3' ends of the Luc gene; and (iv) a CAT-specific probe, to detect D-RNAs containing the CAT gene, consisted of a 305 bp MroINcoI fragment derived from the CAT gene. All the probes were labelled with [32P]dCTP by using the random oligonucleotide-primed synthesis method (Feinberg & Vogelstein, 1983
).
Analysis of reporter gene activities.
CK cells (approximately 2x106) were disrupted in cell medium and centrifuged at 2500 r.p.m. The pelleted cells were resuspended in 1 ml PBSa (Stirrups et al., 2000 ), of which 0·5 ml was used for reporter gene assay and 0·5 ml for RNA extraction. For the luciferase assay, the resuspended cells were centrifuged at 2500 r.p.m. and lysed with 0·5 ml lysis buffer (Promega). Fifty µl of the cell extract was added to 50 µl luciferase assay reagent (Promega) and analysed in a luminometer (Labtech, model Jade 1253). For the CAT assay, the resuspended cells were washed three times in PBSa, lysed in 1 ml lysis buffer (Boehringer Mannheim) and incubated at room temperature for 30 min. CAT protein was detected by ELISA (Boehringer Mannheim, product no. 1363727). Serial dilutions of the cell extracts were made and the amount of CAT protein present in the cell supernatants was determined by comparison with standard amounts of CAT protein.
Sequence analysis of D-RNA-derived mRNAs and IBV gene 5 TASs.
Total cellular RNA was extracted from P6 CK cells infected with a CAT-containing D-RNA. RTPCR was used to amplify the 5' ends of the CAT mRNAs by using oligonucleotides 43 and CATINT- and Pfu polymerase. RTPCR products were sequenced directly or cloned into SmaI-digested pTarget (Promega), from which the D-RNA-derived cDNA was sequenced. D-RNA-derived cDNAs from the cloned PCR products were analysed by A-track sequencing. RTPCR products amplified from virion RNA, derived from IBV strains H120, M41, B1648 and D207, were used to determine the genomic sequences corresponding to the gene 5 TAS region by using oligonucleotides MEND3+ and NSTART-.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression of genes by helper virus-dependent rescue of IBV D-RNAs
In vitro T7-transcribed RNAs corresponding to D-RNAs in which the Luc gene had been inserted into either the PmaCI or SnaBI sites of CD-61 were electroporated into Beaudette-infected CK cells. Luciferase activity was detected in cell lysates from the electroporated cells (P0) when compared with controls in which the Luc-containing D-RNAs were absent (Fig. 2), indicating that the D-RNAs were replicated and that a Luc mRNA was transcribed from the D-RNAs. However, serial passage of the D-RNAs rescued by helper virus resulted in lower luciferase activities, which declined after P4 (Fig. 2
). Northern blot analyses with IBV 5' and 3' probes were carried out on total RNA isolated from the P1P7 cells. No RNA of 7·9 kb, corresponding to a CD-61Luc D-RNA, was detected (data not shown). However, other RNAs of unknown origin, not detectable with the Luc-specific probe, were detected in addition to the IBV helper virus-derived RNAs. The amounts and sizes of the extraneous D-RNAs varied with different rescue experiments. Although luciferase activity was dependent on the presence of IBV helper virus and the spatial position of the Luc gene within the D-RNA did not affect expression, we concluded that the Luc-containing D-RNAs were inherently unstable. Whether the instability was due to the Luc gene sequence specifically, the presence of any heterologous sequence or expression of the luciferase protein was not known.
|
Expression of CAT protein, following rescue of D-RNAs with the CAT gene inserted in either the PmaCI or SnaBI sites of CD-61, was again dependent on the presence of IBV helper virus, a functional IBV TAS and a complete 3' UTR. Analysis of the amounts of CAT protein detected in cells following rescue of the D-RNAs in passages after P0 routinely showed a drop in the amount of CAT protein at P1, followed by a 6-fold increase in the amount of CAT protein detected at P5/P6 compared with that observed at P0 (Fig. 3a). However, the amount of CAT detected routinely decreased on further passage. Similar patterns of CAT expression were observed whether the CAT gene was inserted in the PmaCI site or the SnaBI site of CD-61 (Fig. 3a
). The expression of CAT protein was routinely observed over a higher passage number, up to P12 in one experiment, compared with expression of luciferase activity and the amount of CAT protein produced was observed to be as high as 1·6 µg from 106 cells (Fig. 3b
). It should be noted that the highest levels of CAT expression were observed following electroporation of the T7 D-RNA transcripts into Beaudette-infected Vero cells (P0) followed by serial passage of the D-RNAs in CK cells. No expression of CAT protein was observed from D-RNAs either with the TASCAT sequence inserted in the anti-sense orientation or under the control of a scrambled TAS. There was a minimal amount of CAT protein detected following rescue of the D-RNA containing the CAT gene with a mutated initiation codon, possibly resulting from detection of a truncated CAT protein expressed from a downstream in-frame AUG.
|
RTPCR analysis of RNA extracted from P6 cells containing CD-61 with the CAT gene inserted into the PmaCI site, with oligonucleotides 93/136 and 93/106, resulted in a 910 bp product. This confirmed the presence of a D-RNA with the CD-61-specific domain I/II junction (Pénzes et al., 1996 ). RTPCR analysis of the P6 RNA extracts with a CAT-specific oligonucleotide, CATINT-, and an IBV leader-specific oligonucleotide, 94/155, generated a 353 bp product, indicative of a CAT-specific mRNA containing the IBV leader sequence. Detection of CAT-specific mRNAs was only successful from rescue experiments that yielded the highest levels of CAT protein, supporting the results of Northern blot analysis, which suggested that the amounts of the CAT-specific mRNAs were very low.
Analysis of CAT expression from D-RNAs containing modified TASs
Both of the canonical TASs within the Beaudette gene 5 TAS were retained for expression of genes from CD-61 because it was not known whether one or other or both sequences are utilized for mRNA synthesis. Therefore, a series of gene cassettes with the CAT gene under the control of one TAS with the other sequence scrambled was produced for insertion into CD-61 to determine whether one sequence was sufficient or used preferentially or whether both can be utilized. The D-RNAs were constructed as before except that oligonucleotides IBV5CAT-TAS-1-Scr, IBV5CAT-TAS-2-Scr and IBV5CATScrambled replaced oligonucleotide T7IBV5CATStart for generation of the gene cassettes. The resulting gene cassettes (Fig. 4a) were initially cloned into pBluescript to assess expression of the CAT gene. The TASCAT cassettes containing the modified gene 5 TASs ST/T, T/ST and ST/ST, in which the first canonical consensus sequence (TAS-1), the second canonical sequence (TAS-2) or both sequences scrambled, respectively, were inserted into the PmaCI site in pCD-61-PmaCI. The TASCAT cassette containing both TASs was designated T/T. Sequence analysis confirmed that the sequences incorporated into the D-RNAs were as expected.
|
In order to confirm that RNA isolated from the P3P8 cells contained D-RNAs, two separate RTPCRs were carried out following rescue of D-RNAs CD-61T/TCAT, CD-61ST/TCAT, CD-61T/STCAT and CD-61ST/STCAT. The first RTPCR used oligonucleotides 93/106 and 93/136, which produced a product of 910 bp, confirming the presence of an RNA containing the CD-61 domain I/II-specific junction. The second RTPCR used oligonucleotides 41 and CATINT-. The former anneals proximal to the PmaCI site in CD-61 and the latter corresponds to a sequence within the CAT gene, to confirm the presence of a CAT-containing D-RNA. A product of 400 bp was produced, confirming the presence of an RNA containing the CAT gene within the PmaCI site of a CD-61-derived D-RNA.
Sequence analysis of mRNAs transcribed from CD-61CAT D-RNAs
RTPCR with oligonucleotides 43 and CATINT- was used to analyse the 5' ends of the CAT mRNAs transcribed from CD-61ST/TCAT, CD-61T/STCAT and CD-61T/TCAT. Oligonucleotide 43 corresponded to the 5' end of the IBV leader sequence. The RTPCRs were expected to generate either a 342 or 353 bp product, depending on the canonical TAS used for leader sequence acquisition. The RTPCR products were sequenced directly and after cloning to determine whether there was heterogeneity in the use of the two TASs from CD-61T/TCAT. Sequence analysis of mRNAs transcribed from the D-RNAs showed that leader sequence acquisition occurred on the unmodified TASs in CD-61ST/TCAT and CD-61T/STCAT (Fig. 5a, b
). The sequence between the TAS and the CAT AUG from the mRNA transcribed from CD-61T/STCAT was 11 nt, representing scrambled TAS-2, longer than the corresponding sequence on the mRNA transcribed from CD-61ST/TCAT (Fig. 5a
). No nucleotide substitutions were found between the 11 nt sequence determined from the mRNA and the sequence present on the input D-RNA, indicating that the heterologous sequence did not appear to be detrimental to transcription of the CAT mRNA from CD-61T/STCAT.
|
Sequence analysis of IBV gene 5 TASs
Expression of the CAT gene from IBV D-RNAs using the Beaudette gene 5 TAS or modified versions of the sequence showed that both canonical consensus sequences present in the Beaudette gene 5 TAS were functional. However, when both sequences were present, the 3' canonical sequence, TAS-2, was used for leader sequence acquisition. In addition to the Beaudette sequence, only two other IBV gene 5 sequences have been determined: CU-T2 (Jia & Naqi, 1997 ) and KB8523 (Sutou et al., 1988
). Comparison of the gene 5 sequences from these two strains with the Beaudette sequence showed that CU-T2 and KB8523 only had the canonical sequence equivalent to Beaudette TAS-2. The sequence equivalent to the Beaudette gene 5 TAS-1 site in CU-T2 and KB8523 contained two nucleotide substitutions.
In order to investigate whether other strains of IBV contained one or two canonical TASs for gene 5, we determined the gene 5 TASs from four other IBV strains, H120, M41, B1648 and D207. RTPCRs were carried out on genomic RNA with oligonucleotides MEND3+ and NSTART-, which corresponded to sequences in the 3' end of the M gene and the 5' end of the N gene, respectively. RTPCR products of the expected size, 755 bp, were obtained from the four genomic RNAs and sequenced directly by using oligonucleotides TAS5a+, TAS5a-, TAS5b+, TAS5c+ and TAS5c-. Comparison of the gene 5 TASs determined from the seven IBV strains (Fig. 6) showed that all of the strains except Beaudette contained a single TAS, equivalent to Beaudette TAS-2. The region of the genomic RNAs corresponding to the Beaudette TAS-1 site contained two or three nucleotide substitutions.
|
Sequence analysis of the RTPCR products showed that the TAS-2 site was utilized for acquisition of leader sequence during transcription of mRNA 5. RTPCR products from 22 clones were sequenced, 10 completely and 12 by A-track sequencing. Analysis of the sequences showed that 21 were derived from an mRNA 5 transcribed from TAS-2 and one from an mRNA 5 transcribed from TAS-1. Our results showed that the Beaudette gene 5 TAS-2 canonical sequence is used preferentially for leader sequence acquisition. However, the TAS-1 site can function as a leader sequence acquisition site in the absence of the TAS-2 site.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Other coronavirus-derived D-RNAs have been used for the expression of heterologous genes. D-RNAs based on the D-RNA DIssE (Makino & Lai, 1989 ) from murine hepatitis virus (MHV) have been used to express CAT (Liao & Lai, 1994
), MHV haemagglutininesterase (Liao et al., 1995
; Lai et al., 1997
; Zhang et al., 1998
) and murine IFN-
(Lai et al., 1997
; Zhang et al., 1997
). The D-RNA DI-C from the porcine coronavirus transmissible gastroenteritis virus (TGEV) has been used to express
-glucuronidase (GUS; Izeta et al., 1999
). Expression of genes from MHV D-RNAs was not stable; CAT activity was not observed beyond P2 (Liao & Lai, 1994
), expression of MHV haemagglutininesterase beyond P3 (Liao et al., 1995
) and expression of murine IFN-
beyond P4 (Zhang et al., 1997
). Expression of GUS in the TGEV D-RNA system was also unstable, although expression of GUS was detected up to P10 in some cases. Expression of CAT appeared to be more stable than expression of Luc in the IBV system, but even expression of CAT decreased after P6. The observation that coronavirus D-RNAs expressing heterologous genes are relatively unstable could result from several factors. The most likely explanation may be the presence of non-coronavirus-derived sequences in the D-RNAs. This may have several effects, ranging from some fundamental interference on replication or packaging of the D-RNAs to the natural tendency of D-RNAs to evolve by removal of unnecessary sequences. An effect on packaging is harder to explain, as gene expression was observed after serial passage of the IBV and TGEV D-RNAs. The D-RNAs were sufficiently packaged during passages P1P6 to infect cells on subsequent passage.
The IBV system was demonstrated to be capable of producing CAT protein to levels greater than 1·6 µg per 106 cells and to be capable of producing CAT protein up to P12. The TGEV D-RNA system produced GUS protein to about 1·0 µg per 106 cells. The authors reported that this could be increased 510-fold by using specific transcription regulatory sequences and that the system was capable of expressing GUS up to P10 (Izeta et al., 1999 ). The TGEV D-RNA expression system used a two-step amplification system, in which the D-RNA was expressed initially in P0 cells by Pol II transcription of a transfected cDNA under the control of a cytomegalovirus promoter in the cell nucleus, followed by TGEV helper-dependent replication of the D-RNA in the cytoplasm.
Studies to investigate coronavirus transcription have used either MHV (Makino et al., 1991 ; Joo & Makino, 1992
, 1995
; Makino & Joo, 1993
; van der Most et al., 1994
; van Marle et al., 1995
) or bovine coronavirus (BCoV; Krishnan et al., 1996
) D-RNAs containing one or more TASs for the expression of mRNAs. Insertion of two TASs within an MHV D-RNA resulted in decreased transcription of the larger mRNA if the two TASs were 23 nt apart. The inhibition decreased as the distance between the two TASs was increased, resulting in equal amounts of the mRNAs if the TASs were separated by 124 nt (Joo & Makino, 1995
). The authors concluded that the TAS for the smaller mRNA had some inhibitory effect on the upstream TAS. Further studies on MHV D-RNAs containing combinations of up to three TASs separated by 361761 nt showed that the position of the TASs affected the amounts of the mRNAs produced (van Marle et al., 1995
). The largest mRNA (the 5'-most TAS) was produced in the smallest amount whether it was the only TAS in the D-RNA or it was in combination with one or more TASs. The middle TAS [site B of van Marle et al. (1995)
] produced the largest amount of mRNA whether alone or in conjunction with one or more TASs. The observation that the downstream TASs attenuated upstream TASs but not vice versa led the authors to conclude that their results were consistent with the transcription model proposed by Sawicki & Sawicki (1990)
. Studies on coronavirus transcription using a BCoV D-RNA showed that transcription occurred preferentially at the 3'-most TAS after insertion of either a duplicate or triplicate 27 nt tandem repeat sequence containing the BCoV canonical TAS within a D-RNA (Krishnan et al., 1996
). The BCoV heptameric canonical TASs, UCUAAAC, were separated by 20 nt in the tandem repeats.
The IBV TAS derived from gene 5 of the Beaudette strain of IBV, used in this study, naturally contains a tandem repeat of the octameric IBV canonical TAS, CUUAACAA, separated by 3 nt. We observed that both TASs were functional for the production of a translationally active mRNA in the absence of the other sequence. Analysis of the mRNA produced from a D-RNA containing the CAT gene under the control of the gene 5 TAS showed that the downstream or 3'-most TAS (TAS-2) was used preferentially for transcription of the CAT mRNA. Analysis of subgenomic mRNA 5 derived from gene 5 in Beaudette-infected cells confirmed that TAS-2 was the preferential site for mRNA 5 transcription. Analysis of the gene 5 sequences from other IBV strains showed that only one canonical TAS was present, corresponding to the Beaudette TAS-2.
Our results are consistent with the observation from both the MHV and BCoV experiments that, if two or more canonical TASs are present in close proximity, the 3'-most sequence is used preferentially. The observation that the TAS-2 site of Beaudette gene 5 is used preferentially is in agreement with the conclusions of van Marle et al. (1995) and consistent with the coronavirus transcription model of Sawicki & Sawicki (1990)
. This can be explained if the polymerase terminates preferentially at TAS-2 for synthesis of mRNA 5 and if read-through for synthesis from TAS-1 is rare. Presumably, if both TASs acted as efficient terminators for mRNA 5 synthesis, this would have a detrimental effect on the synthesis of the longer mRNAs.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
c Present address: Departments of Pathology and Cell Biology (BML 342), Yale University School of Medicine, 310 Cedar St, New Haven, CT 06510, USA.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baric, R. S., Stohlman, S. A. & Lai, M. M. C. (1983). Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains.Journal of Virology48, 633-640.[Medline]
Binns, M. M., Boursnell, M. E. G., Cavanagh, D., Pappin, D. J. C. & Brown, T. D. K. (1985). Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV.Journal of General Virology66, 719-726.[Abstract]
Binns, M. M., Boursnell, M. E. G., Tomley, F. M. & Brown, T. D. K. (1986). Comparison of the spike precursor sequences of coronavirus IBV strains M41 and 6/82 with that of IBV Beaudette.Journal of General Virology67, 2825-2831.[Abstract]
Boursnell, M. E. G., Brown, T. D. K., Foulds, I. J., Green, P. F., Tomley, F. M. & Binns, M. M. (1987). Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus.Journal of General Virology68, 57-77.[Abstract]
Cavanagh, D. & Naqi, S. (1997). Infectious bronchitis. In Diseases of Poultry, pp. 511-526. Edited by B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid & H. W. Yoda. Ames, IA: Iowa State University Press.
Darbyshire, J. H., Rowell, J. G., Cook, J. K. A. & Peters, R. W. (1979). Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures.Archives of Virology61, 227-238.[Medline]
Davelaar, F. G., Kouwenhoven, B. & Burger, A. G. (1984). Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands.Veterinary Quarterly6, 114-120.[Medline]
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Analytical Biochemistry132, 6-13.[Medline]
Hiscox, J. A., Mawditt, K. L., Cavanagh, D. & Britton, P. (1995). Investigation of the control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts.Journal of Virology69, 6219-6227.[Abstract]
Izeta, A., Smerdou, C., Alonso, S., Pénzes, Z., Mendez, A., Plana-Durán, J. & Enjuanes, L. (1999). Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.Journal of Virology73, 1535-1545.
Jia, W. & Naqi, S. A. (1997). Sequence analysis of gene 3, gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2.Gene189, 189-193.[Medline]
Joo, M. & Makino, S. (1992). Mutagenic analysis of the coronavirus intergenic consensus sequence.Journal of Virology66, 6330-6337.[Abstract]
Joo, M. & Makino, S. (1995). The effect of two closely inserted transcription consensus sequences on coronavirus transcription.Journal of Virology69, 272-280.[Abstract]
Krishnan, R., Chang, R. Y. & Brian, D. A. (1996). Tandem placement of a coronavirus promoter results in enhanced mRNA synthesis from the downstream-most initiation site.Virology218, 400-405.[Medline]
Kusters, J. G., Niesters, H. G. M., Lenstra, J. A., Horzinek, M. C. & van der Zeijst, B. A. M. (1989). Phylogeny of antigenic variants of avian coronavirus IBV. Virology169, 217-221.[Medline]
Lai, M. M., Zhang, X., Hinton, D. & Stohlman, S. (1997). Modulation of mouse hepatitis virus infection by defective-interfering RNA-mediated expression of viral proteins and cytokines.Journal of Neurovirology3, S33-S34.[Medline]
Lambrechts, C., Pensaert, M. & Ducatelle, R. (1993). Challenge experiments to evaluate cross-protection induced at the trachea and kidney level by vaccine strains and Belgian nephropathogenic isolates of avian infectious bronchitis virus.Avian Pathology22, 577-590.
Liao, C.-L. & Lai, M. M. C. (1994). Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.Journal of Virology68, 4727-4737.[Abstract]
Liao, C.-L., Zhang, X. & Lai, M. M. C. (1995). Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutininesterase.Virology208, 319-327.[Medline]
Makino, S. & Joo, M. (1993). Effect of intergenic consensus sequence flanking sequences on coronavirus transcription.Journal of Virology67, 3304-3311.[Abstract]
Makino, S. & Lai, M. M. C. (1989). High-frequency leader sequence switching during coronavirus defective interfering RNA replication.Journal of Virology63, 5285-5292.[Medline]
Makino, S., Joo, M. & Makino, J. K. (1991). A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. Journal of Virology65, 6031-6041.[Medline]
Meulemans, G., Carlier, M. C., Gonze, M., Petit, P. & Vandenbroeck, M. (1987). Incidence, characterisation and prophylaxis of nephropathogenic avian infectious bronchitis viruses. Veterinary Record120, 205-206.[Medline]
Niesters, H. G. M., Lenstra, J. A., Spaan, W. J. M., Zijderveld, A. J., Bleumink-Pluym, N. M. C., Hong, F., van Scharrenburg, G. J. M., Horzinek, M. C. & van der Zeijst, B. A. M. (1986). The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains.Virus Research5, 253-263.[Medline]
Pause, A., Belsham, G. J., Gingras, A. C., Donze, O., Lin, T. A., Lawrence, J. C.Jr & Sonenberg, N. (1994). Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function.Nature371, 762-767.[Medline]
Pénzes, Z., Tibbles, K., Shaw, K., Britton, P., Brown, T. D. K. & Cavanagh, D. (1994). Characterization of a replicating and packaged defective RNA of avian coronavirus infectious bronchitis virus.Virology203, 286-293.[Medline]
Pénzes, Z., Wroe, C., Brown, T. D., Britton, P. & Cavanagh, D. (1996). Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.Journal of Virology70, 8660-8668.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sawicki, S. G. & Sawicki, D. L. (1990). Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis.Journal of Virology64, 1050-1056.[Medline]
Sawicki, S. G. & Sawicki, D. L. (1998). A new model for coronavirus transcription.Advances in Experimental Medicine and Biology440, 215-219.[Medline]
Shaw, K., Britton, P. & Cavanagh, D. (1996). Sequence of the spike protein of the Belgian B1648 isolate of nephropathogenic infectious bronchitis virus.Avian Pathology25, 607-611.
Stern, D. F. & Kennedy, S. I. T. (1980). Coronavirus multiplication strategy. I. Identification and characterization of virus-specific RNA.Journal of Virology34, 665-674.[Medline]
Stirrups, K., Shaw, K., Evans, S., Dalton, K., Cavanagh, D. & Britton, P. (2000). Leader switching occurs during the rescue of defective RNAs by heterologous strains of the coronavirus infectious bronchitis virus.Journal of General Virology81, 791-801.
Sutou, S., Sato, S., Okabe, T., Nakai, M. & Sasaki, N. (1988). Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus.Virology165, 589-595.[Medline]
van der Most, R. G., de Groot, R. J. & Spaan, W. J. M. (1994). Subgenomic RNA synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation.Journal of Virology68, 3656-3666.[Abstract]
van Marle, G., Luytjes, W., van der Most, R. G., van der Straaten, T. & Spaan, W. J. (1995). Regulation of coronavirus mRNA transcription.Journal of Virology69, 7851-7856.[Abstract]
Zhang, X., Hinton, D. R., Cua, D. J., Stohlman, S. A. & Lai, M. M. (1997). Expression of interferon-gamma by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity.Virology233, 327-338.[Medline]
Zhang, X., Hinton, D. R., Park, S., Parra, B., Liao, C.-L., Lai, M. M. & Stohlman, S. A. (1998). Expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering RNA alters viral pathogenesis.Virology242, 170-183.[Medline]
Received 13 January 2000;
accepted 16 March 2000.