1 Department of Medical Microbiology, Center of Infectious Disease, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
2 Department of Molecular Microbiology, Washington University Medical School, St Louis, MO, USA
Correspondence
Peter Bredenbeek
p.j.bredenbeek{at}lumc.nl
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ABSTRACT |
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Present address: Institute for Animal Science and Health (ID-DLO), 8200AB Lelystad, The Netherlands.
Present address: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, USA.
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INTRODUCTION |
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The YF genome is a positive-stranded RNA molecule of 11·8 kb, with a 5' cap structure and a non-polyadenylated 3' terminus (Rice et al., 1985). The RNA encodes a single open reading frame (ORF) flanked by 5' and 3' non-translated regions (NTRs), which are 118 and 565 bases in length, respectively. Translation of YF RNA results in the production of a precursor protein that is cleaved by host and viral proteases to produce the mature viral proteins (see Lindenbach & Rice, 2001
, for a review). The N-terminal one-third of this polyprotein encompasses the structural proteins (C-prM-E). Proteolytic processing of the remainder of the polyprotein yields the viral non-structural (NS) proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). Replication of the viral genome occurs in the cytoplasm and is associated with cellular membranes (reviewed by Lindenbach & Rice, 2001
).
All the mosquito-borne flaviviruses share conserved RNA sequences and structures (Fig. 1A). Sequence comparison and RNA secondary structure predictions of the 3'-NTR have revealed several short, well-conserved sequences and indicated that the 3'-terminal region (approximately 90 bases) can be folded in a conserved stemloop structure (3'-SS) (Brinton et al., 1986
; Hahn et al., 1987
; Proutski et al., 1997a
; Wengler & Castle, 1986
). Apart from the sequence 5'-CACAG-3' in the bulge at the top and AU and GC base pairs at the very bottom, this stemloop structure is not well conserved in primary sequence. A short conserved sequence (CS1;
26 nucleotides) has been identified upstream of 3'-SS. Complementarity between CS1 and a conserved sequence at the 5' end of the YF ORF (5'-CS) has been proposed to result in a long-range intramolecular RNA interaction (Hahn et al., 1987
). Recent experiments suggest that base-pairing between these sequences is essential for RNA replication of a Kunjin virus (KUN) replicon (Khromykh et al., 2001
).
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Based on computer-assisted RNA folding, phylogenetic sequence comparisons and biochemical and biophysical probing, several models for the RNA structure of the 3' end of the genome of the mosquito-borne flaviviruses have been proposed (Blackwell & Brinton, 1997; Brinton et al., 1986
; Hahn et al., 1987
; Olsthoorn & Bol, 2001
; Proutski et al., 1999
; Shi et al., 1996
)). Although these studies do not yield a consensus model for the flavivirus 3'-NTR structure, it is evident that the folding of the 3'-NTR is complex and involves many stemloop structures and some potential RNA pseudoknots. DEN mutants with deletions in the 3'-UTR have been described (Men et al., 1996
), but their analysis does not favour any particular current RNA structure model.
Attempts to construct a stable, full-length infectious YF cDNA in E. coli plasmid and phage vectors have been unsuccessful due problems with the genetic stability of the full-length clone in the prokaryotic host (Rice et al., 1989
). This problem was circumvented by using two plasmids and an in vitro ligation approach to create a full-length YF cDNA that could be used for the in vitro transcription of infectious YF RNA (Rice et al., 1989
). Although cumbersome, this approach yielded the first functional flavivirus cDNA for in vitro transcription of infectious YF RNA.
In this study, we describe the construction and characterization of full-length YF cDNA in a low-copy-number vector that is stable in several different bacterial strains. The in vitro-transcribed RNA from this clone was shown to be highly infectious. The infectious clone was used to analyse the requirement for the conserved flavivirus RNA elements in YF replication.
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METHODS |
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Plasmid constructions.
Standard nucleic acid methodologies were used (Ausubel et al., 2000; Sambrook et al., 1989
). The E. coli strain MC1061 was used for routine cloning purposes, whereas electro-competent E. coli Sure cells (Stratagene) were used as a host for the construction of full-length YF cDNAs. The cDNA fragments for the construction of the full-length YF cDNA were taken from the plasmids pHYF5'3'IV and pYFM5.2. Plasmid pYFM5.2 has been described (Rice et al., 1989
). pHYF5'3'IV is a phagemid and contains, in addition to the YF sequences of pYF5'3'IV (Rice et al., 1989
), the f1 origin for filamentous phage replication. Plasmid pACNR1181 was created from the low-copy-number vector pACNR1180, which contains the polylinker cassette of pSL1180 (Pharmacia) (Ruggli et al., 1996
). After digestion of pACNR1180 with AatII and NotI, a short spacer sequence was inserted that resulted in the destruction of the AatII site and deleted all the restriction enzyme sites between NotI and SalI in the polylinker cassette. The resulting pACNR1181 was used as a vector to assemble a full-length YF-17D cDNA. pHYF5'3'IV was digested with NotI and XhoI. A 5·1 kb DNA fragment that contained the SP6 RNA polymerase promoter directly fused to the YF 5' end, the YF 5' 2271 bp, a small spacer element and the YF 3' 2586 nucleotides was isolated and cloned in NotI/XhoI-digested pACNR1181. The resulting plasmid, pACNR1181YF5'3'IV, was digested with NsiI and AatII and ligated to the 6747 bp NsiIAatII fragment from pYFM5.2 encompassing the middle part of the YF genome (nt 16558402). This resulted in the construction of pACNR/FLYF-17Dx (Fig. 1B
), which contained a full-length YF-17D cDNA.
Deletion mutagenesis of conserved RNA sequences and structures.
All the deletion mutants were initially created in pHYF5'3'IV. The partial deletion of the YF 5'-CS (Table 1) was constructed by fusion PCR (Landt et al., 1990
). All the other deletion mutants were created using uridylated single-stranded pHYF5'3'IV DNA as a template to introduce additional restriction enzyme sites (Kunkel, 1985
) flanking the conserved RNA sequence and structural elements in the viral 3'-NTR. HindIII sites were inserted immediately 5' and 3' of the RS, CS1 and CS2 sequences. The created plasmids were digested with HindIII and religated to create the
RS,
CS1CS2,
CS1 and
CS2 mutants (Table 1
). Using the same uridylated template and strategy, an additional XhoI site was introduced at nt 10776. This plasmid was cut with XhoI and religated to yield the
SS mutant (Table 1
). The relevant parts of these pHYF5'3'IV derivatives were verified by DNA sequencing and then cloned into the full-length YF cDNA.
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RNA transfections.
A transfection plaque assay (Grakoui et al., 1989) on SW13 cells was used to determine the infectivity of YF-17D transcripts in p.f.u. (µg RNA)-1. In short, almost confluent monolayers of SW13 cells in 35 mm diameter dishes were washed twice with PBS lacking Ca2+ and Mg2+. A mixture of 0·11·0 ng wt or mutant YF-17D RNA transcripts and 4 µg lipofectin (Life Technologies) in 200 µl PBS was added to the cells. After 15 min, the transfection mixture was removed, the cells were washed once with PBS and a DMEM/agarose overlay was applied as described for the plaque assays. Plaques were identified by crystal-violet staining after incubation for 4 days at 37 °C (Rice et al., 1989
).
For direct analysis of experiments in which viral RNA synthesis and protein expression was analysed directly in the transfected cells, BHK-21J cells were electroporated with 5 µg of in vitro-transcribed YF RNA as described previously (Lindenbach & Rice, 1997). Aliquots were taken from the medium of the transfected cells to quantify the virus yields.
Immunofluorescence.
Transfected cells were grown on coverslips. At 24 h post-transfection or infection, the cells were fixed with 3 % paraformaldehyde in PBS (pH 7·4) for at least 30 min and washed with PBS containing 10 mM glycine. Following permeabilization with 0·1 % Triton X-100 in PBS, the cells were incubated in PBS containing 2 % horse serum for 1 h to minimize non-specific immunofluorescence. Indirect immunofluorescence was carried out with a 1 : 1000 dilution of mAb 1A5 (provided by J.J. Schlesinger) in PBS, which is specific for the YF NS1 protein (Schlesinger et al., 1983) and visualized with a secondary Cy3-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted 1 : 1000.
Labelling and analysis of viral RNAs.
In general, 3 ml of the electroporated BHK-21J cell suspension (approximately 1·5x106 cells) was placed in 35 mm plates in DMEM containing 2 % FCS. At the indicated times post-electroporation (p.e.), the medium was replaced with 750 µl medium containing 2 µg actinomycin D ml-1 and 50 µCi [3H]uridine ml-1. At 24 h p.e., RNA was isolated with Trizol (Life Technologies) and resuspended in 21 µl H2O. One-third of the RNA was denatured with glyoxal and DMSO and analysed by electrophoresis in 0·8 % MOPS/agarose gels (Sambrook et al., 1989). Gels were prepared for fluorography as described previously (Bredenbeek et al., 1993
).
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RESULTS |
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XhoI-linearized pACNR/FLYF-17Dx template was used for in vitro RNA transcription and the resulting full-length YF transcripts were electroporated into BHK-21J cells. The transfected cells were fixed at 24 h p.e. and analysed for the expression of YF NS1 by immunofluorescence microscopy. Many of the electroporated cells showed a perinuclear, punctated signal when stained with an NS1-specific antibody (Fig. 2A). As a control, cells were transfected with a truncated YF-17Dx transcript lacking the 3' terminal 155 nucleotides of the YF genome (XbaI site in Fig. 1B
) and therefore unlikely to be replication-competent. The cells transfected with the truncated YF transcripts failed to express any detectable NS1 protein (Fig. 2B
). These results demonstrated that YF RNA transcribed from pACNR/FLYF-17Dx was replication-competent and that the NS1 signal was not merely the result of translation of the input RNA.
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To compare the growth characteristics of the mutants and the parental viruses BHK-21J cells were infected at an m.o.i. of 10 with either the RS or
CS2 mutants or with wt YF-17Dx virus. Virus was harvested at 8 h intervals and the yield was determined by plaque assay (Fig. 6
). All three viruses showed a rapid increase in titre between 8 and 24 h, with virus production peaking at around 32 h and than levelling off. The kinetics of
RS and
CS2 virus production appeared a little slower than wt virus and the maximum titre was also somewhat lower. Both the
RS and
CS2 mutants showed a clear cytopathic effect.
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DISCUSSION |
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The stability of pACNR/FLYF-17Dx was extensively tested in the E. coli strains Sure, DH5 and MC1061. No changes in E. coli colony morphology or growth characteristics were observed during the passaging of pACNR/FLYF-17Dx in these bacteria. More importantly, RNA transcribed from plasmid DNA isolated at passages 5 and 10 showed a similar specific infectivity as RNA derived from the originally isolated plasmids. These results demonstrate the successful construction of a stable full-length YF cDNA in a plasmid vector that can be used for in vitro transcription of highly infectious viral RNA. Apart from the mutagenesis studies that are described in this report, the pACNR/FLYF-17Dx clone has already been used successfully in other studies (Amberg & Rice, 1999
; Kummerer & Rice, 2002
; Lindenbach & Rice, 1999
).
As for all RNA viruses, the 5'- and 3'-NTRs of the YF genome are believed to play a crucial role in the initiation of viral RNA synthesis. Both the YF 5'- and 3'-NTRs contain sequence motifs and/or stemloop structures (Fig. 1) that are well conserved among flaviviruses. It has been suggested that these domains are essential for viral RNA synthesis. In this study, the role of the conserved flavivirus sequences (5'-CS, RS, CS2 and CS1) in virus replication was analysed, as well as the stemloop structure (SS) at the 3' end of the viral genome.
Deleting the RS domain was well tolerated by YF and resulted in a virus with similar biological properties to the YF-17Dx virus. A domain comparable with the YF RS sequences has also been found in the JE virus serogroup, but there is no sequence similarity in the RS domains of YF and JE-like viruses. For KUN, a deletion of 352 nucleotides in the 5'-proximal half of the 3'-NTR resulted in a five- to tenfold reduction in replication (Khromykh & Westaway, 1997). This deletion was upstream of CS2 and included the RS sequences. RS is lacking in DEN. Deletions in this region of DEN type 4 yielded viruses with delayed growth properties and a smaller plaque size (Men et al., 1996
). These deletions always included the DEN CS2B element and are therefore difficult to compare with the YF-17D
RS mutant, which retained CS2.
RNA secondary structure analysis of flaviviruses predicts that the YF CS2 sequence forms an independent stemloop structure within the 3'-NTR (Olsthoorn & Bol, 2001; Proutski et al., 1997b
). The fact that this stemloop structure is well conserved in both pathogenic and vaccine strains of YF suggests that this sequence is essential for virus replication. However, analysis of YF-17D
CS2 showed that this sequence can be deleted with relatively minor effects on virus replication. Compared with YF-17Dx, the rate of RNA synthesis by YF-17D
CS2 was somewhat decreased. However, the kinetics of virus production of YF
CS2 was similar to YF-17Dx. The reason for the observed variability in YF-17D
CS2 plaque size and morphology is unclear. Small and turbid plaques have also been reported for DEN type 4 deletion mutants involving CS2 (Men et al., 1996
). RT-PCR and sequence analysis showed that the deletion was still present in YF-17D
CS2 progeny virus; however, second-site revertants cannot be excluded.
All the mosquito-borne flaviviruses contain a 5' conserved sequence that is located a few nucleotides downstream of the translation initiation codon. This 5'-CS is actually part of the flavivirus coding sequence. It has been suggested that the flavivirus 5'-CS sequence base pairs with CS1 via a long-range RNA interaction (Hahn et al., 1987; Khromykh et al., 2001
). This interaction is predicted to result in a pan-handle-like structure that is hypothesized to be required for virus replication by modulating virus translation (Khromykh et al., 2001
). Recently it was shown, using a KUN replicon and a DEN NS5-based in vitro polymerase assay, that complementarity between 5'-CS and CS1 is a prerequisite for viral RNA synthesis (Khromykh et al., 2001
; You et al., 2001
). The observation that the partial deletion of YF CS1 is lethal for viral RNA synthesis, as reported in this study, is in agreement with a model that requires circularization at some stage of the flavivirus replication cycle. The involvement of CS1 in the cyclization of the viral genomic RNA does not exclude the possibility that the CS1 sequence might also take part in an alternative structure involving base-pairing to other domains within the 3'-NTR. (Proutski et al., 1997a
, b
; Rauscher et al., 1997
; Shi et al., 1996
). It can be hypothesized that these different RNA structures involving CS1 are metastable and in equilibrium with each other. This equilibrium may be influenced by factors like RNAprotein interactions and the cellular environment, thereby regulating negative-strand RNA synthesis versus positive-strand RNA synthesis or availability of the RNA for translation.
Another important element of the 3'-NTR RNA is formed by the 3'-terminal 86 nucleotides that are deleted in the YF-17DSS mutant. In all the models describing the flavivirus 3'-NTR RNA, these nucleotides are involved in the formation of a hairpin structure (Hahn et al., 1987
; Mackenzie et al., 2001
; Proutski et al., 1997b
; Shi et al., 1996
). Deletion of the 3' 86 nucleotides was lethal for YF RNA synthesis as shown by the results with the YF-17D
SS mutant.
Finally, as one of the safest and most effective human vaccines, YF-17D recombinants are being vigorously explored as candidate vaccines for other flavivirus diseases, such as JE (Monath, 2002), DEN (Der Most et al., 2000
) and West Nile virus (Monath, 2001
), as well as for cancer vaccines (McAllister et al., 2000
). The availability of full-length stable YF cDNA clones should help in these efforts.
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ACKNOWLEDGEMENTS |
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Received 26 September 2002;
accepted 10 January 2003.